US 20100151483A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0151483 A1 Hornbeck et al. (43) Pub. Date: Jun. 17, 2010
(54) REAGENTS FOR THE DETECTION OF Related U.S. Application Data PROTEIN PHOSPHORYLATION IN (60) Provisional application No. 60/830,549, filed on Jul. SIGNALNG PATHWAYS 13, 2006. (75) Inventors: Peter Hornbeck, Magnolia, MA Publication Classification (US); Valerie Goss, Seabrook, NH (51) Int. Cl. (US); Kimberly Lee, Seattle, WA GOIN 33/53 (2006.01) (US); Ting-Lei Gu, Woburn, MA C07K 6/8 (2006.01) (US); Albrecht Moritz, Salem, MA (52) U.S. Cl...... 435/7.1:530/387.9 (US) (57) ABSTRACT Correspondence Address: The invention discloses novel phosphorylation sites identi Nancy Chiu Wilker, Ph.D. fied in signal transduction proteins and pathways, and pro Chief Intellectual Property Counsel vides phosphorylation-site specific antibodies and heavy-iso CELL SIGNALING TECHNOLOGY, INC., 3 tope labeled peptides (AQUA peptides) for the selective Trask Lane detection and quantification of these phosphorylated sites/ Danvers, MA 01923 (US) proteins, as well as methods of using the reagents for Such purpose. Among the phosphorylation sites identified are sites (73) Assignee: CELL SIGNALNG occurring in the following protein types: adaptor/scaffold TECHNOLOGY, INC., Danvers, proteins, adhesion/extracellular matrix protein, apoptosis MA (US) proteins, calcium binding proteins, cell cycle regulation pro teins, chaperone proteins, chromatin, DNA binding/repair/ (21) Appl. No.: 12/309,312 replication proteins, cytoskeletal proteins, endoplasmic reticulum or golgi proteins, enzyme proteins, G/regulator (22) PCT Filed: Jul. 13, 2007 proteins, inhibitor proteins, motor/contractile proteins, phos phatase, protease, Ser/Thr protein kinases, protein kinase PCT/US2007/073534 (Tyr)s, receptor/channel/cell surface proteins, RNA binding (86). PCT No.: proteins, transcriptional regulators, tumor suppressor pro S371 (c)(1), teins, ubiquitan conjugating system proteins and proteins of (2), (4) Date: Feb. 19, 2010 unknown function. Patent Application Publication Jun. 17, 2010 Sheet 1 of 22 US 2010/0151483 A1
FIGURE 1
crude protein (e.g., cell extractor cellular organelle) or invirokinase reaction or purified phosphoprotein
obtain protein faction (e.g., lysis, organic extraction, detergent treatment, etc.) immobilize general protein modification-specific antibody to resin denature proteins (e.g., heat treatment)
digest crude protein mixture to peptides withimmobilized trypsin or other protease contact digest with antibody-resin and incubate (e.g., 4, 1-16 hours)
remove unbound peptides by centrifugation, filtration, or column washing
wash antibody-resin extensively t elute bound peptides (e.g., 30% acetic acid, 0.1 M glycine, pH 2.3, or 0.1%trifluoroacetic acid)
desalt and concetrate peptides withmicrocolumn (e.g., reversed-phase ZipTip) (optional)
analyze, e.g., MS for mass, MS after phosphatase treatement to confirm phosphorylation, or tandem MS (MS/MS or MS) for partial sequence
Patent Application Publication Jun. 17, 2010 Sheet 3 of 22 US 2010/0151483 A1
Figure 2
Celine Ng. Protein Type Phosphorylation Site Sequence Disease Tissue Patien ------Adhesion Cr extracellular T cell NP-001085090.1 matrix protein DyEPPSPSPAPGAPPPPPOR leukerTia Jurkat BceAL VEQCPDyr ------mi-n-n NP158852 imatic protein MSEPPWCNL ----- Gel All Adhesion or extracellular NP_0362042 AMENOySPTPGTDC NP 906557.4 matic protein SPSTGOPTNQSMDDTREDIYNNPTFSR Adhesion or extracellular NP-019.72 mali protein DSSCGTGyELTEDNSCs lymphoma transform NP-060217. LEGEWTPNSLSTSyk edkidney NPOO4313. SRSAPPNLWAAQRyGRELRR CML AML NP-004231 Y139 SHLMSLySACSSEVPHGPWDOK T celAll MOTIS - AML Tcel Jurkat NP_004865.1 Y102 leukemia MO-91 T cel Yi SGYGPSDGPSyGR leukemia Jurkat Tce NP_004865, SVPCSGPTVRPCEDAWASPGAyGMGGR leukemia lurkat cell NP_004865. Y302 leukemia Jurkat Teell NP-004365. SGyGPSDGPSYGR leukemia Jurkat KG-1 MKPL.1 NPCO1743. GAGAFGFEWTHDITK AML Monomac 6
NPC55939. CTW-1 AM OLT15 NP_00148, Calcium-binding proten TPWLFDlyEIKEAIK T cell ALL VAL CTW.1 AML H1869 NSCLC HCC1806 Tcell AL MOLT15 breast NCI-N87 cancer R gastric csO01 NP 001148. Calcium-binding protein DAQELyAAGENR cancer csO42 AML CW NP-004030, Y199 DYAGWKR TceALMCLT15 AML CTV-1 NP001145.1 Calcium-binding proten ... Y255 SIPAYLAETLYYAMK Tce AL MOLT15 NP_001145.1 Calcium-binding protein Y297 NFATSLySMIK AMt CW-1 NP-004024.2 NKPLFFADKyk T cell ALL MOLT15 NP004024.2 CalciuT-binding protein LINGLMRPPAYCDAK lymphoma SUPT-13 acute erythrobla stic NP-001731.1 calcium-binding protein SGyIDEHELOALLKDLYEK leukemia HEL 0.04334.1 ng protein I FyALSASFEPFSNK DLBC Pfeifer CTW NP005436.1 Cell cycle regulation hTEEEKEELAOyakWDK AM MKPL-1 NP.005436. Cell cycle regulation CML K562 83-102F NP005436. Cell cycle regulation GALTGGYDTRK NP-00476, Cell cycle regulation ACL S NP0476, Cell cycle regulation WAVEyLDPSPEVOK AM. KG. NP-0046522 Cell cycle regulation ALCL. IIS Tell NP-0574273 Cell cycle regulation LEyFSCDHOELLOR lekema Jurka cell NP0556253 Cell cycle regulator SHIPENFDDVDINEDEDCYSDER leukemia Jurkat Tct NP-955625.3 Ceccle reguation SHPENFDCYWOINEDOCSDER leukemia Jurkat Tcel NP-0556.253. Cell cycle regulation leukemia Jurkat Tce) NP 071394.2 Cell cycle regulation NCyCALKPR leukemia Jurkat EaF3.0ZF A. BaF34ZF NP_003531.1 Cell cycle regulation CML CHRF AML CTW.1 NF-06420.1 Chaperone. WHTVEDYQAMDAEWNLOKLEk Tcell ALL MOLT5 NP 0753.1 ChaperOne KRVEDAyTCNWSLEYEK ALCL. TS
Patent Application Publication Jun. 17, 2010 Sheet 10 of 22 US 2010/0151483 A1
Figure 2
Phospho. Celine Protein Name Accession No. Protein Type Residue Phosphorylation Sita Sequence Disease Tissue Patient KG KG1-A Receptor, channel, transporter AML MO-91
ATPSH NP 008347.1 ...arcel surface Protein-YSE LAALPENPPADWAYYK T ce. AllMOLT15 Receptor, channel, transporter ATP6VA NP-001884.2 or cell surface protein IY485 ALDEYyDKHFTEFVPLR CMBaF3PRTK Receptor, channel, transporter ATP6VC NP-00186.1 or ceasurface protein Y367 HLDSSAAACAPMOPGNLSQQYyPYWYYK CMLBaF3-10ZF Receptor, channel, transporter ATP6VE NP_001687.1 or cell surface protein Y56 IMEyYEK. ---- CMLBaF3-4ZF Receptor, channel, transporter TS ATP6VH legius orcell surface protein Y388 LNEKNyELK ALCL licsO15 Receptor, channel, transporter ATP8B NP-005594.1 or cell surface protein ... Y1217 GyADLISSGR SCLCDMS 53 Receptor, channel, transporter T cell NP-01694.2 or cell surface protein Y655 AVPSADDVQR - leukemia Jurkat Receptor, channel, transporter - NF-008925.1 or celsurface protein Y295 EREMKEMGyAATEGESLREKLQEELK DLBCL OC-ly GDM f HU-3 KG-1 : KG1-A Receptor, channel, transporter Me-F2 -- NP_001020280.1 or cell surface protein Y330 LGEDPYTENGGGOGYSSGPGTSPEAQGK AML hit-44b OND4 ALL GDM-1 AML Jurkat Receptor, channel, transporter T cell KG1-A NP906128.1 or cell surface rotein Y222 DKNSAACWEDMSHSR leukemia lung tumor T57 Receptor, channel, transporter NP-003865.1 or cell surface protein Y264 TYTylMASR ------AMLICHRF AML acute CHRF erythrobla ELF-153 Receptor, channel, transporter stic HEL NP-003865.1 or cell surface grotein Y324 ASTQDSKPPGTSSyEV leukemia gM -1 RC-KS Werona 25S gz68 gzB1 Receptor, channel, transporter ALCL lung tumor T57 - NP-001868.2 or cell surface protein Y1029 WSWDPyNPAS AML patient 6 Receptor, channel, transporter Tell eIF4ENIF NP-062817.1 of cell surface protein Y580 AASADyLRPR -1. Jurkat Receptor, channel, transporter multiple NP-000061 or cell surface protein Y436 FKANDHGONER myelona. KMSB Receptor, channel, transporter -- NP-112571.1 or censurface protein Y924 GENWSEVR AML 255 T cell CPSF6 NP-008938, RNA binding protein Y364 GFPPTDPyGRPPPYDR -m- leukemiarkat AML Tce Jurkat PSF6 . NP-0089381 RNAbinding protein. Y74 GAAPNWTTGKR ... .leukemia. MKPL-1 stF-50 NP_001315.1 RNAbinging protein Y195 TLYDNDEVTCUAFHPTECILASGSRDyTK ALCl. TS . Tce? CsIF-50 NP-001315.1 |Rubids Protein. Y6 LGMENDDTAVCyAGR, ...... leukemia Jurkat CsIF77. NPOO13171 RNAbinging protein Y534 AMLKG-1 AM CTN-1 CUGBP2 NP-006S$2.3 RNA binding protein Y70 ELFEPyGAWONWR Tel AL MOLT15 NP C0407.2.2 RNA indigarotein Y515 OAFPAYPSSPFONTTGy(LPVYNY. ...Cl 223. CL NP C0407.22 RNA binding protein Y520 (AFPAYPSSPFCWTTGYOLPWNY CL 223. C. DA2 NP_0040722 RNA binding protein Y522 IQAFPAYPSSPFONTTGYOPWNy E. 223. Cll
NP-0043092 RNA binding protein Y470 DRIEESDQGPyAILAPTR -- ALCLTS Post: RNAbinding protein Y30 WRPCWYGGADIGQQR K562 NP-0013473. RNAbinding protein. Y53 GEyOKDSSGWSSSKDKDAYSSFGSR leukemia Jurkat DDX5 NP-004387, RNAbinding protein Y58 E. - AML MKPL.1 NP-004387.1 RNAbinding protein Y97 GHNCPKPWLNFyEANFPANYMDVIAR Tcell ALL MOTS Act KG-1 NP-003541RNAbinding protein Y153 SQQSACKEYWGVR . - ... AML TS 303 BAP5 NF-00897.2 RNAbinding protein Y510 NYLDOTNWyGSAORR Tel ALL MOLT15 EAVL1 NP-0014102 RNAbinding protein Y109 WSYARPSSVKDANLySGLPR AM MKPL-1 CML Tce Arkat Al NP_0603261 RNA binding protein Y79 CSYWNADHDyCNR leukemia K562 Tcel
- |NF-005548.1. Transcriptioral regulator. Y522 MLSTSEySOSPK . . . . . leukemia Jurkat T cell --- leopa Transcriptional regulator Y64 ONMLGNyEEWK leukemia Jurkat Tce NP-003968. 1 Transcriptionalegulator Y247 WEEyYEWLDHCSSINK leukemia Jurkat - KG-1 NP-1103942. Transcriptional regulator Y356 GQLNyPLPDFSK AML KG-A NPOO6319.2 Transcriptional regulator Y375 ToyFSSTD6, Tcell AL MOTS Patent Application Publication Jun. 17, 2010 Sheet 11 of 22 US 2010/0151483 A1
Figure 2 B w F G Phospho- Celine 1 Protein Name Accession No. Protein Type Residue Phosphorylation Site Sequence Disease issus Patien ------BaF3. AML FLT3(D842Y) NSCLC 73 breast MCF 311 BAP37 NP-009204.1 Transcriptional regulator Y121 VLSRPNAQELPSMyOR cancer, 10AY969F) 312 BAP37 NP 09204.1 Transcriptional regulator Y34 LLLGAGAVAyGVR T cel AllMOLT15 ALCL MOLTIs 33BAP87 NP-009204.1 TranScriptional regulator Y81 IPWFQYPlyCR T cel All TS CML AML-6246 acute AML-7592 erythrotia BaF3-10ZF stic 34 BCR NP050215.4 Transcriptional regulator Y972 LAKRIANSAGyNGDR leukemia. H Tce 315 Bf NP053554. IranScriptional regulator leukema Jurkat T cell 316 Bf NP-95SSS. Transcriptional regulator. leukina Jurkat 31 CEBPbeta Transcriptional regulator. -A-T- Tce 318 CEBPepsilon NP-0017962 Transcriptional regulator Y107 ALGPGySSPGSYDPR leukemia Jurkat 39 CRSP6 NP9042593 Transcriptional regulator Y256 NTDLDLOKKIPEDYCPLDVQ ALCL. TS 320CTBP2 NP-00320. Transcriptional regulator IY108 GSGyDNDIK 32 NP-001341.1. Trescriptional regulator IY124. PAKLYWYNELCIVLK . K562 . . . 322 NP 00134. Transcriptional regulator Y126 PAKLYWNELCTVLK K562 - 323 OOX7 NP-0063772 Transcriptional regulator Y75 APLPDLyPFGTMR MKP-1 324 TX2 NP-068943.1 Free regulator Y156 GNQLVDLAPLGYNYTVNyTHTQTNK MO-91 BaF3-107F EDG2 NP_0039012 Transcriptional regulator Y97 KOGyENLCCLR. OPM. 326 elonginc NP0056391 Transcriptional regulator Y18 YGGCEGPOAMyWK KG-1 327 ERG NP0440i transcriptional regulator Y276 TEDORPGLDPoLGPTSSR AML MKPl: 32 ERG NPO401 Transcriptional reculator Y345 SKPNMNyDKLS MO-91 plus -- NP_0052341 Transcriptional regulator Y278 QOHPSSMGwyGOESGGFSGPGENR leukemia Jurkat Tce slo - P9052341 Iranscriptional regulator Y41 GOATWSYEDPPTAK leukemia Jurkat Tce 331 FEP NP-0038932 Transcriptional regulator Y628 QOAYYAQTSPQGMPQHPPAPOGQ leukemia Jurkat AML CTW-8 Tce Jurkat FEP3 NP_0039251 Transcriptional regulator Y51 IDSIPHLNNSTPVDPSVyGYGWOK - ekenia RC-K8 333 Fu1 NP_0020032. Transcriptional regulator Y263. NTEQRPPOPOLGPTSSR AML MKPL-1 334 Full NP-902062. Transcriptional egulator .N332 SKPNMyDKLSR AML MO-9 acute erythrobta stic CDAO2NP-1144142 Translational regulator. Y258. TGASYYGECTLHyATNGESAWOL.PK leukemia HEL L13A DX48 NPO55555, Translational regulator Y54 GlyAYGFKPSAOCR AM KY821 33 le:F-2 NP-00952.1 Translational regulator Y443 IMGPNYTPGKKEDLyKPIQR ------T cell AL MOLTS AML CTW-1 se: NP C01952.1 Translational regulator Y760 -- TcellALL MOLT5 339 eF2 NP-0019521. Translational regulator Y9 STASLFYELSENDLNFKQSK MOTs BaF3-OZF BaF342F BaF3. FT3(D842Y BaF3-FLT3(WT) AML a3PRTK eF2A NP-004085.1 Translational regulator Y82 VOKEKGyDLSK CM. EaF3. EaF3-OZF EaF3. FLT3(D842v) aF3. AMt. FLT3(D842Y CML eaf3-FLT3(WT) multiple EaF3 eF2B NP003899.2 Translational regulator Y298 LyFLOCETCHSR - myeloma TelfGFR3. 342 leF2B-alpha NPOO1405.1 Translational regulator... .Y130 DGATILTHAYSR ALCL. TS 343 leF2S3. NP001406.1 Translational regulator Y238 YNEWCEylvK ALC 34 el3-beta NP 903748.1 Translational regulator Y241 TERPVNSAALSPNyDHWLGGGOEAMDWTTTSTR ALCl l BaF3-10ZF EaF3-42F EaF3-PRTK Baf3. effR3 AML KG-1
e3-epsilon NP-003745.1 Translational regulator {Y241 YAyyDTER CM, KGA AML CMK CM CTV. Tce ALL OU45 prostate K562 345 elf3-eta NP-003742.2 Translational regulator Y449 MTLDTLSyETSMGLLDKK cancer MOLT15 AML CTW-1 347 elf3S6P NP 057175. Translational regulator Y72 TWSDLOOKWELOASR Tce ALLIMOLT15
Patent Application Publication Jun. 17, 2010 Sheet 15 of 22 US 2010/0151483 A1
Figure 2
Phospho Celline Accession No. Protein type Residue Phosphorylation Site Sequence Disease tissue Patien Tce NP 060392 Unknown function Y110 LEEEALyAAQR leukemia Jurkat T ce. NP_06:039.2 Unknown function Y37 WOQyHPSNNGEYQSSGPEDDFESCLR leukemia Jurkat T cell NP_060392 Unknown function Y145 VQQYHPSNNGEyGSSGPEDDFESCLR leukemia Jurkat CLL NP689894. ...Y.299 NKAKPEPDLEEEKlyAYPSNITSETGF T cell ALL NF-689894. Unknown function T AKPEPDILEEEKIYAyPSNITSETGFR Cl MEC-2 NP_001073027.1 Unknown function Y222 AYYEFREEAyHSR AML 001073027.1 Y240 AML anaplastic XP943722. Unknown function Y1029 AGCSPSDIGIAPyROQLKINDLLAR ymptoma Kapas 299 AML CTW Unknown function AGRDTLySCAAMPNSASROEFPCGFSPANR T cell ALL MO15 NP-65.3259.3 AM B celALL T cell ALL acute CTV erythrobla HEL Stic Kapas-1106p leukemia MOLS ymphoma SEM neuroast SK-NAS 467 DOCK11 NP-65.3259.3 Unknown function GSLSTDKDTAyGSFQNGHGIK SUPT-43 NP653259.3 Unknown function KTOlySDPR CW NP_982272. Unknown function SSGPEFLQEWTAVTYHNK MOLS NP_001931.2 Unknown function NHPFywPLGAWDPGLLGYNVPALYSSDPAAR B celAL NSCLC 1993 T cell Jurkat NP-004413. Unknown function CWDPSPOAyFTLPR leukemia SEM CML cell leukemia HCT16 Colon Jurkat NP0562613 Unknown function EISEASENYSDVR Carcer K562 NP-055428.1 Unknown function YSwsRPGFR AML MW4-11 NP 055679, Unknown function SRDHWEGEPyAGY AML CMK SFDPASEEWAK AM UT-7 NP_0601.15.3 Tel leukemia Jurkat ...NP038267.1 - Unknown furiction FSARyDAVEAELK
CML breast 477 FAM83A NP-110282 Unknown function Y138 cance NP-1162882 Unknown function Y398 PHDGPPAAVSNLGAYRPTR CML |NP-1121812 Unknown function Y259 TTGNWAR Tce ALL MOLT5 AML CTW Unknown function Y147 NSQLDKNSEWyoEVOAMFDTLGIPK cellAL MOLT5 NP_056448.1 Unknown function AM NP121792 Unknown function KPGADLSDyFNYGFNEDTWK --- lymphoma SUPT-13 Unknown function AFPYGNVAFPHLPGSAPSWPSLVDTSKQWDYyAR AML MKPL-1 NP-1121792 anaplastic l Unknown function la CAEEySFAFEHCHR lymphoma Kapas 299 NP 9687532 Unknown function Y164 HELPK AML CTV-1 T celALL MCF. breast 10AY969F) NP-90273. Vesicle protein DSDYyNMLK...... NP_001273.1 Vesicle protein LONNNWAK MOLT15 CRAPEWSOYlyQWDSILKN T cell ALL MOLT5 --- NP_001273. Vesicle protein --w cell NP_005859. Y18 AGLGEGWPPGNYGNyGYANSGYSACEEENERLTESLR leukemia Jurkat NP_004850.1 Y1096 Ay:FAER VAL
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Figure 2
iPhospho Protein Name Accession No. Residue Phosphorylation Site Sequence ------8. BaF-PRTK CTV. H1869 HCC1808 HCC1937 K562 KOPT.K. MC-116 MOLT15
INP 004350 Vesicle protein Y 1211 CYEKMyDAK II: Wesicle protein Y10 VHMFEAHSDIR NP009942 vesicle protein Y202 LODAYYIFCEMADK NP-004936.2 Vesicle protein Y125 VySPHVLNLTLDLPG|TK NP_006.786.2 Vesicle protein Y29 EPELFOTWAEGROlyAOK NP_006.7862 Vesicle protein Y448 DKPTyDEFYTLSPWNGK DU145 NP 037465.2 Vesicle protein NIWHNySEAEK Mom 14 Patent Application Publication dCIÐdOJLSSCIRISNESASTAX
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REAGENTS FOR THE DETECTION OF to gain a finer appreciation of cellular regulation. In spite of PROTEIN PHOSPHORYLATION IN the importance of protein modification, phosphorylation is SIGNALNG PATHWAYS not yet well understood due to the extraordinary complexity of signaling pathways, and the slow development of the tech RELATED APPLICATIONS nology necessary to unravel it. 0008. In many instances, such knowledge is likely to pro 0001 Pursuant to 35 U.S.C. S119(e) this application vide valuable tools useful to evaluate, and possibly to claims the benefit of, and priority to, provisional application manipulate target pathways, ultimately altering the functional U.S. Ser. No. 60/830,549, filed Jul. 13, 2006, the disclosure of status of a given cell for a variety of purposes. which is incorporated herein, in its entirety, by reference 0009. The importance of protein kinase-regulated signal TECHNICAL FIELD transduction pathways is underscored by a number of drugs designed to treat various cancer types by the inhibition of 0002 The invention relates generally to a variety of moi target protein kinases at the apex or intermediary levels of eties and tools for the detection of protein phosphorylation. pathways implicated in cancer development. See Sternet al., Moreover, the invention relates to the use of the same for Expert Opin. Ther. Targets 9(4):851-60 (2005). diagnostic and therapeutic purposes. 0010 Leukemia, a disease in which a number of underly ing signal transduction events have been elucidated, has BACKGROUND become a disease model for phosphoproteomic research and 0003. The activation of proteins by post-translational development efforts. As such, it represent a paradigm leading modification is an important cellular mechanism for regulat the way for many other programs seeking to address many ing most aspects of biological organization and control, classes of diseases (See, Harrison's Principles of Internal including growth, development, homeostasis, and cellular Medicine, McGraw-Hill, New York, N.Y.) communication. Cellular signal transduction pathways 0011 Depending on the cell type involved and the rate by involve protein kinases, protein phosphatases, and phosphop which the disease progresses leukemia can be defined as acute rotein-interacting domain (e.g., SH2, PTB, WW, FHA, 14-3- or chronic myelogenous leukemia (AML or CML), or acute 3) containing cellular proteins to provide multidimensional, and chronic lymphocytic leukemia (ALL or CLL). dynamic and reversible regulation of many biological activi 0012 Most varieties of leukemia are generally character ties. See e.g., Sawyer et al., Med. Chem. 1(3): 293-319 ized by genetic alterations e.g., chromosomal translocations, (2005). deletions or point mutations resulting in the constitutive acti 0004 Protein phosphorylation on a proteome-wide scale Vation of protein kinase genes, and their products, particularly is extremely complex as a result of three factors: the large tyrosine kinases. The most well known alteration is the onco number of modifying proteins, e.g. kinases, encoded in the genic role of the chimeric BCR-Abl gene. See Nowell, Sci genome, the much larger number of sites on Substrate proteins ence 132: 1497 (1960)). The resulting BCR-Abl kinase pro that are modified by these enzymes, and the dynamic nature tein is constitutively active and elicits characteristic signaling of protein expression during growth, development, disease pathways that have been shown to drive the proliferation and states, and aging. The human genome, for example, encodes survival of CML cells (see Daley, Science 247: 824-830 over 520 different protein kinases, making them the most (1990); Raitano et al., Biochim. Biophys. Acta. December 9: abundant class of enzymes known. See Hunter, Nature 411: 1333(3): F201-16 (1997)). 355-65 (2001). Most kinases phosphorylate many different 0013 The recent success of Imanitib (also known as Substrate proteins, at distinct tyrosine, serine, and/or threo STI571 or GleevecR), the first molecularly targeted com nine residues. Indeed, it is estimated that one-third of all pound designed to specifically inhibit the tyrosine kinase proteins encoded by the human genome are phosphorylated, activity of BCR-Abl, provided critical confirmation of the and many are phosphorylated at multiple sites by different central role of BCR-Abl signaling in the progression of CML kinases. See Graves et al., Pharmacol. Ther. 82: 111-21 (see Schindleret al., Science 289; 1938-1942 (2000): Nardiet (1999). al., Curr. Opin. Hematol. 11:35-43 (2003)). 0005. Many of these phosphorylation sites regulate criti 0014. The success of GleevecR) now serves as a paradigm cal biological processes and may prove to be important for for the development of targeted drugs designed to block the diagnostic or therapeutic modalities useful in the treatment activity of other tyrosine kinases known to be involved in and management of many pathological conditions and dis many diseased including leukemias and other malignancies eases, including interalia cancer, developmental disorders, as (see, e.g., Sawyers, Curr. Opin. Genet. Dev. February; 12(1): inflammatory, immune, metabolic and bone diseases. 111-5 (2002); Druker, Adv. Cancer Res. 91:1-30 (2004)). For 0006 For example, of the more than 100 dominant onco example, recent studies have demonstrated that mutations in genes identified to date, 46 are protein kinases. See Hunter, the FLT3 gene occur in one third of adult patients with AML. Supra. Understanding which proteins are modified by these FLT3 (Fms-like tyrosine kinase 3) is a member of the class III kinases will greatly expand our understanding of the molecu receptor tyrosine kinase (RTK) family including FMS, plate lar mechanisms underlying oncogenic transformation. There let-derived growth factor receptor (PDGFR) and c-KIT (see fore, the identification of, and ability to detect, phosphoryla Rosnet et al., Crit. Rev. Oncog. 4:595-613 (1993). In 20-27% tion sites on a wide variety of cellular proteins is crucially of patients with AML, an internal tandem duplication in the important to understanding the key signaling proteins and juxta-membrane region of FLT3 can be detected (see Yokota pathways implicated in the progression of many disease et al., Leukemia 11: 1605-1609 (1997)). Another 7% of States. patients have mutations within the active loop of the second 0007 Understanding reversible protein phosphorylation kinase domain, predominantly Substitutions of aspartate resi and its role in the operation and interrelationship between due 835 (D835), while additional mutations have been cellular components and functions provides the opportunity described (see Yamamoto et al., Blood 97: 2434-2439 (2001): US 2010/015 1483 A1 Jun. 17, 2010
Abu-Duhier et al., Br. J. Haematol. 113: 983-988 (2001)). at long last provides the elements necessary to attain those Expression of mutated FLT3 receptors results in constitutive much needed proteomics tools and modalities. tyrosine phosphorylation of FLT3, and Subsequent phospho 0020. The invention discloses novel phosphorylation sites rylation and activation of downstream molecules Such as identified in signal transduction proteins and pathways under STAT5, Akt and MAPK, resulting in factor-independent lying various disease states including for example human growth of hematopoietic cell lines. leukemias. The invention thus provides new reagents, includ 0015. Altogether, FLT3 is the single most common acti ing phosphorylation-site specific antibodies and AQUA pep vated gene in AML known to date. This evidence has trig tides, for the selective detection and quantification of these gered an intensive search for FLT3 inhibitors for clinical use phosphorylated sites/proteins. Also provided are methods of leading to at least four compounds in advanced stages of using the reagents of the invention for the detection and clinical development, including: PKC412 (by Novartis), quantification of the disclosed phosphorylation sites. CEP-701 (by Cephalon), MLN518 (by Millenium Pharma ceuticals), and SU5614 (by Sugen/Pfizer) (see Stone et al., BRIEF DESCRIPTION OF THE DRAWINGS Blood (in press) (2004); Smith et al., Blood 103: 3669-3676 (2004); Clark et al., Blood 104: 2867-2872 (2004); and 0021 FIG. 1—Is a diagram broadly depicting the immu Spiekerman et al., Blood 101: 1494-1504 (2003)). noaffinity isolation and mass-spectrometric characterization 0016. There is also evidence indicating that kinases such methodology (IAP) employed to identify the novel phospho as FLT3, c-KIT and Abl are implicated in some cases of ALL rylation sites disclosed herein. (see Cools et al., Cancer Res. 64: 6385-6389 (2004); Hu, Nat. 0022 FIG. 2 Is a table (corresponding to Table 1) enu Genet. 36: 453-461 (2004); and Graux et al., Nat. Genet. 36: merating the Leukemia signaling protein phosphorylation 1084-1089 (2004)). In contrast, very little is know regarding sites disclosed herein: any causative role of protein kinases in CLL, except for a high 0023 Column A the name of the parent protein; Column correlation between high expression of the tyrosine kinase B-the SwissProt accession number for the protein (human ZAP70 and the more aggressive form of the disease (see sequence); Column C the protein type/classification; Col Rassenti et al., N. Eng. J. Med. 351: 893-901 (2004)). umn D the tyrosine residue (in the parent protein amino acid 0017 Despite the identification of a few key molecules sequence) at which phosphorylation occurs within the phos involved in progression of leukemia, the vast majority of phorylation site: Column E=the phosphorylation site signaling protein changes underlying this disease remains sequence encompassing the phosphorylatable residue (resi unknown. There is, therefore, relatively scarce information due at which phosphorylation occurs (and corresponding to about kinase-driven signaling pathways and phosphorylation the respective entry in Column D) appears in lowercase; sites relevant to the different types of leukemia. This has Column F-the type of leukemia in which the phosphorylation hampered a complete and accurate understanding of how site was discovered; and Column G=the cell type(s), tissue(s) protein activation within signaling pathways is driving these and/or patient(s) in which the phosphorylation site was dis complex cancers. Accordingly, there is a continuing and covered. pressing need to unravel the molecular mechanisms of 0024 FIG. 3 is an exemplary mass spectrograph depict kinase-driven oncogenesis in leukemia by identifying the ing the detection of the tyrosine 48 phosphorylation site in downstream signaling proteins mediating cellular transfor CRKL (see Row 37 in FIG.2/Table 1), as further described in mation in this disease. Identifying particular phosphorylation Example 1 (red and blue indicate ions detected in MS/MS sites on Such signaling proteins and providing new reagents, spectrum);Y indicates the phosphorylated tyrosine (shown Such as phospho-specific antibodies and AQUA peptides, to as lowercase “y” in FIG. 2). detect and quantify them remains particularly important to 0025 FIG. 4 is an exemplary mass spectrograph depict advancing our understanding of the biology of this disease. ing the detection of the tyrosine 83 phosphorylation site in 0018 Presently, diagnosis of leukemia is made by tissue Catalase (see Row 59 in FIG.2/Table 1), as further described biopsy and detection of different cell surface markers. How in Example 1 (red and blue indicate ions detected in MS/MS ever, misdiagnosis can occur since some leukemia cases can spectrum);Y indicates the phosphorylated tyrosine (shown be negative for certain markers, and because these markers as lowercase “y” in FIG. 2). may not indicate which genes or protein kinases may be 0026 FIG. 5 is an exemplary mass spectrograph depict deregulated. Although the genetic translocations and/or ing the detection of the tyrosine 365 phosphorylation site in mutations characteristic of a particular form of leukemia can ANXA11 (see Row 62 in FIG.2/Table 1), as further described be sometimes detected, it is clear that other downstream in Example 1 (red and blue indicate ions detected in MS/MS effectors of constitutively active kinases having potential spectrum);Y indicates the phosphorylated serine (shown as diagnostic, predictive, or therapeutic value, remain to be elu lowercase “y” in FIG. 2). cidated. Accordingly, identification of downstream signaling 0027 FIG. 6 is an exemplary mass spectrograph depict molecules and phosphorylation sites involved in different ing the detection of the tyrosine 24 phosphorylation site in types of leukemia and development of new reagents to detect ENO2 (see Row 186 in FIG.2/Table 1), as further described and quantify these sites and proteins may lead to improved in Example 1 (red and blue indicate ions detected in MS/MS diagnostic/prognostic markers, as well as novel drug targets, spectrum);Y indicates the phosphorylated tyrosine (shown for the detection and treatment of this disease. as lowercase “y” in FIG. 2) 0028 FIG. 7 is an exemplary mass spectrograph depict SUMMARY OF THE INVENTION ing the detection of the tyrosine 208 phosphorylation site in 0019. Several novel protein phosphorylation sites have Fgr (see Row 262 in FIG. 2/Table 1), as further described in been identified in a variety of cell lines. Such novel phospho Example 1 (red and blue indicate ions detected in MS/MS rylation sites (tyrosine), and their corresponding parent pro spectrum);Y indicates the phosphorylated tyrosine (shown teins are reported (see Table 1). The elucidation of these sites as lowercase “y” in FIG. 2). US 2010/015 1483 A1 Jun. 17, 2010
0029 FIG. 8 is an exemplary mass spectrograph depict phorylated Bad apoptosis protein, the AQUA peptide com ing the detection of the tyrosine 89 phosphorylation site in prising the phosphorylatable peptide sequence listed in Col eIF3S6IP (see Row 348 in FIG. 2/Table 1), as further umn E. Row 52, of Table 1/FIG. 2 (which encompasses the described in Example 1 (red and blue indicate ions detected in phosphorylatable tyrosine at position 110). MS/MS spectrum);Y indicates the phosphorylated tyrosine 0034. In one embodiment, the invention provides an iso (shown as lowercase “y” in FIG. 2). lated phosphorylation site-specific antibody that specifically binds a target signaling protein/polypeptide selected from DETAILED DESCRIPTION Column A of Table 1 (Rows 2-498) only when phosphory 0030 Several novel protein phosphorylation sites have lated at the tyrosine residue listed in corresponding ColumnD been identified in a variety of cell lines. Such novel phospho of Table 1, comprised within the phosphorylatable peptide rylation sites (tyrosine), and their corresponding parent pro sequence listed in corresponding Column E of Table 1 (SEQ teins are reported (see Table 1). The elucidation of these sites ID NOs: 1-278, 280-289, 291-499), wherein said antibody at long last provides the elements necessary to attain those does not bind said signaling protein when not phosphorylated much needed proteomics tools and modalities. at said tyrosine. In another embodiment, the invention pro 0031. The disclosure of the phosphorylation sites provides vides an isolated phosphorylation site-specific antibody that the key to the production of new moieties, compositions and specifically binds a target signaling protein/polypeptide methods to specifically detect and/or to quantify these phos selected from Column A of Table 1 only when not phospho phorylated sites/proteins. Such moieties include for example rylated at the tyrosine residue listed incorresponding Column reagents. Such as phosphorylation site-specific antibodies and D of Table 1, comprised within the peptide sequence listed in AQUA peptides (heavy-isotope labeled peptides). Such corresponding Column E of Table 1 (SEQ ID NOs: 1-278, reagents are highly useful, interalia, for studying signal trans 280-289,291-499), wherein said antibody does not bind said duction events underlying the progression of many diseases signaling protein when phosphorylated at said tyrosine. Such known or Suspected to involve protein phosphorylation e.g., reagents enable the specific detection of phosphorylation (or leukemia in a mammal. Accordingly, the invention provides non-phosphorylation) of a novel phosphorylatable site dis novel reagents phospho-specific antibodies and AQUA closed herein. The invention further provides immortalized peptides—for the specific detection and/or quantification of a cell lines producing Such antibodies. In one embodiment, the target signaling protein/polypeptide (e.g., a signaling protein/ immortalized cell line is a rabbit or mouse hybridoma. polypeptide implicated in leukemia) only when phosphory 0035. In another embodiment, the invention provides a lated (or only when not phosphorylated) at a particular phos heavy-isotope labeled peptide (AQUA peptide) for the quan phorylation site disclosed herein. The invention also provides tification of a target signaling protein/polypeptide selected methods of detecting and/or quantifying one or more phos from Column A of Table 1, said labeled peptide comprising phorylated target signaling protein/polypeptide using the the phosphorylatable peptide sequence listed in correspond phosphorylation-site specific antibodies and AQUA peptides ing Column E of Table 1 (SEQ ID NOs: 1-278, 280-289, of the invention. 291-499), which sequence comprises the phosphorylatable 0032. These phosphorylation sites correspond to numer tyrosine listed in corresponding Column D of Table 1. In ous different parent proteins (the full sequences (human) of certain embodiments, the phosphorylatable tyrosine within which are all publicly available in SwissProt database and the labeled peptide is phosphorylated, while in other embodi their Accession numbers listed in Column B of Table 1/FIG. ments, the phosphorylatable residue within the labeled pep 2), each of which are have been linked to specific functions in tide is not phosphorylated. the literature and thus may be organized into discrete protein 0036 Reagents (antibodies and AQUA peptides) provided type groups, for example adaptor/scaffold proteins, cytoskel by the invention may conveniently be grouped by the type of etal proteins, protein kinases, and DNA binding proteins, etc. target signaling protein/polypeptide in which a given phos (see Column C of Table 1), the phosphorylation of which is phorylation site (for which reagents are provided) occurs. The relevant to signal transduction activity (e.g., underlying AML. protein types for each respective protein (in which a phos CML, CLL, and ALL), as disclosed herein. phorylation site has been discovered) are provided in Column 0033. In part, the invention provides an isolated phospho C of Table 1/FIG. 2, and include: adaptor/scaffold proteins, rylation site-specific antibody that specifically binds a given adhesion/extracellular matrix protein, apoptosis proteins, target signaling protein/polypeptide only when phosphory calcium binding proteins, cell cycle regulation proteins, lated (or not phosphorylated, respectively) at a particular chaperone proteins, chromatin, DNA binding/repair/replica tyrosine enumerated in Column D of Table 1/FIG. 2 com tion proteins, cytoskeletal proteins, endoplasmic reticulum or prised within the phosphorylatable peptide site sequence enu golgi proteins, enzyme proteins, G/regulator proteins, inhibi merated in corresponding Column E. In further part, the tor proteins, motor/contractile proteins, phosphatase, pro invention provides a heavy-isotope labeled peptide (AQUA tease, Ser/Thr protein kinases, protein kinase (Tyr)S, receptor/ peptide) for the detection and quantification of a given target channel/cell surface proteins, RNA binding proteins, signaling protein/polypeptide, the labeled peptide compris transcriptional regulators, tumor Suppressor proteins, ubiq ing a particular phosphorylatable peptide site/sequence enu uitan conjugating system proteins and proteins of unknown merated in Column E of Table 1/FIG. 2 herein. For example, function. Each of these distinct protein groups is a Subset of among the reagents provided by the invention is an isolated target signaling protein/polypeptide phosphorylation sites phosphorylation site-specific antibody that specifically binds disclosed herein, and reagents for their detection/quantifica the AFAPadaptor/scaffold protein only when phosphorylated tion may be considered a Subset of reagents provided by the (or only when not phosphorylated) at tyrosine 501 (see Row invention. 6 (and Columns D and E) of Table 1/FIG. 2). By way of 0037 Subsets of the phosphorylation sites (and their cor further example, among the group of reagents provided by the responding proteins) disclosed herein are those occurring on invention is an AQUA peptide for the quantification of phos the following protein types/groups listed in Column C of US 2010/015 1483 A1 Jun. 17, 2010
Table 1/FIG. 2 adaptor/scaffold proteins, calcium binding listed in corresponding Column E. Rows 61-69, of Table 1 proteins, chromatin or DNA binding/repair/replication pro (SEQ ID NOs: 60-68), which sequence comprises the phos teins, cytoskeletal proteins, enzyme proteins, protein kinases phorylatable tyrosine listed in corresponding Column D. (Tyr), protein kinases (Ser/Thr), receptor/channel/trans Rows 61-69, of Table 1. porter/cell Surface proteins, transcriptional regulators and translational regulators. Accordingly, among Subsets of 0042 Among this subset of reagents, antibodies and reagents provided by the invention are isolated antibodies and AQUA peptides for the detection/quantification of the follow AQUA peptides useful for the detection and/or quantification ing calcium binding protein phosphorylation sites are: of the foregoing protein/phosphorylation site Subsets. ANXA11 (Y365), ANXA2 (Y199) and ANXA5 (Y256) (see 0038. The patents, published applications, and scientific SEQ ID NOs: 61, 62 and 63). literature referred to herein establish the knowledge of those 0043 In another subset of embodiments there is provided: with skill in the art and are hereby incorporated by reference (i) An isolated phosphorylation site-specific antibody that in their entirety to the same extent as if each was specifically specifically binds a chromatin or DNA binding/repair/repli and individually indicated to be incorporated by reference. cation protein selected from Column A. Rows 86-96, of Table Any conflict between any reference cited herein and the spe 1 only when phosphorylated at the tyrosine listed in corre cific teachings of this specification shall be resolved in favor sponding Column D. Rows 86-96, of Table 1, comprised of the latter. Likewise, any conflict between an art-understood within the phosphorylatable peptide sequence listed in corre definition of a word or phrase and a definition of the word or sponding Column E. Rows 86-96, of Table 1 (SEQ ID NOs: phrase as specifically taught in this specification shall be 85-95), wherein said antibody does not bind said protein resolved in favor of the latter. when not phosphorylated at said tyrosine. 0039. In one subset of embodiments, there is provided: (ii) An equivalent antibody to (i) above that only binds the (i) An isolated phosphorylation site-specific antibody that chromatin or DNA binding/repair/replication protein when specifically binds an adaptor/scaffold protein selected from not phosphorylated at the disclosed site (and does not bind the Column A. Rows 2-44, of Table 1 only when phosphorylated protein when it is phosphorylated at the site). at the tyrosine listed in corresponding Column D. Rows 2-44. (iii) A heavy-isotope labeled peptide (AQUA peptide) for the of Table 1, comprised within the phosphorylatable peptide quantification of a signaling protein that is a chromatin or sequence listed in corresponding Column E. Rows 2-44, of DNA binding/repair/replication protein selected from Col Table 1 (SEQID NOs: 1-43), wherein said antibody does not umn A. Rows 86-96, said labeled peptide comprising the bind said protein when not phosphorylated at said tyrosine. phosphorylatable peptide sequence listed in corresponding (ii) An equivalent antibody to (i) above that only binds the Column E. Rows 86-96, of Table 1 (SEQ ID NOs: 85-95), adaptor/scaffold protein when not phosphorylated at the dis which sequence comprises the phosphorylatable tyrosine closed site (and does not bind the protein when it is phospho listed in corresponding Column D. Rows 86-96, of Table 1. rylated at the site). 0044 Among this subset of reagents, antibodies and (iii) A heavy-isotope labeled peptide (AQUA peptide) for the AQUA peptides for the detection/quantification of the follow quantification of an adaptor/scaffold protein selected from ing chromatin or DNA binding/repair/replication protein Column A. Rows 2-44, said labeled peptide comprising the phosphorylation sites are: APE1 (Y45) and APTX (Y200) phosphorylatable peptide sequence listed in corresponding (see SEQID NOs: 85 and 87). Column E. Rows 2-44, of Table 1 (SEQID NOs: 1-43), which sequence comprises the phosphorylatable tyrosine listed in 0045 In still another subset of embodiments there is pro corresponding Column D. Rows 2-44, of Table 1. vided: 0040. Among this subset of reagents, antibodies and (i) An isolated phosphorylation site-specific antibody that AQUA peptides for the detection/quantification of the follow specifically binds a cytoskeletal protein selected from Col ing adaptor/scaffold protein phosphorylation sites are: 14-3-3 umn-A, Rows 97-125, of Table 1 only when phosphorylated at Zeta (Y82), AKAP2 (Y507), ARRB2 (Y48) and CrkL (48) the tyrosine listed in corresponding Column D. Rows 97-125, (see SEQID NOs: 1, 8, 26 and 36). of Table 1, comprised within the phosphorylatable peptide 0041. In a second subset of embodiments there is pro sequence listed in corresponding Column E. Rows 97-125, of vided: Table 1 (SEQID NOs: 96-124), wherein said antibody does (i) An isolated phosphorylation site-specific antibody that not bind said protein when not phosphorylated at said specifically binds a calcium binding protein selected from tyrosine. Column A. Rows 61-69, of Table 1 only when phosphorylated (ii) An equivalent antibody to (i) above that only binds the at the tyrosine listed in corresponding Column D. Rows cytoskeletal protein when not phosphorylated at the disclosed 61-69, of Table 1, comprised within the phosphorylatable site (and does not bind the protein when it is phosphorylated peptide sequence listed in corresponding Column E. Rows at the site). 61-69, of Table 1 (SEQ ID NOs: 60-68), wherein said anti (iii) A heavy-isotope labeled peptide (AQUA peptide) for the body does not bind said protein when not phosphorylated at quantification of a signaling protein that is a cytoskeletal said tyrosine. protein selected from Column A. Rows 97-125, said labeled (ii) An equivalent antibody to (i) above that only binds the peptide comprising the phosphorylatable peptide sequence calcium binding protein when not phosphorylated at the dis listed in corresponding Column E. Rows 97-125, of Table 1 closed site (and does not bind the protein when it is phospho (SEQID NOs: 96-124), which sequence comprises the phos rylated at the site). phorylatable tyrosine listed in corresponding Column D. (iii) A heavy-isotope labeled peptide (AQUA peptide) for the Rows 97-125, of Table 1. quantification of a signaling protein that is a calcium binding 0046 Among this subset of reagents, antibodies and protein selected from Column A. Rows 61-69, said labeled AQUA peptides for the detection/quantification of the follow peptide comprising the phosphorylatable peptide sequence ing cytoskeletal protein phosphorylation sites are: ACTN1 US 2010/015 1483 A1 Jun. 17, 2010
(Y582), Arp2 (Y72), Arp3 (Y16), cofilin 1 (Y117), ezrin at the tyrosine listed in corresponding Column D. Rows 257 (Y116) and FLII (Y737) (see SEQID NOs: 99, 101,104,108, 267, of Table 1, comprised within the phosphorylatable pep 120 and 124). tide sequence listed in corresponding Column E. Rows 257 0047. In still another subset of embodiments there is pro 267, of Table 1 (SEQ ID NOs: 256-266), wherein said vided: antibody does not bind said protein when not phosphorylated (i) An isolated phosphorylation site-specific antibody that at said tyrosine. specifically binds an enzyme protein selected from Column (ii) An equivalent antibody to (i) above that only binds the A. Rows 130-195, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D. Rows 130-195, of protein kinase (Tyr) when not phosphorylated at the disclosed Table 1, comprised within the phosphorylatable peptide site (and does not bind the protein when it is phosphorylated sequence listed in corresponding Column E. Rows 130-195, at the site). of Table 1 (SEQ ID NOs: 129-194), wherein said antibody (iii) A heavy-isotope labeled peptide (AQUA peptide) for the does not bind said protein when not phosphorylated at said quantification of a signaling protein that is a protein kinase tyrosine. (Tyr) selected from Column A. Rows 257-267, said labeled (ii) An equivalent antibody to (i) above that only binds the peptide comprising the phosphorylatable peptide sequence enzyme protein when not phosphorylated at the disclosed site listed in corresponding Column E. Rows 257-267, of Table 1 (and does not bind the protein when it is phosphorylated at the (SEQ ID NOS: 256-266), which sequence comprises the site). phosphorylatable tyrosine listed in corresponding Column D. (iii) A heavy-isotope labeled peptide (AQUA peptide) for the Rows 257-267, of Table 1. quantification of a signaling protein that is a enzyme protein 0.052 Among this subset of reagents, antibodies and selected from Column A. Rows 130-195, said labeled peptide AQUA peptides for the detection/quantification of the follow comprising the phosphorylatable peptide sequence listed in ing protein kinase (Tyr) phosphorylation sites are: Abl (70), corresponding Column E. Rows 130-195, of Table 1 (SEQID Btk(Y40), CSK(Y416), Fgr(Y208) and FGFR3 (Y577) (see NOs: 129-194), which sequence comprises the phosphorylat SEQID NOs: 256, 258, 259,261 and 264). able tyrosine listed in corresponding Column D. Rows 130 0053 In yet another subset of embodiments, there is pro 195, of Table 1. vided: 0048. Among this subset of reagents, antibodies and (i) An isolated phosphorylation site-specific antibody that AQUA peptides for the detection/quantification of the follow specifically binds a receptor/channel/transporter/cell surface ing enzyme protein phosphorylation sites are: ADA (Y67), protein selected from Column A. Rows 268-287, of Table 1 ASS (Y133), ENO2 (Y25) and FASN (Y222) (see SEQ ID only when phosphorylated at the tyrosine listed in corre NOs: 145, 159, 185 and 194). sponding Column D. Rows 268-287, of Table 1, comprised 0049. In still another subset of embodiments there is pro within the phosphorylatable peptide sequence listed in corre vided: sponding Column E. Rows 268-287, of Table 1 (SEQIDNOs: (i) An isolated phosphorylation site-specific antibody that 267-278, 280-287), wherein said antibody does not bind said specifically binds a protein kinase (Ser/Thr) selected from protein when not phosphorylated at said tyrosine. Column A. Rows 235-256, of Table 1 only when phosphory (ii) An equivalent antibody to (i) above that only binds the lated at the tyrosine listed in corresponding Column D. Rows receptor/channel/transporter/cell Surface protein when not 235-256, of Table 1, comprised within the phosphorylatable phosphorylated at the disclosed site (and does not bind the peptide sequence listed in corresponding Column E. Rows protein when it is phosphorylated at the site). 235-256 of Table 1 (SEQ ID NOs: 234-255), wherein said antibody does not bind said protein when not phosphorylated (iii) A heavy-isotope labeled peptide (AQUA peptide) for the at said tyrosine. quantification of a signaling protein that is a receptor/chan (ii) An equivalent antibody to (i) above that only binds protein nel/transporter/cell surface protein selected from Column A, kinase (Ser/Thr) when not phosphorylated at the disclosed Rows 268-287, said labeled peptide comprising the phospho site (and does not bind the protein when it is phosphorylated rylatable peptide sequence listed in corresponding Column E. at the site). Rows 268-287, of Table 1 (SEQID NOs: 267-278,280-287), (iii) A heavy-isotope labeled peptide (AQUA peptide) for the which sequence comprises the phosphorylatable tyrosine quantification of a signaling protein that is a protein kinase listed in corresponding Column D. Rows 268-287, of Table 1. (Ser/Thr) selected from Column A. Rows 235-256, said 0054 Among this subset of reagents, antibodies and labeled peptide comprising the phosphorylatable peptide AQUA peptides for the detection/quantification of the follow sequence listed in corresponding Column E. Rows 235-256, ing receptor/channel/transporter/cell Surface protein phos of Table 1 (SEQ ID NOs: 234-255), which sequence com phorylation sites are: CD34 (Y330) and CR2 (Y1029) (see prises the phosphorylatable tyrosine listed in corresponding SEQID NOs: 280 and 284). Column D, Rows 235-256, of Table 1. 0055 In yet another subset of embodiments, there is pro 0050. Among this subset of reagents, antibodies and vided: AQUA peptides for the detection/quantification of the follow (i) An isolated phosphorylation site-specific antibody that ing protein kinase (Ser/Thr) phosphorylation sites are: ATM specifically binds a transcriptional regulator selected from (Y2019), Bcr(Y513), DNA-PK(Y779) and ERK2 (Y36) (see Column A. Rows 306–334, of Table 1 only when phosphory SEQID NO. 237, 240,251 and 253). lated at the tyrosine listed in corresponding Column D. Rows 0051. In yet another subset of embodiments, there is pro 306–334, of Table 1, comprised within the phosphorylatable vided: peptide sequence listed in corresponding Column E. Rows (i) An isolated phosphorylation site-specific antibody that 306–334, of Table 1 (SEQ ID NOs: 307-335), wherein said specifically binds a protein kinase (Tyr) selected from Col antibody does not bind said protein when not phosphorylated umn A. Rows 257-267, of Table 1 only when phosphorylated at said tyrosine. US 2010/015 1483 A1 Jun. 17, 2010
(ii) An equivalent antibody to (i) above that only binds the (iii) A heavy-isotope labeled peptide (AQUA peptide) for the transcriptional regulator when not phosphorylated at the dis quantification of a protein selected from the group consisting closed site (and does not bind the protein when it is phospho of catalase (Y83), ACP (Y87), ataxin-3 (Y58), CRMP-2 rylated at the site). (Y499) and CLH-17 (Y1205) (Column-A, Rows 59,225,386, (iii) A heavy-isotope labeled peptide (AQUA peptide) for the 445 and 491 of Table 1), said labeled peptide comprising the quantification of a signaling protein that is a transcriptional phosphorylatable peptide sequence listed in corresponding regulator selected from Column A. Rows 306–334, said Column E of Table 1 (SEQ ID NOs: 58, 224, 387, 446 and labeled peptide comprising the phosphorylatable peptide 492), which sequence comprises the phosphorylatable sequence listed in corresponding Column E. Rows 306–334, tyrosine listed in corresponding Column D. Rows 59, 225, of Table 1 (SEQ ID NOs: 307-335), which sequence com 386, 445 and 491 of Table 1. prises the phosphorylatable tyrosine listed in corresponding 0060. The invention also provides an immortalized cell Column D, Rows 306–334, of Table 1. line producing an antibody of the invention, for example, a 0056 Among this subset of reagents, antibodies and cell line producing an antibody within any of the foregoing AQUA peptides for the detection/quantification of the follow subsets of antibodies. In an embodiment, the immortalized ing transcriptional regulator phosphorylation sites are: cell line is a rabbit hybridoma or a mouse hybridoma. BAP37 (Y 121) and CR2C/EBP-beta (Y137) (see SEQ ID 0061. In other embodiments, a heavy-isotope labeled pep NO:312 and 318). tide (AQUA peptide) of the invention (for example, an AQUA 0057. In still another subset of embodiments, there is pro peptide within any of the foregoing Subsets of AQUA pep vided: tides) comprises a disclosed site sequence wherein the phos (i) An isolated phosphorylation site-specific antibody that phorylatable tyrosine is phosphorylated. In yet other embodi specifically binds a translational regulator selected from Col ments, a heavy-isotope labeled peptide of the invention umn A. Rows 335-357, of Table 1 only when phosphorylated comprises a disclosed site sequence wherein the phosphory at the tyrosine listed in corresponding Column D. Rows 335 latable tyrosine is not phosphorylated. 357, of Table 1, comprised within the phosphorylatable pep 0062. The foregoing subsets of reagents of the invention tide sequence listed in corresponding Column E. Rows 335 should not be construed as limiting the scope of the invention, 357, of Table 1 (SEQ ID NOs: 336-358), wherein said which, as noted above, includes reagents for the detection antibody does not bind said protein when not phosphorylated and/or quantification of disclosed phosphorylation sites on at said tyrosine. any of the other protein type/group Subsets (each a Subset) (ii) An equivalent antibody to (i) above that only binds the listed in Column C of Table 1/FIG. 2. translational regulator when not phosphorylated at the dis 0063 Also provided by the invention are methods for closed site (and does not bind the protein when it is phospho detecting or quantifying a target signaling protein/polypep rylated at the site). tide that is tyrosine phosphorylated, said method comprising (iii) A heavy-isotope labeled peptide (AQUA peptide) for the the step of utilizing one or more of the above-described quantification of a signaling protein that translational regula reagents of the invention to detect or quantify one or more tor selected from Column A, Rows 335-357, said labeled target Signaling Protein(s)/Polypeptide(s) selected from Col peptide comprising the phosphorylatable peptide sequence umn A of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column E. Rows 335-357, of Table 1 listed in corresponding Column D of Table 1. In certain (SEQ ID NOs: 336-358), which sequence comprises the embodiments of the methods of the invention, the reagents phosphorylatable tyrosine listed in corresponding Column D. comprise a Subset of reagents as described above. The anti Rows 335-357, of Table 1. bodies according to the invention maybe used in standard 0058 Among this subset of reagents, antibodies and (e.g., ELISA or conventional cytometric assays). The inven AQUA peptides for the detection/quantification of the follow tion thus, provides compositions and methods for the detec ing a translational regulator phosphorylation sites are: elF2B tion and/or quantitation of a given target signaling protein or (Y298), elF3-eta (Y449), elF3-theta (Y32) and EIF5A (Y97) polypeptide in a sample, by contacting the sample and a (see SEQID NO:342, 347, 350 and 358). control sample with one or more antibody of the invention 0059. In yet a further subset of embodiments, there is under conditions favoring the binding and thus formation of provided: the complex of the antibody with the protein or peptide. The (i) An isolated phosphorylation site-specific antibody that formation of the complex is then detected according to meth specifically binds a protein selected from the group consisting ods well established and known in the art. of catalase (Y83), ACP (Y87), ataxin-3 (Y58), CRMP-2 0064. Also provided by the invention is a method for (Y499) and CLH-17 (Y1205) (Column-A, Rows 59,225,386, obtaining a phosphorylation profile of a certain target protein 445 and 491 of Table 1) only when phosphorylated at the group, for example adaptor/scaffold proteins or cell cycle tyrosine listed in corresponding Column D of Table 1), said regulation proteins (Rows 2-44 and Rows 70-81, respectively, tyrosine comprised within the phosphorylatable peptide of Table 1), that is phosphorylated in a disease signaling sequence listed in corresponding Column E of Table 1 (SEQ pathway, said method comprising the step of utilizing one or ID NOs: 58, 224, 387, 446 and 492), wherein said antibody more isolated antibody that specifically binds the protein does not bind said protein when not phosphorylated at said group selected from Column A of Table 1 only when phos tyrosine. phorylated at the tyrosine listed in corresponding Column D. (ii) An equivalent antibody to (i) above that only binds a of Table 1, comprised within the phosphorylation site protein selected from the group consisting of catalase (Y83), sequence listed in corresponding Column E, to detect the ACP (Y87), ataxin-3 (Y58), CRMP-2 (Y499) and CLH-17 phosphorylation of one or more of said protein group, thereby (Y1205) (ColumnA, Rows 59,225,386,445 and 491 of Table obtaining a phosphorylation profile for said protein group. 1) when not phosphorylated at the disclosed site (and does not 0065. The invention further contemplates compositions, bind the protein when it is phosphorylated at the site). foremost pharmaceutical compositions, containing one or a US 2010/015 1483 A1 Jun. 17, 2010 more antibody according to the invention formulated together improvement in the treatment of the pulmonary inflammation with a pharmaceutically acceptable carrier. One of skill will according to standard methodologies. Such evaluation will appreciate that in certain instances the composition of the aid and inform in evaluating whether to increase, reduce or invention may further comprise other pharmaceutically continue a particular treatment dose, mode of administration, active moieties. The compounds according to the invention etc. The term “therapeutic composition” refers to any com are optionally formulated in a pharmaceutically acceptable pounds administered to treat or prevent a disease. It will be understood that the Subject to which a compound (e.g., an vehicle with any of the well-known pharmaceutically accept antibody) of the invention is administered need not suffer able carriers, including diluents and excipients (see Reming from a specific traumatic state. Indeed, the compounds (e.g., ton's Pharmaceutical Sciences, 18th Ed., Gennaro, Mack antibodies) of the invention may be administered prophylac Publishing Co., Easton, Pa. 1990 and Remington. The Science tically, prior to any development of symptoms. The term and Practice of Pharmacy, Lippincott, Williams & Wilkins, “therapeutic.” “therapeutically, and permutations of these 1995). While the type of pharmaceutically acceptable carrier/ terms are used to encompass therapeutic, palliative as well as vehicle employed in generating the compositions of the prophylactic uses. Hence, as used herein, by “treating or invention will vary depending upon the mode of administra alleviating the symptoms” is meant reducing, preventing, tion of the composition to a mammal, generally pharmaceu and/or reversing the symptoms of the individual to which a tically acceptable carriers are physiologically inert and non compound of the invention has been administered, as com toxic. Formulations of compositions according to the pared to the symptoms of an individual receiving no Such invention may contain more than one type of compound of the administration. invention), as well any other pharmacologically active ingre 0068. The term “therapeutically effective amount” is used dient useful for the treatment of the symptom/condition being to denote treatments at dosages effective to achieve the thera treated. peutic result sought. Furthermore, one of skill will appreciate 0066. The invention also provides methods of treating a that the therapeutically effective amount of the compound of mammal comprising the step of administering Such a mam the invention may be lowered or increased by fine tuning mal a therapeutically effective amount of a composition and/or by administering more than one compound of the according to the invention. invention, or by administering a compound of the invention 0067. As used herein, by “treating is meant reducing, with another compound. See, for example, Meiner, C. L., preventing, and/or reversing the symptoms in the individual “Clinical Trials. Design, Conduct, and Analysis,' Mono to which a compound of the invention has been administered, graphs in Epidemiology and Biostatistics, Vol.8 Oxford Uni as compared to the symptoms of an individual not being versity Press, USA (1986). The invention therefore provides treated according to the invention. A practitioner will appre a method to tailor the administration/treatment to the particu ciate that the compounds, compositions, and methods lar exigencies specific to a given mammal. As illustrated in described herein are to be used in concomitance with con the following examples, therapeutically effective amounts tinuous clinical evaluations by a skilled practitioner (physi may be easily determined for example empirically by starting cian or veterinarian) to determine Subsequent therapy. Hence, at relatively low amounts and by step-wise increments with following treatment the practitioners will evaluate any concurrent evaluation of beneficial effect.
TABL E 1. Phosphorylation Sites
A. Protein B C hospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEO ID NO 2 14-3-3 NPOO3397.1 Adaptor/scaffold Y82 KQQMAREyREKIETELR SEO ID NO: 1 Zeta
3 Abi-1 NPO O54 61.2 Adaptor/scaffold Y484 NDDGWyEGVCNR SEO ID NO: 2
4 adaptin, NPOO1273.1 Adaptor/scaffold Y136 CLKDEDPyVR SEO ID NO: 3 beta
5 adaptin, NPOO1273.1 Adaptor/scaffold Y888 NVEGQDMLyOSLK SEO ID NO: 4 beta
6 AFAP NPO67651.2 Adaptor/scaffold YSO1 VISANPYLGGTSNGYAHPSGTALHyDD SEQ ID NO: 5 WPCINGSLK
7 AKAP2 NP_00100.4065. 2 Adaptor/scaffold Y939 WEAGIyANQEEEDNE SEO ID NO: 6 8 AKAP2 NP_00100.4065. 2 Adaptor/scaffold Y293 LFEDDEHEKEQy CIR SEO ID NO: 7 9 AKAP2 NP_00100.4065. 2 Adaptor/scaffold Ys Of SPGALETPSAAGSQGNTASQGKEGPyS SEQ ID NO: 8 EPSKR
10 AKAP8 NP OO5849.1 Adaptor/scaffo c Y229 SDPFVPPAASSEPLSTPWNELNyVGGR SEQ ID NO: 9
11 AKAP9 NPOO5742. 4 Adaptor/scaffold Y1233 CEVNAEDKENSGDyISENEDPELODYR SEQ ID NO: 10 US 2010/015 1483 A1 Jun. 17, 2010
TABLE 1- Continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H Name Accession No. Protein Type Residue Site Sequence SEQ NO 12 AKAP9 POO5742. Adaptor/scaffo Y2482 SLENOTyFK SEQ NO :
13 AKAP9 POO5742. Adaptor/scaffo Y34 61 RILyoNLNEPTTWSLTSDR SEQ NO :
14 AKAP9 POO5742. Adaptor/scaffo HDWSAHHDLNIDQSQCNEMyINSSQR SEQ NO :
15 AMOTL1 P 570899. Adaptor/scaffo Y219 GQQQQQQQQGAVGHGYyMAGGTSQK SEQ NO :
16 ANK1 POOOO28. Adaptor/scaffo Y1259 LRCyCMTDDKVDK SEQ NO :
17 ANK1 POOOO28. Adaptor/scaffo PySVGFR SEQ NO :
18 ANK1 POOOO28. Adaptor/scaffo ELVNyGANVNAQSQK SEQ NO :
19 POO39 O8. Adaptor/scaffo VGyLGAMLLLDER SEQ NO :
AP3D1 POO3929. Adaptor/scaffo Y365 ALDLLyGMVSK SEQ NO :
21 AP3M1 PO3 6227. Adaptor/scaffo Y31 SVVSQSVCDyFFEAQEK SEQ NO :
22 APBA2 POO5494. Adaptor/scaffo Y193 EGYODYYPEEANGNTGASPYR SEQ NO : 21
23 APBB1IP PO61916. Adaptor/scaffo Y225 THCDCNVDWCLYEIyPELQIER SEQ NO : 22
24 APBB1IP P 061916. Adaptor/scaffo Y34 O SEQ NO : 23
25 APBB1IP PO61916. Adaptor/scaffo SEQ NO : 24
26 APPL PO3 6228. Adaptor/scaffo Y522 SDDHPDWyETMR SEQ NO : 25
27 ARRB2 POO 43 O4. Adaptor/scaffo SEQ NO : 26
28 axin 1 POO3493. Adaptor/scaffo TRSQRKVGGGSAQPCDSIVVAyYFCGE SEQ NO : 27 PIPYR
29 axin 1 POO3493. Adaptor/scaffo Y788 TRSQRKVGGGSAQPCDSIVVAYyFCGE SEQ NO : 28 PIPYR
3 O axin 1 POO3493. Adaptor/scaffo Y796 TRSORKVGGGSAOPCDSIVVAYYFCGE SEQ NO : 29 PIPyR
31 BCAP P 689522. Adaptor/scaffo Y23 O VENEyTISVK SEQ NO :
32 BIN2 P O57377. Adaptor/scaffo Y232 LNHNLyEVMSK SEQ NO : 31
33 Cas-L POO 6394. Adaptor/scaffo Y164 TGHGyVYEYPSR SEQ NO : 32
34 CD2AP PO3 6252. Adaptor/scaffo Y541 DTCySPKPSVYLSTPSSASK SEQ NO : 33
35 CGN PO65821. Adaptor/scaffo Y1 ASTyGVAVR SEQ NO : 34
36 CrkL POO51.98. Adaptor/scaffo Y198 GKHGNRNSNSyGIPEPAHAY SEQ NO : 35
37 CrkL POO 51.98. Adaptor/scaffo DSSTCPGDyVLSVSENSR SEQ NO : 36
38 CrkL POO51.98. Adaptor/scaffo IHyLDTTTLIEPAPR SEQ NO : 37
39 diaphanous POO521 O. Adaptor/scaffo Y13 O EMVSQYLyTSK SEQ NO : 38 1.
4 O DNMBP PO5 6.O36. Adaptor/scaffo Y515 HHTSSVySISER SEQ NO : 39
41 DOCK2 POO4937. Adaptor/scaffo Y1434 SEQ NO : 4 O
42 Dok2 POO3965. Adaptor/scaffo Y357 SLyDSPQEPRGEAW SEQ NO : 41
43 FCRL3 P 443171. Adaptor/scaffo Y161 QLPNSYNLEKITVNSVSRDNSKyHCTA SEQ NO : 42 YR US 2010/015 1483 A1 Jun. 17, 2010
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
44. FCRL3 NP 443171.2 Adaptor/scaffold Y166 OLPNSYNLEKITVNSWSRDNSKYHCTA SEO ID NO : 43 yR
45 afadin NPOO 1035.090.1 Adhesion or Y17 O2 DyEPPSPSPAPGAPPPPPQR SEQ ID NO: 44
46 afadin NP OO 5927.2 Adhesion or Y568 VEQQPDyR SEQ ID NO: 45
47 ARHGAP9 NP 15885.2 Adhesion or Y191 MSEPPVyCNL SEQ ID NO: 46
48 CD93 NPO3 6204.2 Adhesion or Y644 AMENOySPTPGTDC SEO ID NO : 47
49 DNAM-1 NP OO6557.1 Adhesion or Y325 SPISTGOPTNQSMDDTREDIYVNyPTF SEQ ID NO : 48 extracellular SR
SO FBLN1 NP OO1987.2 Adhesion or Y252 DSSCGTGyELTEDNSCK SEQ ID NO: 49 extracellular matrix protein 51. ANKHD1 NP O60217. Apoptosis Y1661 LEGEVTPNSLSTSyK SEO ID NO : 50
52 Bad NPOO 4313. Apoptosis Y11 O SRSAPPNLWAAQRyGRELRR SEQ ID NO: 51
53 Bag2 NPOO4273. Apoptosis Y139 SHLMSLySACSSEVPHGPVDOK SEO ID NO : 52
54 BAG4 NPOO4865. Apoptosis Y1O2 AGGSHOEQPPYPSYNSNyWNSTAR SEO ID NO : 53
55 BAG4 NPOO4865. Apoptosis Y17 SGYGPSDGPSyGR SEQ ID NO: 54
56 BAG4 NP OO 4865. Apoptosis Y249 SVPQSGPTVRPQEDAWASPGAyGMGGR SEQ ID NO. 55
57 BAG4 NPOO4865. Apoptosis Y3 O2 DSSYPySQSDQSMNR SEO ID NO : 56
58 BAG4 NPOO4865. Apoptosis Y9 SGyGPSDGPSYGR SEO ID NO : 57
59 catalase NP OO1743. Apoptosis Y84 GAGAFGyFEVTHDITK SEO ID NO : 58
6 O DIP NPO55939. Apoptosis Y39 O. AKYDTPyIIWR SEO ID NO : 59
61. ANXA11 NP OO1148. Calcium-binding Y279 TPWLFDIyEIKEAIK SEQ ID NO: 60 protein
62 ANXA11 NP OO1148. Calcium-binding Y365 DAQELyAAGENR SEQ ID NO : 61 protein
63 ANXA2 NPOO4 O3 O. Calcium-binding Y199 DLyDAGVKR SEQ ID NO: 62 protein
64 ANXA5 NP OO1145. Calcium-binding Y256 SIPAYLAETLyYAMK SEQ ID NO: 63 protein
65 ANXA5 NP OO1145. Calcium-binding Y297 NFATSLySMIK SEQ ID NO: 64 protein
66 ANXA6 NPOO4 O24.2 Calcium-binding Y603 NKPLFFADKLyK SEO ID NO : 65 protein
67 ANXA6 NP OO4 O24.2 Calcium-binding Y95 LIVGLMRPPAyCDAK SEQ ID NO: 66 protein US 2010/015 1483 A1 Jun. 17, 2010 10
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
68 CALB2 NP OO1731. Calcium-binding Y214 SGyIDEHELDALLKDLYEK SEO ID NO : 67 prorein
69 calreticulin NPOO4334. Calcium-binding Y75 FyALSASFEPFSNK SEQ ID NO: 68 protein
70 bamacan NPOO543 6. Ce cycle Y213 LHTLEEEKEELAQyOKWDK SEQ ID NO: 69 regulation
71 bamacan NPOO543 6. Ce cycle Y560 LFyHIVDSDEVSTK SEO ID NO : 7 O regulation
72 bamacan NPOO543 6. Ce cycle Y668 GALTGGyYDTRK SEO ID NO : 71 regulation
73 Bulo NPOO4716. Ce cycle Y141 TPCNAGTFSQPEKVyTLSVSGDR SEO ID NO : 72 regulation
74 Bullo NPOO4716. Ce cycle Y2O7 VAVEyLDPSPEVOK SEO ID NO : 73 regulation
75 Coc23 NPOO4 6 52.2 Ce cycle Y353 AALyFQR SEO ID NO : 74 regulation
76 CENPF NPO57427.3 Ce cycle Y1848 LEyFSCDHQELLQR SEO ID NO : 75 regulation
77 CEP3s O NP O55,625.3 Ce cycle Y2572 ISHIPENFDDyVDINEDEDCYSDER SEO ID NO : 76 regulation
78 CEP350 NP O55,625.3 Ce cycle Y2582 ISHIPENFDDYVDINEDEDCySDER SEO ID NO : 77 regulation
79 CEP350 NP O55,625.3 Ce cycle Y261O EKDVSEyFYEK SEO ID NO : 78 regulation
80 claspin NPO71394.2 Ce cycle Y887 NQyOALKPR SEO ID NO : 79 regulation
81 CUL-3 NP 0035.81.1 Ce cycle Y512 VLTTGyWPTQSATPK SEQ ID NO: 80 regulation
82 CCT7 NP OO6420.1 Chaperone Y27s VHTVEDYQAIVDAEWNILyDKLEK SEQ ID NO: 81
83 CCT- NPOO1753.1 Chaperone Y229 KRVEDAyILTCNVSLEYEK SEQ ID NO: 82 Zeta
84 CCT- NPOO1753.1 Chaperone Y239 SLEyEKTEVNSG SEQ ID NO: 83 Zeta
85 FKBP4. NPOO2 OO5.1 Chaperone Y411 EKKLyANMFER SEQ ID NO: 84
86 APE1 NP OO1632.2 Chromatin, DNA- Y45 EAAGEGPALyEDPPDQK SEO ID NO : 85 binding, DNA repair or DNA replication protein
87 APTX NP 778243.1 Chromatin, DNA- Y195 ISMODPKMOVYKDEQVVVIKDKyPK SEQ ID NO: 86 binding, DNA repair or DNA replication protein
88 APTX NP 778243.1 Chromatin, DNA- Y2OO ARyHWLVLPWTSISSLK SEO ID NO : 87 binding, DNA repair or DNA replication protein US 2010/015 1483 A1 Jun. 17, 2010 11
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
89 ARID1A NP OO6OO6.3 Chromatin, DNA- Y1506 NDMTyNYANR SEQ ID NO: 88 binding, DNA repair or DNA replication protein
90 ARID1A NP OO6 OO6.3 Chromatin, DNA- Y1508 NDMTYNyANR SEO ID NO : 89 binding, DNA repair or DNA replication protein
91 BAF155 NPOO3 O65.2 Chromatin, DNA- Y171 TLVONNCLTRPNIyLIPDIDLK SEO ID NO ; 9 O binding, DNA repair or DNA replication protein
92 BAZ1A NPO38.476.2 Chromatin, DNA- Y964 SSNAyDPSQMCAEK SEQ ID NO: 91 binding, DNA repair or DNA replication protein
93 ESCO2 NP OO101742O. 1 Chromatin, DNA- Y15.2 yRHIKPVSRNSR SEQ ID NO: 92 binding, DNA repair or DNA replication protein
94 EST1A NPO60045.4 Chromatin DNA- Y146 TKKPDLQIyQPGR SEQ ID NO: 93 binding, DNA repair or DNA replication protein
95 EST1A NPO6 OO 45.4 Chromatin, DNA- YSO 9 FONSDNPYyYPR SEQ ID NO : 94 binding, DNA repair or DNA replication protein
96 FBXL10 NP 115979.3 Chromatin, DNA- Y739 yASNLPGSLLKEQK SEO ID NO : 95 binding, DNA repair or DNA replication protein
97 aloIIM NPOO23 O4.3 Cytoskeleta Y383 VDNEILDyKDLAAIPK SEQ ID NO: 96 protein
98 aloIIM NPOO23 O4.3 Cytoskeleta Y678 NGLHRPVSTDFAQYNSygDVSGGVR SEO ID NO : 97 protein
99 aloIIM NP 002304.3 Cytoskeleta Y688 DyOTLPDGHMPAMR SEO ID NO : 98 protein
OO ACTN1 NP OO1 O93.1 Cytoskeleta Y582 IVQTyHVNMAGTNPYTTITPQEINGK SEQ ID NO: 99 protein
O1 ACTN4 NPOO 4915.2 Cytoskeleta Y693 ISIEMNGTLEDQLSHLKQyER SEQ ID NO : 100 protein
O2 ARP2 NPOO5713.1 Cytoskeleta Y72 SMLEVNyPMENGIVR SEQ ID NO : 101 protein
O3 ARP2 NPOO5713.1 Cytoskeleta Y91 NWDDMKHLWDyTFGPEKLNIDTR SEQ ID NO : 102 protein
O4 Airp3 NPOO5712.1 Cytoskeleta Y1OO yLRAEPEDHYFL.LTEPPLNTPENR SEQ ID NO : 103 protein
O5 Arp3 NPOO5712.1 Cytoskeleta Y16 LPACVVDCGTGyTK SEQ ID NO : 104 protein
O6 ARPC3 NPOO5710.1 Cytoskeleta Y48 ETKDTDIVDEAIYyFK SEQ ID NO : 105 protein US 2010/015 1483 A1 Jun. 17, 2010 12
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
Of CAPZA1 NP OO6126.1 Cytoskeleta Y198 IQVHyYEDGNVQLVSHK SEQ ID NO : 106 protein
O8 CLIM1 NPO 66272.1 Cytoskeleta Y197 PLDHAQPPSSLVIDKESEVyK SEO ID NO : 107 protein
09 cofilin 1 NPOO5498.1 Cytoskeleta Y117 MIyASSKDAIK SEQ ID NO : 108 protein
O coronin NP O5514 O. 1 Cytoskeleta Y3O4. YFEITDESPYVHyLNTFSSKEPOR SEQ ID NO. 109 1C protein
1 DBNL NP_001014436. 1 Cytoskeleta Y224 REQRyQEQGGEASPQR SEQ ID NO : O protein
2 Destrin NP OO6861.1 Cytoskeleta Y117 MIyASSKDAIK SEQ ID NO : 1. protein
3 DST NPO563 63.2 Cytoskeleta Y1726 TLDDIVGRyEDLSK SEQ ID NO : 2 protein
4. Emerin NPOOO1 O8.1 Cytoskeleta Y41 IFEyETQR SEQ ID NO : 3 protein
5 Emerin NPOOO1 O8.1 Cytoskeleta Y9 LSPPSSSAASSysFSDLNSTR SEQ ID NO : 4. protein
6 EPB41 NPOO4428.1 Cytoskeleta Y13 VSLLDDTVyECVVEK SEQ ID NO : 5 protein
7 EPB41 NPOO 4428.1 Cytoskeleta Y525 TITyEAAQTVK SEQ ID NO : 6 iso 4 protein
8 EPB41L5 NPO6596O2 Cytoskeleta Y353 YSGKTEyQTTK SEQ ID NO : 7 protein
9 EWL NPO57421.1 Cytoskeleta Y18 ASVMVyDDTSKK SEQ ID NO : 8 protein
2O EWL NPO57421.1 Cytoskeleta Y41 INIyHNTASNTFR SEQ ID NO : 9 protein
21 ezrin. NPOO3370.2 Cytoskeleta Y116 EGILSDEIyCPPETAVLLGSYAVQAK SEQ ID NO: 120 protein
22 Fascin NPOO3O79.1 Cytoskeleta Y314 THTGKyWTLTATGGVOSTASSK SEQ ID NO: 121 protein
23 Fascin NPOO3O79.1 Cytoskeleta Y335 NASCyFDIEWR SEQ ID NO: 122 protein
24 FLII NPOO2 OO9.1 Cytoskeleta Y117s CSNEKGyFAVTEK SEQ ID NO: 123 protein
25 FLII NPOO2O09.1 Cytoskeleta Y737 VGLGLGyLELPQINYK SEQ ID NO: 124 protein
26 CHERP NP OO 63 78.3 Endoplasmic Y714 NSEGWEQNGLyEFFR SEQ ID NO: 125 reticulum or golgi 27 CHERP NPOO6378.3 Endoplasmic Y894 GVGVALDDPyENYRR SEQ ID NO: 126 reticulum or golgi 28 CHERP NPOO6378.3 Endoplasmic Y897 GVGVALDDPYENyRR SEO ID NO : 127 reticulum or golgi
29 FCMD NP OO6722.2 Endoplasmic Y31 HyLSTKNGAGLSK SEQ ID NO: 128 reticulum or golgi
US 2010/015 1483 A1 Jun. 17, 2010 15
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
198 ARF CAI2O95O1 G protein or Y356 KKYNDDSDDSYFTSSSSyFDEPVELR SEQ ID NO : 197 GAP 3 regulator
199 ARF NPO55385.2 G protein or Y408 RKPDyEPVENTDEAQKK SEQ ID NO : 198 GAP 3 regulator
2OO ARF NPO55385.2 G protein or Y432 AISSDMyFGR SEO ID NO : 199 GAP 3 regulator
2O1 ARF NPO60679.1 G protein or Y17s VSGQPQSVTASSDKAFEDWLNDDLGSy SEQ ID NO. 200 GAP1 regulator QGAOGNR
2O2 ARHGEF6 NPOO 4831.1 G protein or Y719 SLVDTVyALKDEVR SEQ ID NO: 2O1 regulator
2O3 centaurin- NP O56O57.1 G protein or Y264 SFDLTTPyR SEQ ID NO: 2O2 delta 2 regulator
204 centaurin- NPO56O57.1 G protein or Y543 WCVLGDGVLSyFENER SEQ ID NO: 2O3 delta 2 regulator
2O5 ECT2 NPO6 O568.3 G protein or Y213 VAVSLGTPIMKPEWIyKAWER SEQ ID NO: 2O4 regulator
2O6 ARPP-21 NPO57384.2 Inhibitor protein Y288 SIEEREEEyORVR SEQ ID NO: 2O5 207 calpastatin NP OO1741.4 Inhibitor protein Y422 CGEDDETIPSEyR SEQ ID NO: 2O6
2O8 AK2 NP OO1616.1 Kinase (non- Y12 APSVPAAEPEyPK SEO ID NO : 2O7 protein)
209 AK2 NP OO1616.1 Kinase (non- Y2O1 LQAYHTQTTPLIEYyR SEQ ID NO: 2O8 protein)
210 endolfin NP O55548.2 Lipid binding Y73 DQECVNSCASSETSyGTNESSLNEK SEQ ID NO: 209 protein
211 ATPSO NP OO1688.1 itochondrial Y35 LVRPPVOVyGIEGR SEQ ID NO: 210 protein
212 DNAH5 NP OO13 6 O. 1 cotto or Y16 OO yNMPFKAQIOK SEQ ID NO: 211 contractile protein
213 DNAH8 NP OO13 62.2 cotto or Y3631 MKELEDNLLyK SEQ ID NO: 212 contractile protein
214 DNCH1 NP OO1367.2 cotto or Y1010 YOVGVHyELTEEEKFYR SEQ ID NO: 213 contractile protein
215 DNCH1 NPOO1367.2 cotto or Y1283 TKPVTGNLRPEEALOALTIyEGKFGR SEQ ID NO: 214 contractile protein
216 DNCH1 NPOO1367.2 cotto or Y31 O3 CVLNWFGDWSTEALyoVGK SEQ ID NO: 215 contractile protein
217 DNCH1 NPOO1367.2 cotto or Y3377 NYMSNPsyNYEIVNR SEQ ID NO: 216 contractile protein 218 dynactin1 NPOO4O73.2 cotto or Y1149 LSHEGPGSELPAGALyR SEO ID NO : 217 contractile protein 219 dynactin1 NPOO4O73.2 cotto or Y1244 AKEEQODDTVyMGK SEQ ID NO: 218 contractile protein 22O dynactin2 NPOO 6391.1 cotto or Y318 VHQLyETIQR SEQ ID NO: 219 contractile protein
221 dynactin4 NPO573 O5.1 cotto or Y1Of KAyYLACGFCR SEQ ID NO: 220 contractile protein US 2010/015 1483 A1 Jun. 17, 2010 16
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
222 dynactin4 NP O573 O5.1 Motor or Y108 KAYyLACGFCR SEQ ID NO: 221 contractile protein 223 Eg5 NPOO 4514.2 Motor or Y211 GLEEITVHNKDEVyQILEK SEQ ID NO: 222 contractile protein 224 Eg5 NPOO 4514.2 Motor or Y399 NGVyISEENFR SEQ ID NO: 223 contractile protein
225 ACP1 NPOO 42.91.1 Phosphatase Y88 QITKEDFATFDyILCMDESNLR SEQ ID NO: 224
226 DUSP7 NP OO1938.1 Phosphatase Y118 LRDDGCQAyYLQGGFNK SEQ ID NO: 225
227 APEH NP OO1631.3 Protease Y209 LKKPDQAIKGDOFVFyEDWGENMVSK SEQ ID NO: 226 228 calpain 2 NP OO1739.1 Protease Y377 GSTAGGCRNyPNTFWMNPQYLIK SEO ID NO : 227
229 CAPN1 NPOO5177.2 Protease Y371 KWNTTLyEGTWR SEQ ID NO: 228
23 O CAPN1 NPOO5177.2 Protease Y387 GSTAGGCRNyPATFWVNPQFK SEQ ID NO: 229
231 CASP1 O NPOO 1221.2 Protease Y9 SQGQHWySSSDKNCK SEQ ID NO: 23 O
232 Bullo 1 NPOO 43.27.1 protein kinase Y219 SEySVHSSLASK SEQ ID NO: 231
233 DYRK1A NP OO1387.2 protein kinase, Y111 HINEVyYAK SEO ID NO. 232 dual-specificity
234 DYRK1A NPOO1387.2 protein kinase, Y1.47 WYNDGYDDDNYDyIVK SEQ ID NO: 233 dual-specificity
235 AAK1 NPO55726.2 protein kinase, Y34 REQGGSGLGSGSSGGGGSTSGLGSGyI SEQ ID NO: 234 Ser A Thr (non- GR receptor)
236 ATM NPOOOO 42.3 protein kinase, Y1753 TGHSFWEIyKMTTDPMLAYLOPFRTSR SEQ ID NO: 235 Ser A Thr (non receptor)
237 ATM NPOOOO42.3 protein kinase, Y1763 TGHSFWEIYKMTTDPMLAyLQPFRTSR SEQ ID NO: 236 Ser A Thr (non receptor)
238 ATM NPOOOO 42.3 protein kinase, Y2019 SIGEPDSLyGCGGGK SEO ID NO. 237 Ser A Thr (non receptor)
239 BCr NPOO 4318.3 protein kinase, Y279 DNLIDANGGSRPPWPPLEYQPyOSIYV SEQ ID NO. 238 Ser A Thr (non- GGMMEGEGK receptor)
24 O Bor NPOO4318.3 protein kinase, Y455 HQDGLPyIDDSPSSSPHLSSK SEQ ID NO. 239 Ser A Thr (non receptor)
241 BCr NPOO 4318.3 protein kinase, Y513 KWVLSGILASEETyLSHLEALLLPMKP SEQ ID NO: 24 O Ser A Thr (non- LK receptor)
242 BIKE NP 94.2595.1 protein kinase, Y813 ySDMSSVYR SEQ ID NO: 241 Ser A Thr (non receptor)
243 BIKE NP 94.2595.1 protein kinase, Y82O YSDMSSVyR SEQ ID NO: 242 Ser A Thr (non receptor) US 2010/015 1483 A1 Jun. 17, 2010 17
TABLE 1- Continued Phosphorylation Sites
E Protein C Phospho-Phosphorylation Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
244 CAMKK2 NP OO6540.3 protein kinase, Y234 SEQ ID NO : 243 Ser A Thr (non receptor)
245 CDK3 NP OO1249.1 protein kinase, Y19 IGEGTYGVVyKAK SEQ ID NO : 244 Ser A Thr (non receptor)
246 CK1-A CAA5671.O. 1 protein kinase, Y17 yKLVRKIGSGSFGDIYLAINITNGEEV SEQ ID NO. 245 Ser A Thr (non ALKLESOK receptor)
247 CK1-A CAA5671.O. 1 protein kinase, YKLVRKIGSGSFGDIyLAINITNGEEV SEQ ID NO : 246 Ser A Thr (non ALKLESOK receptor)
248 CK2 NPOO1887.1 protein kinase, Y13 ARVyAEVNSLR SEQ ID NO : 247 alpha2 Ser A Thr (non receptor)
249 CPNE3 NPOO39 OO ... 1 protein kinase, ISLNSLCyGDMDK SEQ ID NO : 248 Ser A Thr (non receptor)
250 CRIK NPOO9105.1 protein kinase, Y1417 CSTCLPATCGLPAEyATHFTEAFCR SEQ ID NO : 249 Ser/Thr (non receptor)
251 DNA-PK NPOO8835.5 protein kinase, Y2936 SIGEyDVLR SEQ ID NO : 25 O Ser A Thr (non receptor)
252 DNA-PK NPOO8835.5 protein kinase, SIyIDRHVMOPY SEQ ID NO : 251 Ser A Thr (non receptor)
253 DNA-PK NPOO8835.5 protein kinase, Y883 SyVAWDREK SEQ ID NO : 252 Ser A Thr (non receptor)
254 ERK2 NPOO2736.3 protein kinase, YTNLSYIGEGAyGMVCSAYDNVNKVR SEQ ID NO. 253 Ser A Thr (non receptor)
255 ERK2 NPOO2736.3 protein kinase, YTNLSYIGEGAYGMVCSAyDNVNKVR SEQ ID NO : 254 Ser A Thr (non receptor)
256 ERK3 NPOO2739.1 protein kinase, Y209 LLLSPNNy TK SEQ ID NO : 255 Ser A Thr (non receptor)
257 Abil NPOO5148.2 protein kinase, ENLLAGPSENDPNLFVALyDFVASGON SEQ ID NO. 256 Tyr (non TLSITK receptor)
258 Btk NPOOOO52.1 protein kinase, Y334 SEQ ID NO : 257 Tyr (non receptor)
259 Btk NPOOOO52.1 protein kinase, LSYyEYDFER SEQ ID NO : 258 Tyr (non receptor)
26 O CSK NPOO4374.1 protein kinase, Y416 MDAPDGCPPAVyEVMK SEQ ID NO : 259 Tyr (non receptor) US 2010/015 1483 A1 Jun. 17, 2010 18
TABLE 1- Continued Phosphorylation Sites
D E Protein B C Phospho-Phosphorylation Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
261 Fer NPOO5237. protein kinase, Y734 WTAPEALNyGR SEQ ID NO : 26 O Tyr (non receptor)
262 NPOO5239. protein kinase, Y208 KLDMGGyYITTR SEQ ID NO : 261 Tyr (non receptor)
263 NPOO5239. protein kinase, Y209 KLDMGGYyITTR SEQ ID NO : 262 Tyr (non receptor)
264 NPOOO133. protein kinase, Y552 HKNIINLLGACTQGGPLyVLVEYAAK SEQ ID NO : 263 Tyr (receptor)
265 NPOOO133. protein kinase, Y77 RPPGLDySFDTCKPPEEQLTFK SEQ ID NO : 264 Tyr (receptor)
266 NPOOO133. protein kinase, Y647 DVHNLDyYKKTTNGRLPVK SEQ ID NO : 265 Tyr (receptor)
267 NPOOO133. protein kinase, Y648 DVHNLDYy KKTTNGRLPVK SEQ ID NO : 266 Tyr (receptor)
268 ABCC4 NPOO5836. Receptor, Y1255 EyDEPYWLLQNK SEQ ID NO : 267 channel transporter or cell surface protein
269 ABCC4 NPOO5836. Receptor, Y1259 SEQ ID NO : 268 channel, transporter or cell surface protein
27 O ATPSH NP OO 6347. Receptor, Y15 O yPYWPHOPIENL SEQ ID NO : 269 channel, transporter or cell surface protein
271 ATPSH NPOO 6347. Receptor, Y15.2 YPyWPHOPIENL SEQ ID NO : 27 O channel, transporter or cell surface protein
272 ATPSH NPOO 6347. Receptor, LAALPENPPAIDWAyYK SEQ ID NO : 271 channel, transporter or cell surface protein
273 NP OO1681. Receptor, Y465 ALDEYyDKHFTEFVPLR SEQ ID NO : 272 channel, transporter or cell surface protein
274 NP OO1686. Receptor, Y367 HLDSSAAAIIDAPMDIPGLNLSQQEYy SEQ ID NO : 273 channel, transporter or cell surface protein
NPOO1687. Receptor, IMEyYEK SEQ ID NO : 274 channel, transporter or cell surface protein
276 Receptor, Y388 LNEKNyELLK SEQ ID NO : channel, transporter or cell surface protein US 2010/015 1483 A1 Jun. 17, 2010 19
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO 277 ATP8B1 NPOO5594.1 Receptor, Y1217 GyADLISSGR SEO ID NO : 276 channel, transporter or cell surface protein
278 BAI2 NP OO1694.2 Receptor, Y655 ATyVPSADDVQR SEO ID NO : 277 channel, transporter or cell surface protein 279 BTN3A3 NPOO8925.1 Receptor, Y295 EREMKEMGyAATEQEISLREKLQEELK SEQ ID NO: 278 channel, transporter or cell surface protein 280 CD34 NP_00102028 O. 1 Receptor, Y33 O LGEDPYyTENGGGQGYSSGPGTSPEAQ SEQ ID NO: 280 channel, GK transporter or cell surface protein
281 CD7 NP OO6128.1 Receptor, Y222 DKNSAACVVyEDMSHSR SEQ ID NO: 281 channel, transporter or cell surface protein
282 CD84 NPOO3865.1 Receptor, Y264 TIYTyIMASR SEQ ID NO: 282 channel transporter or cell surface protein
283 CD84 NPOO3865.1 Receptor, Y324 ASTQDSKPPGTSSyEIVI SEQ ID NO: 283 channel, transporter or cell surface protein
284 CR2 NP OO1868.2 Receptor, Y1O29 EVYSVOPyNPAS SEQ ID NO: 284 channel, transporter or cell surface protein
285 eIF4ENI NPO 62817.1 Receptor, Y58O AASADyLRPR SEO ID NO : 285 F1 channel, transporter or cell surface protein
2.86 ETB NPOOO1 O6.1 Receptor, Y43 O. FKANDHGyDNFR SEQ ID NO: 286 channel, transporter or cell surface protein
287 FCRL5 NP 112571.1 Receptor, Y924 GENVVySEVR SEO ID NO : 287 channel, transporter or cell surface protein 288 CPSF6 NPOO8938.1 RNA binding Y384 GPPPTOPygRPPPYDR SEQ ID NO: 288 protein
289 CPSF6 NPOO8938.1 RNA binding Y74 GAAPNVVyTYTGKR SEQ ID NO: 289 protein
29 O CStP-SO NP OO1315.1 RNA binding Y195 TLYDHVDEVTCLAFHPTEOILASGSR SEQ ID NO : 291 protein DyTLK
291 CStP-SO NP OO1315.1 RNA binding Y67 LGMENDDTAVOyAIGR SEQ ID NO : 292 protein
292 CStP-77 NP OO1317.1 RNA binding Y534 FMDLyPCSASELK SEQ ID NO : 293 protein US 2010/015 1483 A1 Jun. 17, 2010 20
TABLE 1- Continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation 1. Name Accession No. Protein Type Residue Site Sequence SEQ NO 293 CUGBP2 NPOO6552. RNA binding Y70 ELFEPygAVYOINVLR SEQ NO : 294 protein
294 DAZ1. NPOO4O72. RNA binding Y515 QAFPAYPSSPFQVTTGyOLPVYNY SEQ NO : 295 protein
295 DAZ1. NPOO4O72. RNA binding QAFPAYPSSPFQVTTGYOLPVyNY SEQ NO : 296 protein
296 DAZ1. NPOO4O72. RNA binding Y522 QAFPAYPSSPFQVTTGYOLPVYNy SEQ NO : 297 protein
297 DDX23 NPOO4809. RNA binding Y47 O IDRIEESDQGPyAIILAPTR SEQ NO : 298 protein
298 DDX3 NPOO1347. RNA binding VRPCVVyGGADIGQQIR SEQ NO : 299 protein
299 DDX3 NPOO1347. RNA binding GFyDKOSSGWSSSKDKDAYSSFGSR SEQ NO : protein
3 OO DDX5 NPOO4387. RNA binding Y518 DRENYDRGySSLLKR SEQ NO : 3O1 protein
301 DDX5 NPOO4387. RNA binding GHNCPKPVLNFyEANFPANVMDVIAR SEQ NO : protein
302 dyskerin NP OO1354. RNA binding Y153 SQQSAGKEyVGIVR SEQ NO : protein
3O3. E1B-APs NPOO8971. RNA binding Y510 SEQ NO : protein
3O4 ELAWL1 NP OO141O. RNA binding Y109 VSYARPSSEVIKDANLyISGLPR SEQ NO : 305 protein
305 FAM51A1 NPO6 O326. RNA binding Y179 LDSYVNADHDLyCNTR SEQ NO : 3 O 6 protein
306 53BP1 NPOO5648. Transcriptiona Y522 LMLSTSEySQSPK SEQ NO : 3. Of regulator
3. Of AF-4 NPOO5926. Transcriptiona IONMLGNyEEVK SEQ NO : regulator
3O8 AIP NPOO3968. Transcriptiona Y247 LVVEEyYEVLDHCSSILNK SEQ NO : 309 regulator
309 AKNA. NP 11 O394. Transcriptiona Y356 GQLNyPLPDFSK SEQ NO : regulator
310 ASCC3 NPOO 6819. Transcriptiona TGyFSSTDLGR SEQ NO : regulator
311 BAP37 NPOO92O4. Transcriptiona Y121 VLSRPNAQELPSMyQR SEQ NO : regulator
312 BAP37 NPOO92O4. Transcriptiona LLLGAGAVAyGVR SEQ NO : regulator
313 BAP37 NPOO92O4. Transcriptiona IPWFQYPIIyDIR SEQ NO : regulator
314 BCoR NPO 6 O215. Transcriptiona Y972 LAKRIANSAGyVGDR SEQ NO : regulator
315 Btf NPO55554. ranscriptiona Y408 FNDSEGDDTEETEDyR SEQ NO : regulator US 2010/015 1483 A1 Jun. 17, 2010 21
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
316 Btf NP O55554.1 Transcriptiona Y786 EySGFAGVSR SEO ID NO : 317 regulator 317 CAEBP- NPOO51852 Transcriptiona Y1.37 KPAEyGYVSLGR SEQ ID NO : 318 beta regulator 318 CAEBP- NPOO1796.2 Transcriptiona Y1Of ALGPGIySSPGSYDPR SEQ ID NO : 319 epsilon regulator
319 CRSP6 NPOO4259.3 Transcriptiona Y256 NTDLDLDKKIPEDYCPLDVOIPSDLE SEO ID NO : 32 O regulator GSAyIK
32O CTBP2 NP OO132O. 1 Transcriptiona Y108 IGSGyDNVDIK SEQ ID NO: 321 regulator
321. Daxx NP OO1341.1 Transcriptiona Y124 PAKLyVYINELCTVLK SEQ ID NO: 322 regulator
322 Daxx NP OO1341.1 Transcriptiona Y126 PAKLYVyINELCTVLK SEQ ID NO: 323 regulator
323 DDX17 NPOO6377.2 Transcriptiona Y75 APLPDLyPFGTMR SEQ ID NO: 324 regulator
324 DTX2 NP O65943.1 Transcriptiona Y156 GNOLVDLAPLGYNYTVNyTTHTOTNK SEQ ID NO: 325 regulator
325 EDG2 NPOO3901.2 Transcriptiona Y97 KQGyENLCCLR SEQ ID NO: 326 regulator 326 elongin NPOO5639.1 Transcriptiona Y18 TYGGCEGPDAMyVK SEO ID NO : 327 C regulator
327 ERG NPOO 44 4 O. 1 Transcriptiona Y276 TEDQRPQLDPyOILGPTSSR SEQ ID NO: 328 regulator
328 ERG NPOO 44 4 O. 1 Transcriptiona Y345 SKPNMNyDKLSR SEQ ID NO: 329 regulator
3.29 EWS NPOO5234.1 Transcriptiona Y278 QDHPSSMGVyGQESGGFSGPGENR SEQ ID NO : 330 regulator
3.30 EWS NPOO5234.1 Transcriptiona Y417 GDATVSyEDPPTAK SEQ ID NO : 331 regulator
331. FBP1 NPOO3893.2 Transcriptiona Y626 QQAAYyAQTSPQGMPQHPPAPQGQ SEQ ID NO : 332 regulator
332 FBP3 NPOO3925.1 Transcriptiona Y1 IDSIPHLNNSTPLVDPSVyGYGVOK SEQ ID NO : 333 regulator
333 FLI1 NPOO2 OO8.2 Transcnptional Y263 NTEQRPQPDPyoILGPTSSR SEQ ID NO. 334 regulator
334 FILI1 NPOO2OO8.2 Transcriptional Y332 SKPNMNyDKLSR SEO ID NO : 335 regulator
335 CDAO2 NP 114414.2 Translational Y258 TGASYYGEQTLHyIATNGESAVVOLPK SEQ ID NO : 336
336 DDX48 NPO55555.1 Translational Y54 GIyAYGFEKPSAIQQR SEO ID NO : 337
337 eBEF-2 NP OO1952.1 Translational Y443 IMGPNYTPGKKEDLyLKPIQR SEQ ID NO : 338
338 eBEF-2 NP OO1952.1 Translational Y76 O LMEPIYLVEIQCPEQVVGGIygVLNR SEQ ID NO : 339 US 2010/015 1483 A1 Jun. 17, 2010 22
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO 339 eBEF-2 NP OO1952.1 Translationa Y79 STAISLFyELSENDLNFIKQSK SEQ ID NO: 34 O regulator
34 O eIF2A NPOO4 O85.1 Translationa Y82 VDKEKGyIDLSK SEQ ID NO: 341 regulator
341 eIF2B NPOO3899.2 Translationa Y298 LyFLQCETCHSR SEQ ID NO: 342 regulator
342 eIF2B- NP OO14 O5.1 Translationa Y13 O DGATILTHAySR SEQ ID NO: 343 alpha regulator
343 eIF2S3 NP OO1406.1 Translationa Y238 YNIEVVCEyIVK SEQ ID NO: 344 regulator
344 eIF3- NPOO3748.1 Translationa Y241 TERPVNSAALSPNYDHVVLGGGOEAMD SEO ID NO. 345 beta regulator WTTTSTR
345 eIF3- NPOO3745.1 Translationa Y241 YAyYDTER SEQ ID NO: 346 epsilon regulator
346 eIF3 - et a NPOO3742.2 Translationa Y449 MTLDTLSIyETPSMGLLDKK SEO ID NO : 347 regulator
347 eIF3S 6IP NP O571.75. Translationa Y72 TVSDLIDQKVyELQASR SEQ ID NO: 348 regulator
348 eIF3S 6IP NPO571.75. Translationa Y89 VSSDVIDQKVyEIODIYENSWTK SEQ ID NO: 349 regulator
349 eIF3- NPOO3741. Translationa Y32 KQPALDVLyDVMK SEO ID NO : 350 theta regulator
350 eIF3- NPOO3741. Translationa Y382 FNVLQyVVPEVK SEQ ID NO: 351 theta regulator
351 eIF3- NPOO3744. Translationa Y3 O DMPyOPFSKGDR SEQ ID NO: 352 Zeta regulator
352 eIF4A1 NP OO14 O7. Translationa Y48 GIyAYGFEKPSAIQQR SEO ID NO : 353 regulator
353 eIF4A1 NPOO14 O7. Translationa Y70 GyDVIAQAQSGTGK SEQ ID NO: 354 regulator
354 eIF4B NP OO14 O8.2 Translationa Y141 LKGFGyAEFEDLDSLLSALSLNEESLG SEQ ID NO: 355 regulator NRR
355 eIF4B NP OO14 O8.2 Translationa Y285 AFGSGyR SEO ID NO : 356 regulator
356 eIF4B NP OO1408. 2 Translationa Y63 WHSNDDDVyRAPPIDRSILPT SEO ID NO : 357 regulator
357 eIESA NP OO1961.1 Translationa Y98 RNDFQLIGIQDGyLSLLQDSGEVR SEO ID NO : 358 regulator
358 AIM2 NPOO 4824.1 Tumor Y74 LNyMLLAKRLQEEKEKVDKQYK SEO ID NO : 359 suppressor
359 AIM2 NPOO 4824.1 Tumor Y92 LNYMLLAKRLQEEKEKVDKQyK SEQ ID NO: 360 suppressor
36O apollon NPO57336.2 Ubiquitin Y4461 TAEIVyAATTSLR SEQ ID NO: 361 conjugating system
361 ATG5 NPOO 484 O. 1 Ubiguitin Y35 IPTCFTLYODEITEREAEPyYLLLPR SEQ ID NO: 362 conjugating system US 2010/015 1483 A1 Jun. 17, 2010 23
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
362 FAF-X NP_001034679.2 Ubiquitin Y2439 RPYTGNPQYTyNNWSPPVOSNETSNGY SEQ ID NO: 363 conjugating FLER system
363 A2LP NPO59867.2 Unknown function Y118 GQSTGKGPPQSPVFEGVyNNSR SEQ ID NO: 364
364 A.2LP NPO59867.2 Unknown function Y14.5 NGTTyEGIFK SEO ID NO : 365
365 A2LP NPO59867.2 Unknown function Y27 O TTyDSSLSSYTVPLEKDNSEEFR SEQ ID NO: 366
366 A.2LP NPO59867.2 Unknown function Y277 TTYDSSLSSyTVPLEKDNSEEFR SEO ID NO : 367
367 ACBD6 NP 115736.1 Unknown function Y69 EQLLYLyAR SEQ ID NO: 368
368 AF15q14 NP 653 O91.2 Unknown function Y103 KLEDNy CEITGMNTLLSAPIHTOMOOK SEQ ID NO: 369
369 AF15q14 NP 653 O91.2 Unknown function Y1438 VYVDDIyVIPQPHFSTDQPPLPK SEO ID NO : 37 O
370 AF15q14 NP 653 O91.2 Unknown function Y417 ILAMTPESIySNPSIQGCK SEO ID NO : 371
371 AF15q14 NP 653 O91.2 Unknown function Y463 MyCNPDAMSSLTEK SEO ID NO : 372
372 ALKBH3 NP 631917.1 Unknown function Y127 TGIREDITyQQPR SEO ID NO : 373
373 ANKRD13 NP 1491.1.2.1 Unknown function Y52O SQELSGPASNGGISQTNTyDAQYER SEO ID NO : 374
374. ANKRD13 NP 1491.1.2.1 Unknown function Y524 SQELSGPASNGGISQTNTYDAQyER SEO ID NO : 375
375 ANKRD13 NP 1491.1.2.1 Unknown function Y91 QGWTVLHEAVSTGDPEMVyTVLOHR SEO ID NO : 376
376. ANKRD26 NPO55730.1 Unknown function Y296 NLEATyGTVR SEO ID NO : 377
377 ANKS1 NPO56 O 6 O2 Unknown function Y834 IIASLADRPyEEPPQKPPR SEO ID NO : 378
378 ANKS1 NPO 56060.2 Unknown function Y922 LTLRPPSLAAPyAPVQSWQHQPEK SEO ID NO : 379
379 ARID3B NPOO 64 56.1 Unknown function Y295 TQyMKYLYAYECEKKALSSPAELQAAI SEQ ID NO: 380 DGNRR
380 ARID3B NPOO 64 56.1 Unknown function Y298 TOYMKyLYAYECEKKALSSPAELQAAI SEQ ID NO: 381 DGNRR
381 ARID3B NP OO6456.1 Unknown function Y3 OO TOYMKYLyAYECEKKALSSPAELQAAI SEQ ID NO: 382 DGNRR
382 ARID3B NPOO 64 56.1 Unknown function Y3 O2 TOYMKYLYAyECEKKALSSPAELQAAI SEQ ID NO: 383 DGNRR
383 ARMET NP OO6 OO1.2 Unknown function Y79 LCyYIGATDDAATK SEQ ID NO: 384
384 ARS2 NP O56992.4 Unknown function Y63 O IVHSLDYYNTCEyPNEDEMPNR SEO ID NO : 385
385 ataxin-2 NPOO2964.2 Unknown function Y471 VALENDDRSEEEKyTAVQR SEQ ID NO: 386
386 ataxin-3 NPOO4984.2 Unknown function Ys MAEGGVTSEDyR SEO ID NO : 387
387 ATG9A NPO76990 .. 4 Unknown function Y209 ELTELDIyHR SEQ ID NO: 388
388 ATP11C NP 775965.2 Unknown function Y38 TVFVGNHPVSETEAyIAQR SEO ID NO : 389
389 BAT2 NPOO4629.2 Unknown function Y1OO3 RDYSyER SEO ID NO : 39 O
390 BAT2 NPOO4629.2 Unknown function Y2O 97 VDLyOOASPPDALR SEQ ID NO: 391
391 BAT2 NPOO4629.2 Unknown function Y1082 SEGSEyEEIPKR SEQ ID NO: 392 iso2
392 BAT2D1 NPO55987.2 Unknown function Y164 EKETNDDNyGPGPSLRPPNVACWR SEO ID NO : 393 US 2010/015 1483 A1 Jun. 17, 2010 24
TABLE 1- Continued Phosphorylation Sites
A. D E Protein Phospho Phosphorylation H Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
393 BAT2D1 N P O55987. Un KOW function Y1971 LPSAQTPNGTDyVASGK SEQ ID NO: 394
394 BAT2D1 N P O55987. Un KOW function Y241 LNGQQAALASQyR SEO ID NO : 395
395 BAT2D1 P O55987. Un KOW function Y253 AMMPPYMFOOyPR SEQ ID NO: 396
396 BAT2D1 P O55987. Un KOW function Y663 PAVLSGyFK SEO ID NO : 397
397 BCDIN3 P 062552. Un KOW function Y418 KFQYGNyCK SEO ID NO : 398
398 BK1048 P 06517 O. Un KOW function Y101 LyHNLQEYAK SEO ID NO : 399 E9 6
399 BK1048 P 06517 O. Un KOW function LYHNLQEyAK SEQ ID NO: 4OO E9 6
4 OO N P O55568. Un KOW function Y283 SEQ ID NO: 401
BRWD2 N P O60587. 8 Un KOW function Y1086 YLOTyGEWNR SEQ ID NO: 402
L.A. AAP44 OO3.1 Un KOW function Y257 MQEGSEVCSNPCLEENKPGIVyASLNH SEQ ID NO: 403 SWIGLNSR
P 861445. Un KOW function Y282 NVKEAPTEyASICVR SEQ ID NO: 404
PO6O791. Un KOW function Y127 KKQDLyMWLSNSPHGPSAK SEQ ID NO: 405
4 OS PO37397. Un KOW function Yst 9 LLTHyVKVOGLVISQMLRKSVETR SEQ ID NO: 406
PO714 O1. Un KOW function Y6O2 FIKVyWHR SEO ID NO : 4O7
4. Of 4orf112 PO57552. Un KOW function Y67 LKENKISLESEyEKIK SEQ ID NO: 408
408 4 orf13 O PO6 O578. Un KOW function Y271 RVDGMOyYCS SEQ ID NO: 409
409 4 orf13 O PO6 O578. Un KOW function Y272 RVDGMOYyCS SEQ ID NO: 410
4 of 172 P 68952 O. Un KOW function Y67 SEQ ID NO: 411
orf123 PO6 O357. Un KOW function Y45 CGNCGEISDKWOyIR SEQ ID NO: 412
orf73 PO5 6249. Un KOW function Y935 SLEDPySQQIR SEQ ID NO: 413
C PO76414. Un KOW function Y146 HKIRNIEKKKLKLEDyKDR SEQ ID NO: 414
PO76414. 2 Un KOW function Y849 GLPSISNGNySQLQFQAR SEQ ID NO: 415
C2Oorf27 P OO1034229.1 Un KOW function Y188 CVGAELEyDSEHSDWHGFD SEQ ID NO: 416
P 443097. 1. Un KOW function Y342 NONIQKPEySE SEO ID NO : 417
C21orfs POO5119. Un KOW function Y1330 SYYPCyLKVSHR SEQ ID NO: 418
POO 6322. Un KOW function Y222 TEKMVSISNyPLSAAL SEQ ID NO: 419
P OO6322. Un KOW function Y61 VGKTyELLNCDKHK SEQ ID NO: 42O
P 777.568. Un KOW function Y146 AYADSYYyEDGGMKPR SEQ ID NO: 421
421 PO78857. Un KOW function MKQQALTEFEAyKHR SEQ ID NO: 422
422 PO56437. Un KOW function Y437 SEQ ID NO: 423 A.
423 P O55427. Un KOW function Y418 NLTEQNSySNIPHEGK SEQ ID NO: 424
424 P 848543. Un KOW function Y76 QVAPASGyLTFER SEQ ID NO: 425
425 CALCO POO 5822. Un KOW function WKEQKDyWETELLQLK SEQ ID NO: 426 CO2 US 2010/015 1483 A1 Jun. 17, 2010 25
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO 426 CAMSA NPOs 62 62. Unknown function Y1259 CYYPDTEEIyKL SEO ID NO : 427 P1
427 CANP NP 945185. Unknown function Y348 IRNyYFCSLPR SEQ ID NO: 428
428 CANP NP 945185. Unknown function Y349 IRNYyFCSLPR SEQ ID NO: 429
429 CAP1 NPOO6358. Unknown function Y164 EMNDAAMFyTNR SEQ ID NO : 43 O
43 O CAP1 NPOO6358. Unknown function Y354 AYIyKCVNT SEQ ID NO : 431
431 CapZIP NP 443 O94.2 Unknown function Y156 SRPSEAEEVPVSFDQPPEGSHLPCyNK SEQ ID NO : 432
432 CASKIN2 NPO65804. Unknown function Y336 IGyFPPGIVEVVSKR SEQ ID NO : 433
433 CCDC59 NPO54886. Unknown function Y176 AQEEyEQIQAK SEQ ID NO : 434
434 CCDC64 NP 9971.94.2 Unknown function Y131 QyEQMHKELTDKLEHLEQEK SEQ ID NO : 435
435 CCDC8 NP 1144.29. Unknown function Y197 WREyVSQVSWGKLKRR SEQ ID NO : 436
436 CDW-3 NPO6 OO18. Unknown function Y169 TAPVQAPPAPVIVTETPEPAMTSGVyR SEQ ID NO : 437 PPGAR
437 CDW-3 NP 060018. Unknown function Y257 LQLDNQYAVLENQKSSHSQyN SEQ ID NO : 438
438 CHCHD2 NPO57223. Unknown function Y99 SGGSNAEPARPDITyOEPQGTQPAQQQ SEQ ID NO : 439 OPCLY
439 CHD-6 NP 15597.3 Unknown function Y15.4 O AARTLyRIELLR SEQ ID NO: 440
440 CKAP2L NP 6897.28.2 Unknown function Y113 RLTSECVSSNPySKPSSK SEQ ID NO: 441
441. CKAP2L NP 6897.28.2 Unknown function Y31 O SoyERPNETK SEQ ID NO: 442
4 42 COPG2 NPO3 62 65.3 Unknown function Y826 NSHSLyLAGIFR SEQ ID NO: 443
443 CRMP-2 NPOO1377. Unknown function Y32 IVNDDQSFyADIYMEDGLIK SEQ ID NO: 444
444 CRMP-2 NPOO1377. Unknown function Y431 THNSSLEyNIFEGMECR SEQ ID NO: 445
445 CRMP-2 NP OO1377. Unknown function Y499 GLyDGPVCEVSVTPK SEQ ID NO: 446
446 CWF19L2 NP 689647. Unknown function Y479 FMGKTDGDyYTLDDMFVSK SEO ID NO : 447
447 DCBLD1 NP 775945. Unknown function YSO 6 QIKyPFAR SEQ ID NO: 448
4 48 DDX29 NP 061903.2 Unknown function Y811 YQEyIPVOTGAHADLNPFYOK SEQ ID NO: 449
449 DDX48 NPO55555. Unknown function Y2O2 MLVLDEADEMLNKGFKEQIyDVYR SEQ ID NO: 450
450 DDX6 NPOO 4388. Unknown function Y3 O1 GVTOyYAYVTER SEQ ID NO: 451
451 DDX6 NPOO4388. Unknown function Y429 NLITyDDRFNLKSIEEQLGTEIKPIPS SEQ ID NO: 452
452. DHX3 O NPO55781.2 Unknown function Y929 ENYLEENLLyAPSLR SEQ ID NO: 453
453 DHX57 NP 945,314. Unknown function Y1261 NDGyVHIHPSSVNYOVR SEQ ID NO: 454
454 DHX57 NP 94.5314. Unknown function Y1271 NDGYVHIHPSSVNyQVR SEO ID NO : 455
455 DJ-1 NPOO 91.93.2 Unknown function Y139 MMNGGHyTYSENRVEK SEQ ID NO: 456
456 DKFZP4 NPOs 62 68. Unknown function Y187 TSHALDSHILDyYENPAIKEDVSTL SEO ID NO : 457 34FO91
457 DKFZP4 NPOs 62 68. Unknown function Y188 TSHALDSHILDYyENPAIKEDVSTL SEQ ID NO: 458 US 2010/015 1483 A1 Jun. 17, 2010 26
TABLE 1 - continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
458 DKFZp5 NPO61039.2 Unknown function Y11 O LEEEALyAAQR SEO ID NO : 459 64C182
459 DKFZp5 NPO 61O39.2 Unknown function Y1.37 IVQoyHPSNNGEYQSSGPEDDFESCLR SEQ ID NO: 46 o 64C182
460 DKFZp5 NPO 61O39.2 Unknown function Y14.5 IVOQYHPSNNGEyOSSGPEDDFESCLR SEQ ID NO: 461 64 O182
461 DKFZp6 NP 689894.1 Unknown function Y209 NKAKPEPDILEEEKIyAYPSNITSETG SEQ ID NO: 462 86KO275
462 DKFZp6 NP 689894.1 Unknown function Y211 AKPEPDILEEEKIYAyPSNITSETGFR SEQ ID NO: 463 86KO275
463 DKFZp7 NP 001073027.1. Unknown function Y222 AYYEFREEAyHSR SEQ ID NO: 464 62N1910
464 DKFZp7 NPOO1073027.1. Unknown function Y74. O YYRNyYGYOGYR SEQ ID NO: 465 62N1910
465 DNA2I XP 94.3722.1 Unknown function Y1O29 AGCSPSDIGIIAPyRQQLKIINDLLAR SEQ ID NO: 466
466 DOCK11 NP 6532 59.3 Unknown function Y1.188 LAGRDTLySCAAMPNSASRDEFPCGFT SEQ ID NO: 467 SPANR
467 DOCK11 NP 6532 59.3 Unknown function Y1223 GSLSTDKDTAyGSFONGHGIK SEQ ID NO: 468
468 DOCK11 NP 6532 59.3 Unknown function Y7 KTQIySDPLR SEQ ID NO: 469
469 DOCK8 NP 9822721 Unknown function Y47 SSGPEFLQEVyTAVTYHNK SEO ID NO : 47 O
470 DRPLA NP OO1931.2 Unknown function Y905 NHPFyVPLGAVDPGLLGYNVPALYSSD SEQ ID NO: 471 PAAR
471 DWL2 NPOO 4413.1 Unknown function Y362 CWDPSPQAyFTLPR SEO ID NO : 472
472 ELYS NPOs 62 61.3 Unknown function Y1790 EISEASENIySDVR SEO ID NO : 473
4.73 ETEA NPO55428.1 Unknown function Y81 IYSyVVSRPQPR SEO ID NO : 474
474 FAM2OB NPO55679.1 Unknown function Y138 SRDHWEGEPyAGY SEO ID NO : 475
475 FAM29A NPO 6O115.3 Unknown function Y418 SFDPASEEVyAK SEO ID NO : 476
476 FAMSOB NPO3 62 67.1 Unknown function YS3 FSAHyDAVEAELK SEO ID NO : 477
477 FAM83A NP 116288 .2 Unknown function Y138 SSATVYFQTVK SEO ID NO : 478
478 FAM83A NP 116288 .2 Unknown function Y398 PHDGPPAAVySNLGAYRPTR SEO ID NO : 479
479 FAM83D NP 112181.2 Unknown function Y259 TITGNIyYAR SEQ ID NO : 48O
480 FAM98B NP 775882.2 Unknown function Y1.47 NSQLDKNSEVyOEVOAMFDTLGIPK SEQ ID NO : 481
481 EGER1O NPOs 64 48.1 Unknown function Y46 RVEAMKQyOEEIQELNEVAR SEQ ID NO : 482 P2
482 FIP1L1 NP 112179.2 Unknown function Y17s KPGADLSDyFNYGFNEDTWK SEQ ID NO : 483
483 FIP1L1 NP 112179.2 Unknown function Y454 AFPYGNVAFPHLPGSAPSWPSLVDTSK SEO ID NO : 484 QWDYyAR
484 FLJ11305 NPO6 O856.1 Unknown functibn Y286 QAEEyLSFAFEHCHR SEQ ID NO : 485
485 FLJ11773 NP O68753.2 Unknown function Y164 HEyLPK SEQ ID NO : 486
486 adaptin, NP OO1273.1 Vesicle protein Y277 DSDYyNMLLK SEO ID NO : 487 beta US 2010/015 1483 A1 Jun. 17, 2010 27
TABLE 1- Continued Phosphorylation Sites
A. D E Protein B C Phospho-Phosphorylation H 1. Name Accession No. Protein Type Residue Site Sequence SEQ ID NO
487 adaptin, NP OO1273. Vesicle protein Y874 LONNNVyTIAK SEQ ID NO : 488 beta
488 adaptin, NPOO1273. Vesicle protein Y928 CRAPEVSQYIyOVYDSILKN SEQ ID NO : 489 beta
489 BET1 NPOO5859. Vesicle protein Y18 AGLGEGVPPGNYGNyGYANSGYSACEE SEQ ID NO: 490 ENERLTESLR
490 CLH-17 NPOO4850. Vesicle protein Y1096 AyEFAER SEQ ID NO: 491
491 CLH-17 NPOO4850. Vesicle protein Y12O6 LAELEEFINGPNNAHIQQVGDRCyDEK SEQ ID NO: 492
492 CLH-17 NPOO4850. Vesicle protein Y1211 CYDEKMyDAAK SEQ ID NO: 493 493 COP NP OO 4757. Vesicle protein Y101 VHMFEAHSDyIR SEQ ID NO: 494 beta prime
494 COPE NPOO91.94.2 Vesicle protein Y2O2 LQDAyYIFQEMADK SEO ID NO : 495
495 DYN2 NPOO 493 6.2 Vesicle protein Y125 Vy SPHVLNLTLIDLPGITK SEQ ID NO: 496 496 EHD1 NP OO6786.2 Vesicle protein Y29 EPELFQTVAEGLRQLyAQK SEO ID NO : 497
497 EHD1 NPOO6786.2 Wesicle rotein Y448 DKPTyDEIFYTLSPVNGK SEQ ID NO: 498
498 eps in 1 NPO374 65.2 Vesicle protein Y17 NIVHNySEAEIK SEQ ID NO: 499
0069. The short name for each protein in which a phos 4: 2081-7 (1995)). Decreased expression of ADA protein phorylation site has presently been identified is provided in correlates with increased response to drug associated with Column A, and its SwissProt accession number (human) is stomach ulcer (Eur J Pharmacol 205: 101-3 (1991)). Poly provided Column B. The protein type/group into which each morphism in the ADA gene correlates with early onset form protein falls is provided in Column C. The identified tyrosine of type II diabetes mellitus (Proc Natl Acad Sci USA 88: residue at which phosphorylation occurs in a given protein is 1484-8 (1991)). Splice site mutation in the ADA gene causes identified in Column D, and the amino acid sequence of the severe combined immunodeficiency (Am J Hum Genet 55: phosphorylation site encompassing the tyrosine residue is 59-68(1994)). Mutation in the ADA gene causes autosomal provided in Column E (lower case y=the tyrosine (identified recessive form of severe combined immunodeficiency (J in Column D)) at which phosphorylation occurs. Table 1 Immunol 166: 1698-702 (2001)). Increased expression of above is identical to FIG. 2, except that the latter includes the ADA protein correlates with peritoneal tuberculosis (Gut 36: disease and cell type(s) in which the particular phosphoryla 419-21 (1995)). Polymorphism in the ADA gene correlates tion site was identified (Columns F and G). with abnormal response to nutrient associated with type II 0070. One of skill in the art will appreciate that, in many diabetes mellitus (Proc Natl Acad Sci USA 88: 1484-8 instances the utility of the instant invention is best understood (1991)). Absence of the adenosine deaminase activity of ADA in conjunction with an appreciation of the many biological causes severe combined immunodeficiency (Am J Hum roles and significance of the various target signaling proteins/ Genet 63: 1049-59 (1998)). Deletion mutation in the ADA polypeptides of the invention. The foregoing is illustrated in gene causes immunologic deficiency syndromes (Genomics the following paragraphs summarizing the knowledge in the 7: 486-90 (1990)). Nonsense mutation in the ADA gene art relevant to a few non-limiting representative peptides con causes severe combined immunodeficiency (Hum Mol Genet taining selected phosphorylation sites according to the inven 4: 2081-7 (1995)). Decreased expression of ADA protein tion. correlates with increased response to drug associated with (0071 ADA (P00813), phosphorylated at Y28,Y66, Y307, stomach ulcer (EurJ Pharmacol 243: 301-3 (1993)). Absence Y347, is among the proteins listed in this patent. ADA, of the adenosine deaminase activity of ADA causes late onset Adenosine deaminase, plays a role in immune response, form of severe combined immunodeficiency (Am J Hum binds to CD26 (DPP4), altered activity or expression is asso Genet 63: 1049-59 (1998)). Loss of function mutation in the ciated with various cancers and autoimmune diseases; gene ADA gene causes decreased immune system function asso polymorphism is associated with autism. This protein has ciated with immunologic deficiency syndromes (J Immunol potential diagnostic and/or therapeutic implications based on 153: 2331-9 (1994)). Increased expression of ADA in blood the following findings. Abnormal mRNA splicing of ADA correlates with autoimmune thyroiditis (J Cell Biochem 89: causes severe combined immunodeficiency (Hum Mol Genet 550-5 (2003)). Absence of the adenosine deaminase activity US 2010/015 1483 A1 Jun. 17, 2010 28 of ADA correlates with severe combined immunodeficiency may regulate actin polymerization: MLLT4-ALL-1 (MLL) (Science 296: 24.10-3 (2002)). Loss of function mutation in fusion variant is associated with acute myeloid leukemia. the ADA gene causes late onset form of immunologic defi This protein has potential diagnostic and/or therapeutic ciency syndromes (J Immunol 153: 2331-9 (1994)). Splice implications based on the following findings. MLLT4 map site mutation in the ADA gene correlates with late onset form position may correlate with carcinoma tumors associated of severe combined immunodeficiency (J Clin Invest 92: with ovarian neoplasms (Cancer Res 56: 5586-9 (1996)). 2291-302 (1993)). Decreased adenosine deaminase activity Translocation of the MLLT4 gene correlates with acute of ADA causes late onset form of immunologic deficiency myelocytic leukemia (Cancer Res 53: 5624-8 (1993)). Trans syndromes (JImmunol 153: 2331-9 (1994)). Deletion muta location of the MLLT4 gene correlates with acute monocytic tion in the ADA gene correlates with late onset form of severe leukemia (Blood 87: 2496-505 (1996)). Translocation of the combined immunodeficiency (J Clin Invest 92: 2291-302 MLLT4 gene correlates with acute myelocytic leukemia (1993)). Missense mutation in the ADA gene correlates with (Blood 87: 2496-505 (1996)). (PhosphoSite(R), CellSignaling late onset form of severe combined immunodeficiency (JClin Technology (Danvers, Mass.), Human PSDTM, Biobase Cor Invest 92: 2291-302 (1993)). Decreased adenosine deami poration, (Beverly, Mass.)). nase activity of ADA causes decreased immune system func 0073 ANXA11 (P50995), phosphorylated at Y365, is tion associated with immunologic deficiency syndromes (J among the proteins listed in this patent. ANXA11, Annexin Immunol 153: 2331-9 (1994)). ADA map position may cor A11 (annexin XI), member of the annexin family of calcium relate with disease susceptibility associated with type II dia dependent phospholipid-binding proteins, binds ALG-2 betes mellitus (Hum Mol Genet 6: 1401-8 (1997)). Absence (PDCD6), may play roles in phagocytosis and mitosis: of the adenosine deaminase activity of ADA causes severe autoantibodies are detected in Sera of patients with autoim combined immunodeficiency (J Biol Chem 273: 5093-100 mune disease. This protein has potential diagnostic and/or (1998)). Polymorphism in the ADA gene correlates with therapeutic implications based on the following findings. abnormal response to nutrient associated with type II diabetes (PhosphoSite(R), Cell Signaling Technology (Danvers, mellitus (PNAS 88: 1484-8 (1991)). Deletion mutation in the Mass.), Human PSDTM, Biobase Corporation, (Beverly, ADA gene causes severe combined immunodeficiency (J Mass.)). Immunol 149: 3107-12 (1992)). Polymorphism in the ADA (0074 ANXA2 (P07355), phosphorylated at Y198, is gene correlates with early onset form of type II diabetes among the proteins listed in this patent. ANXA2. Annexin A2, mellitus (Proc Natl Acad Sci USA 88: 1484-8 (1991)). plasmin reductase and tissue-type plasminogen activator Absence of the adenosine deaminase activity of ADA causes (PLAT) receptor, regulates plasmin activity and cell migra autosomal recessive form of severe combined immunodefi tion, marker for various cancers (prostate, brain, breast, lung, ciency (JImmunol 166: 1698-702 (2001)). Polymorphism in pancreas, colorectal) and for heart failure. This protein has the ADA gene correlates with early onset form of type II potential diagnostic and/or therapeutic implications based on diabetes mellitus (PNAS 88: 1484-8 (1991)). Abnormal the following findings. Increased expression of ANXA2 expression of ADA protein correlates with non-Hodgkin’s mRNA may correlate with drug-resistant form of colorectal lymphoma (Cancer 70: 20-7 (1992)). Absence of the adenos neoplasms (Cancer Res 63: 4602-6 (2003)). Viral exploitation ine deaminase activity of ADA causes lymphopenia (Am J of the ANXA2 protein may correlate with cytomegalovirus Hum Genet 63: 1049-59 (1998)). Abnormal enhancer splic infections (Biochemistry Usa38: 5089-95 (1999)). Abnormal ing of ADA correlates with early onset form of severe com nucleus localization of ANXA2 correlates with astrocytoma bined immunodeficiency (Hum Mol Genet 4:2081-7 (1995)). (Oncol Res 6:561-7 (1994)). Increased presence of ANXA2 Increased expression of ADA in blood correlates with autoimmune antibody correlates with squamous cell carci Graves’ disease (JCell Biochem 89: 550-5 (2003)). Missense noma associated with lung neoplasms (PNAS 98: 9824-9 mutation in the ADA gene causes severe combined immuno (2001)). Increased expression of ANXA2 protein correlates deficiency (Hum Mol Genet 6: 2271-8 (1997)). Absence of with increased occurrence of death associated with colorectal the adenosine deaminase activity of ADA causes severe com neoplasms (Cancer 92: 1419-26 (2001)). Increased expres bined immunodeficiency (JBC 273: 5093-100 (1998)). sion of ANXA2 mRNA correlates with glioblastoma tumors Increased expression of ADA protein correlates with Graves associated with brain neoplasms (Cancer Res 52: 6871-6 disease (Endocr Res 28: 207-15 (2002)). ADA map position (1992)). Abnormal expression of ANXA2 mRNA may corre correlates with obesity (J Clin Invest 100: 1240-7 (1997)). late with B-cell lymphoma (Biochim Biophys Acta 1313: Increased expression of ADA protein correlates with autoim 295-301 (1996)). Increased expression of ANXA2 protein mune thyroiditis (Endocr Res 28: 207-15 (2002)). Increased correlates with increased severity of carcinoma associated expression of ADA protein correlates with more severe form with colorectal neoplasms (Cancer 92: 1419-26 (2001)). of stomach neoplasms (Cancer Lett 109: 199-202 (1996)). Decreased expression of ANXA2 mRNA correlates with Decreased expression of ADA protein correlates with inborn esophageal neoplasms associated with Squamous cell carci errors of purine-pyrimidine metabolism (J Clin Invest 103: noma (IntJ Cancer 106:327-33 (2003)). Increased presence 833-41 (1999)). Polymorphism in the ADA gene correlates of ANXA2 autoimmune antibody correlates with adenocar with abnormal response to nutrient associated with type II cinoma tumors associated with lung neoplasms (PNAS 98: diabetes mellitus (Proc Natl Acad Sci USA 88: 1484-8 9824-9 (2001)). Increased expression of ANXA2 mRNA may (1991)). (PhosphoSite(R), Cell Signaling Technology (Dan correlate with increased response to drug associated with vers, Mass.), Human PSDTM, Biobase Corporation, (Beverly, breast neoplasms (Cancer Res 63: 4602-6 (2003)). Increased Mass.)). presence of ANXA2 autoimmune antibody correlates with 0072 Afadin (P55196), phosphorylated at Y568, Y1675, squamous cell carcinoma associated with lung neoplasms is among the proteins listed in this patent. Afadin, Mixed (Proc Natl AcadSci USA 98:9824-9 (2001)). Viral exploita lineage-leukemia translocation to 4 homolog (afadin), inter tion of the ANXA2 protein may correlate with cytomegalovi cellular junction protein, negatively regulates cell adhesion, rus infections (Biochemistry 38: 5089-95 (1999)). Increased US 2010/015 1483 A1 Jun. 17, 2010 29 presence of ANXA2 autoimmune antibody correlates with peutic implications based on the following findings. Mutation adenocarcinoma tumors associated with lung neoplasms in the ATXN3 protein may cause abnormal protein folding (Proc Natl Acad Sci USA 98: 9824-9 (2001)). Increased associated with Machado-Joseph disease (Hum Mol Genet 8: presence of ANXA2 autoimmune antibody correlates with 673-82 (1999)). Trinucleotide repeat instability in the squamous cell carcinoma associated with lung neoplasms ATXN3 gene may cause decreased myelination associated (Proc Natl Acad Sci USA 98: 9824-9 (2001)). Decreased with Machado-Joseph disease (Hum Mol Genet 11: 1075-94 expression of ANXA2 mRNA correlates with squamous cell (2002)). Trinucleotide repeat instability in the ATXN3 gene carcinoma associated with esophageal neoplasms (IntJ Can may cause decreased peripheral nervous system function cer 106: 327-33 (2003)). Increased phosphorylation of associated with Machado-Joseph disease (Hum Mol Genet ANXA2 may correlate with B-cell lymphoma (Biochim Bio 11: 1075-94 (2002)). Mutation in the ATXN3 gene causes phys Acta 1313: 295-301 (1996)). Increased presence of increased incidence of familial form of Machado-Joseph dis ANXA2 autoimmune antibody correlates with adenocarci ease (Am J Hum Genet 68: 523-8 (2001)). Trinucleotide noma tumors associated with lung neoplasms (Proc Natl repeat instability in the ATXN3 gene causes Machado-Joseph Acad Sci USA 98: 9824-9 (2001)). Increased expression of disease (Nat Genet 8: 221-8 (1994)). Trinucleotide repeat ANXA2 protein may correlate with malignant form of col instability in the ATXN3 gene may cause decreased axono orectal neoplasms (Cancer 92: 1419-26 (2001)). Increased genesis associated with Machado-Joseph disease (Hum Mol expression of ANXA2 protein correlates with glioblastoma Genet 11: 1075-94 (2002)). Increased nuclear inclusion body tumors associated with brain neoplasms (Oncol Res 6:561-7 localization of ATXN3 may cause Machado-Joseph disease (1994)). Decreased expression of ANXA2 protein may cause (Neuron 19:333-44 (1997)). Trinucleotide repeat instability increased cell migration associated with prostatic neoplasms in the ATXN3 gene may cause defective dentate gyrus devel (Oncogene 22: 1475-85 (2003)). Increased expression of opment associated with Machado-Joseph disease (Hum Mol ANXA2 protein correlates with malignant form of pancreatic Genet 11: 1075-94 (2002)). Trinucleotide repeat instability in neoplasms (Carcinogenesis 14: 2575-9 (1993)). Increased the ATXN3 protein may cause increased induction of apop expression of ANXA2 mRNA correlates with astrocytoma tosis by intracellular signals associated with Machado-Jo tumors associated with brain neoplasms (Cancer Res 52: seph disease (Nat Genet 13: 196-202 (1996)). Trinucleotide 6871-6 (1992)). Increased expression of ANXA2 protein cor repeat instability in the ATXN3 gene may cause decreased relates with more severe form of glioblastoma (Oncol Res 6: cerebellar cortex function associated with Machado-Joseph 561-7 (1994)). Increased expression of ANXA2 protein cor disease (Hum Mol Genet 11: 1075-94 (2002)). Trinucleotide relates with pancreatic neoplasms (Oncogene 16: 625-33 repeat instability in the ATXN3 gene may cause defective (1998)). Increased expression of ANXA2 mRNA correlates cerebellum development associated with Machado-Joseph with more severe form of astrocytoma (Cancer Res 52: disease (Hum Mol Genet 11: 1075-94 (2002)). Trinucleotide 6871-6 (1992)). Increased expression of ANXA2 protein cor repeat instability in the ATXN3 gene may cause defective relates with carcinoma tumors associated with colorectal neo pons development associated with Machado-Joseph disease plasms (Cancer 92: 1419-26 (2001)). Decreased expression (Hum Mol Genet 11: 1075-94 (2002)). Abnormal cleavage of of ANXA2 mRNA correlates with small cell carcinoma asso ATXN3 may cause abnormal protein folding associated with ciated with lung neoplasms (Genomics 61: 5-14 (1999)). Machado-Joseph disease (J Neurochem 89:908-18 (2004)). Increased expression of ANXA2 protein correlates with (PhosphoSite(R), Cell Signaling Technology (Danvers, adenocarcinoma tumors associated with pancreatic neo Mass.), Human PSDTM, Biobase Corporation, (Beverly, plasms (Carcinogenesis 14: 2575-9 (1993)). Decreased Mass.)). expression of ANXA2 mRNA correlates with small cell car 0.077 ATM (Q13315), phosphorylated at Y1753, Y1763, cinoma (Genomics 61: 5-14 (1999)). Decreased expression of Y2019, is among the proteins listed in this patent. ATM, ANXA2 mRNA correlates with prostatic neoplasms (Cancer Ataxia telangiectasia mutated, a serine/threonine kinase Res 61: 6331-4 (2001)). (PhosphoSite R, CellSignaling Tech involved in apoptosis, DNA stability, cell cycle, and radiation nology (Danvers, Mass.), Human PSDTM, Biobase Corpora response; gene mutation is associated with ataxiatelangiecta tion, (Beverly, Mass.)). sia and implicated in B cell chronic lymphocytic leukemia. 0075 ARRB2 (P32121), phosphorylated atY48, is among This protein has potential diagnostic and/or therapeutic the proteins listed in this patent. ARRB2, Arrestin beta 2, an implications based on the following findings. Mutation in the adaptor that regulates GPCR desensitization by targeting ATM gene may correlate with chronic lymphocytic leukemia GPCRs to clathrin-coated pits, abnormal thyroid expression (Blood 100: 603-9 (2002)). Loss of heterozygosity at the correlates with thyroid nodules; mouse Arrb2 plays a role in ATM gene correlates with chronic B-cell leukemia (Cancer the development of allergic asthma. This protein has potential Res 58: 4552-7 (1998)). Decreased expression of ATM pro diagnostic and/or therapeutic implications based on the fol tein correlates with decreased response to ionizing radiation lowing findings. Abnormal expression of ARRB2 in thyroid associated with chronic B-cell leukemia (Blood 98: 814-22 correlates with thyroid nodule (FEBS Lett 486: 208-212 (2001)). Abnormal mRNA splicing of ATM causes prolym (2000)). (PhosphoSite(R), Cell Signaling Technology (Dan phocytic leukemia (Blood 91: 3920-6 (1998)). Mutation in vers, Mass.), Human PSDTM, Biobase Corporation, (Beverly, the ATM gene causes ataxia telangiectasia (Science 268: Mass.)). 1749-53 (1995)). Decreased expression of ATM protein cor 0076 Ataxin-3 (P54252), phosphorylated at Y58, is relates with increased occurrence of death associated with among the proteins listed in this patent. ataxin-3, Ataxin 3, a chronic B-cell leukemia (Cancer Res 58: 4552-7 (1998)). ubiquitin protease that inhibits histone acetylation and may Splice site mutation in the ATM gene causes increased inci mediate ubiquitinated protein degradation; variants with an dence of familial form of breast neoplasms (Cancer Res 63: expanded polyglutamine region are associated with 3325-33 (2003)). Nonsense mutation in the ATM gene causes Machado-Joseph (spinocerebellar ataxia 3) and Parkinson prolymphocytic leukemia (Blood 91: 3920-6 (1998)). Point disease. This protein has potential diagnostic and/or thera mutation in the ATM gene causes idiopathic form of mantle US 2010/015 1483 A1 Jun. 17, 2010 30 cell lymphoma (PNAS 97:2773-8 (2000)). Splice site muta mantle-cell lymphoma (Proc Natl AcadSci USA 100: 5372-7 tion in the ATM gene causes increased incidence of non (2003)). Deletion mutation in the ATM gene causes idiopathic familial form of breast neoplasms (Am J Hum Genet 66: form of mantle-cell lymphoma (Proc Natl AcadSci USA97: 494-500 (2000)). Gain of function mutation in the ATM gene 2773-8 (2000)). Frameshift mutation in the ATM gene corre may cause increased incidence of familial form of breast lates with T-cell lymphoma (PNAS 100: 5372-7 (2003)). neoplasms (JNatl Cancer Inst94: 205-15 (2002)). Absence of Deletion mutation in the ATM gene causes idiopathic form of the protein kinase activity of ATM may cause increased inci mantle-cell lymphoma (PNAS97:2773-8 (2000)). Abnormal dence of familial form of ovarian neoplasms (Cancer Res 63: mRNA splicing of ATM causes idiopathic form of mantle-cell 3325-33 (2003)). Missense mutation in the ATM gene corre lymphoma (PNAS97:2773-8 (2000)). Absence of the protein lates with decreased response to radiation associated with kinase activity of ATM may cause increased incidence of Hodgkin's disease (Blood 103: 283-90 (2004)). Mutation in familial form of breast neoplasms (Cancer Res 63: 3325-33 the ATM gene causes ataxiatelangiectasia (Am J Hum Genet (2003)). Mutation in the ATM gene causes increased severity 66: 494-500 (2000)). Decreased expression of ATM mRNA of necrosis associated with breast neoplasms (BrJCancer 76: correlates with carcinoma tumors associated with breast neo 1546-9 (1997)). Frameshift mutation in the ATM gene corre plasms (IntJ Cancer 78: 306-9 (1998)). Missense mutation in lates with T-cell lymphoma (Proc Natl Acad Sci USA 100: the ATM gene may cause increased incidence of non-familial 5372-7 (2003)). Deletion mutation in the ATM gene causes form of breast neoplasms (Proc Natl AcadSci USA99: 925 prolymphocytic leukemia (Blood 91: 3920-6 (1998)). Point 30 (2002)). Frameshift mutation in the ATM gene correlates mutation in the ATM gene causes idiopathic form of mantle with T-cell lymphoma (Proc Natl AcadSci USA 100: 5372-7 cell lymphoma (Proc Natl AcadSci USA97:2773-8 (2000)). (2003)). Decreased protein kinase activity of ATM may cause Loss of heterozygosity at the ATM locus correlates with increased incidence of familial form of breast neoplasms breast neoplasms (Oncogene 14: 339-47 (1997)). Abnormal (PNAS 99: 925-30 (2002)). Missense mutation in the ATM protein binding of ATM may cause increased incidence of gene correlates with increased incidence of familial form of familial form of ovarian neoplasms (Cancer Res 63: 3325-33 breast neoplasms (Cancer 92: 479-87 (2001)). Decreased (2003)). Mutation in the ATM gene causes increased inci protein serine/threonine kinase activity of ATM causes dence of non-familial form of breast neoplasms (Am J Hum decreased protein amino acid phosphorylation associated Genet 66: 494-500 (2000)). Decreased expression of ATM with acute lymphocytic leukemia (L1) (Blood 101: 3622-7 protein correlates with increased severity of disease progres (2003)). Deletion mutation in the ATM gene may correlate sion associated with chronic B-cell leukemia (Cancer Res 58: with disease susceptibility associated with leukemia (Am J 4552-7 (1998)). Missense mutation in the ATM gene causes Hum Genet 62: 334-45 (1998)). Decreased expression of diffuse large-cell lymphoma (Blood 100: 1430-7 (2002)). ATM mRNA may cause malignant form of breast neoplasms Missense mutation in the ATM gene causes increased inci (Int J Cancer 78: 306-9 (1998)). Decreased protein kinase dence of familial form of ovarian neoplasms (Cancer Res 63: activity of ATM may cause increased incidence of familial 3325-33 (2003)). Missense mutation in the ATM gene may form of breast neoplasms (Proc Natl AcadSci USA99: 925 correlate with lymphoma (Am J Hum Genet 62: 334-45 30 (2002)). Missense mutation in the ATM gene may cause (1998)). Polymorphism in the ATM gene may correlate with increased incidence of non-familial form of breast neoplasms abnormal response to radiation associated with breast neo (PNAS 99: 925-30 (2002)). Mutation in the ATM gene cor plasms (Cancer Res 63: 8717-25 (2003)). Missense mutation relates with mantle-cell lymphoma (PNAS 100: 5372-7 in the Phosphatidylinositol 3- and 4-kinase domain of ATM (2003)). Nonsense mutation in the ATM gene causes idio causes decreased protein amino acid phosphorylation associ pathic form of mantle-cell lymphoma (Proc Natl Acad Sci ated with acute lymphocytic leukemia (L1) (Blood 101: USA97:2773-8 (2000)). Missense mutation in the ATM gene 3622-7 (2003)). Abnormal mRNA splicing of ATM causes may correlate with breast neoplasms (Proc Natl Acad Sci diffuse large-cell lymphoma (Blood 100: 1430-7 (2002)). USA99: 925-30 (2002)). Abnormal mRNA splicing of ATM Abnormal mRNA splicing of ATM causes idiopathic form of causes idiopathic form of mantle-cell lymphoma (Proc Natl mantle-cell lymphoma (Proc Natl AcadSci USA97:2773-8 AcadSci USA97:2773-8 (2000)). Decreased protein serine/ (2000)). Deletion mutation in the ATM gene may correlate threonine kinase activity of ATM correlates with increased with lymphoma (Am J Hum Genet 62:334-45 (1998)). Mis occurrence of disease Susceptibility associated with sense mutation in the ATM gene correlates with increased Hodgkin's disease (Blood 103: 283-90 (2004)). Abnormal incidence of early onset form of breast neoplasms (Cancer 92: mRNA splicing of ATM correlates with increased occurrence 479-87 (2001)). Nonsense mutation in the ATM gene causes of disease Susceptibility associated with Hodgkin's disease idiopathic form of mantle-cell lymphoma (PNAS97:2773-8 (Blood 103: 283-90 (2004)). Missense mutation in the ATM (2000)). Missense mutation in the ATM gene may correlate gene causes prolymphocytic leukemia (Blood 91: 3920-6 with breast neoplasms (Proc Natl AcadSci USA99: 925-30 (1998)). Single nucleotide polymorphism in the ATM gene (2002)). Decreased expression of ATM protein causes pro correlates with increased occurrence of disease Susceptibility lymphocytic leukemia (Blood 91: 3920-6 (1998)). Point associated with Hodgkin’s disease (Blood 103: 283-90 mutation in the ATM gene causes idiopathic form of mantle (2004)). Missense mutation in the ATM gene correlates with cell lymphoma (Proc Natl AcadSci USA97:2773-8 (2000)). non-familial form of breast neoplasms (Am J Hum Genet 62: Missense mutation in the ATM gene may correlate with dis 334-45 (1998)). Nonsense mutation in the ATM gene causes ease susceptibility associated with leukemia (Am J Hum idiopathic form of mantle-cell lymphoma (Proc Natl AcadSci Genet 62: 334-45 (1998)). Mutation in the ATM gene corre USA97:2773-8 (2000)). Deletion mutation in the ATM gene lates with mantle-cell lymphoma (Proc Natl Acad Sci USA causes idiopathic form of mantle-cell lymphoma (Proc Natl 100: 5372-7 (2003)). Abnormal protein binding of ATM may AcadSci USA97:2773-8 (2000)). Loss of heterozygosity at cause increased incidence of familial form of breast neo the ATM gene causes prolymphocytic leukemia (Blood 91: plasms (Cancer Res 63: 3325-33 (2003)). Splice site mutation 3920-6 (1998)). Mutation in the ATM gene correlates with in the ATM gene causes increased incidence of familial form US 2010/015 1483 A1 Jun. 17, 2010
of ovarian neoplasms (Cancer Res 63: 3325-33 (2003)). with acute form of leukemia (Leukemia 9: 1483-6 (1995)). Splice site mutation in the ATM gene may correlate with Amplification of the BCR gene may correlate with drug genomic instability associated with colonic neoplasms (Int J resistant form of leukemia (Blood 95: 1758-66 (2000)). Cancer 86: 262-8 (2000)). Deletion mutation in the ATM gene Translocation of the BCR gene correlates with recurrence may correlate with genomic instability associated with associated with acute T-cell leukemia (Leukemia 8: 889-94 colonic neoplasms (IntJ Cancer 86: 262-8 (2000)). Mutation (1994)). Translocation of the BCR gene correlates with in the ATM gene causes increased incidence of familial form chronic form of Philadelphia-negative myeloid leukemia of ovarian neoplasms (Cancer Res 63: 3325-33 (2003)). (Blood 88: 2236-40 (1996)). Translocation of the BCR gene Mutation in the ATM gene correlates with decreased response correlates with acute myelocytic leukemia associated with to ionizing radiation associated with chronic B-cell leukemia acute L2 lymphocytic leukemia (Cancer 73: 1526-32 (1994)). (Blood 98: 814-22 (2001)). Mutation in the ATM gene causes Decreased expression of BCR mutant protein may cause increased incidence of familial form of breast neoplasms increased apoptosis associated with chronic myeloid leuke (Cancer Res 63: 3325-33 (2003)). Missense mutation in the mia (Oncogene 21: 5716-24 (2002)). Translocation of the ATM gene may correlate with breast neoplasms (PNAS 99: BCR gene correlates with chronic-phase myeloid leukemia 925-30 (2002)). Induced stimulation of the protein kinase (Blood 98: 3778-83 (2001)). Translocation of the BCR gene activity of ATM may correlate with increased response to correlates with Philadelphia chromosome associated with drug associated with myeloid leukemia (Blood 101: 4589-97 chronic myeloid leukemia (Leukemia 13: 2007-11 (1999)). (2003)). Nonsense mutation in the ATM gene causes diffuse Translocation of the BCR gene correlates with increased large-cell lymphoma (Blood 100: 1430-7 (2002)). Decreased response to drug associated with acute promyelocytic leuke protein kinase activity of ATM may cause increased incidence mia (Oncogene 22:6900-8 (2003)). Amplification of the BCR of familial form of breast neoplasms (Proc Natl Acad Sci gene correlates with Sezary syndrome associated with skin USA99: 925-30 (2002)). Missense mutation in the ATM gene neoplasms (Blood 101: 1513-9 (2003)). Induced inhibition of may cause increased incidence of non-familial form of breast BCR mutant protein may prevent decreased apoptosis asso neoplasms (Proc Natl Acad Sci USA 99: 925-30 (2002)). ciated with chronic myeloid leukemia (Blood 91: 641-8 Missense mutation in the ATM gene causes increased inci (1998)). Translocation of the BCR gene correlates with pre dence of familial form of breast neoplasms (Cancer Res 63: B-cell leukemia associated with chronic myeloid leukemia 3325-33 (2003)). (PhosphoSite R, Cell Signaling Technology (Leukemia 13: 2007-11 (1999)). Induced inhibition of BCR (Danvers, Mass.), Human PSDTM, Biobase Corporation, mutant protein may prevent increased cell proliferation asso (Beverly, Mass.)). ciated with chronic myeloid leukemia (Blood 91: 3414-22 0078 Bcr (P11274), phosphorylated at Y513, is among (1998)). Induced inhibition of BCR mutant protein may cause the proteins listed in this patent. Bcr, Breakpoint cluster increased apoptosis associated with chronic myeloid leuke region, GTPase-activating protein for p21 rac with serine mia (Leukemia 15: 1537-43 (2001)). Induced inhibition of threonine kinase activity; BCR-ABL gene fusion is associ BCR mutant protein may prevent decreased cell cycle arrest ated with several types of leukemia and multiple myeloma, associated with chronic myeloid leukemia (Blood 91: 641-8 variants may be associated with bipolar disorder. This protein (1998)). Decreased expression of BCR mutant protein may has potential diagnostic and/or therapeutic implications cause increased apoptosis associated with acute L2 lympho based on the following findings. Translocation of the BCR cytic leukemia (Blood 104: 356-63 (2004)). Increased gene correlates with acute B-cell leukemia (Leukemia 15: expression of BCR mutant protein correlates with increased 1834-40 (2001)). Amplification of the BCR gene correlates incidence of disease progression associated with chronic with mycosis fungoides associated with skin neoplasms myeloid leukemia (Blood86:2371-8 (1995)). Mutation in the (Blood 101: 1513-9 (2003)). Amplification of the BCR gene BCR gene correlates with chronic form of Philadelphia-nega correlates with drug-resistant form of leukemia (Cancer 100: tive myeloid leukemia (Cancer 75: 464-70 (1995)). Translo 1459-71 (2004)). Translocation of the BCR gene correlates cation of the BCR gene correlates with decreased incidence with early onset form of acute L2 lymphocytic leukemia of death associated with Philadelphia-positive myeloid leu (Cancer 73: 1526-32 (1994)). Decreased expression of BCR kemia (Leukemia 4:448-9 (1990)). Methylation of the BCR mutant protein may prevent chronic-phase myeloid leukemia gene correlates with Philadelphia-negative myeloid leukemia (Blood 87: 4770-9 (1996)). Increased expression of BCR (Leukemia 6: 35-41 (1992)). Translocation of the BCR gene mutant protein correlates with early onset form of acute T-cell correlates with increased incidence of death associated with leukemia (Leukemia 8: 1124-30 (1994)). Translocation of the acute B-cell leukemia (Blood 102: 2014-20 (2003)). Deletion BCR gene correlates with decreased cell differentiation asso mutation in the BCR gene correlates with acute L2 lympho ciated with chronic myeloid leukemia (Leukemia 13: 2007 cytic leukemia (Blood 97:3581-8 (2001)). Translocation of 11 (1999)). Translocation of the BCR gene causes acute lym the BCR gene correlates with chronic myeloid leukemia phocytic leukemia (Leukemia 4: 397-403 (1990)). (Hum Mol Genet 11: 1391-7 (2002)). Decreased expression Translocation of the BCR gene correlates with chronic of BCR mutant protein may prevent increased incidence of myeloid leukemia associated with Philadelphia-positive recurrence associated with chronic-phase myeloid leukemia myeloid leukemia (Leukemia 13: 2007-11 (1999)). (Blood 93: 284-92 (1999)). Translocation of the BCR gene Decreased expression of BCR mutant protein prevents may cause multiple myeloma (Nucleic Acids Res 28: 4865 increased occurrence of recurrence associated with acute L2 72 (2000)). Translocation of the BCR gene correlates with lymphocytic leukemia (Blood 100: 2357-66 (2002)). Trans pre-B-cell leukemia (Leukemia 13: 2007-11 (1999)). Trans location of the BCR gene correlates with advanced stage or location of the BCR gene correlates with pre-B-cell leukemia high grade form of acute lymphocytic leukemia (L1) (Leu (Leukemia 15: 1834-40 (2001)). Decreased expression of kemia 9: 1689-93 (1995)). Deletion mutation in the BCR BCR mutant protein may prevent recurrence associated with gene correlates with chronic myeloid leukemia (Blood 97: Philadelphia-negative myeloid leukemia (Leukemia 13:999 3581-8 (2001)). Translocation of the BCR gene correlates 1008 (1999)). Translocation of the BCR gene may correlate US 2010/015 1483 A1 Jun. 17, 2010 32 with Philadelphia chromosome associated with acute T-cell syndromes associated with agammaglobulinemia (PNAS 91: leukemia (Leukemia 8: 889-94 (1994)). Decreased expres 9062-6 (1994)). Nonsense mutation in the BTK gene causes sion of BCR mutant protein may prevent increased incidence agammaglobulinemia (Mol Med 2: 619-23 (1996)). of recurrence associated with chronic-phase myeloid leuke Decreased phosphatidylinositol-3,4,5-triphosphate binding mia (Blood 87: 2588-93 (1996)). Translocation of the BCR of BTK may cause agammaglobulinemia (EMBO J. 16: gene correlates with chronic form of Philadelphia-negative 3396–404 (1997)). Decreased phosphatidylinositol-3,4,5-tri myeloid leukemia (Leukemia 6: 385-92 (1992)). Transloca phosphate binding of BTK may cause agammaglobulinemia tion of the BCR gene correlates with Philadelphia chromo (EMBO J 16: 3396–404 (1997)). Decreased expression of some associated with pre-B-cell leukemia (Leukemia 13: BTK in monocytes correlates with abnormal B-lymphocytes 2007-11 (1999)). Alternative form of BCR mutant protein function associated with agammaglobulinemia (Blood 91: correlates with increased response to drug associated with 595-602 (1998)). Missense mutation in the protein kinase Philadelphia-positive myeloid leukemia (Leukemia 6: 948 domain of BTK causes agammaglobulinemia (Proc Natl 51 (1992)). Translocation of the BCR gene correlates with Acad Sci USA 91: 12803-7 (1994)). Nonsense mutation in late onset form of acute B-cell leukemia (Leukemia 7: 10547 the BTK gene causes agammaglobulinemia (PNAS 91: (1993)). Translocation of the BCR gene correlates with 9062-6 (1994)). Absence of the protein binding of BTK may chronic myeloid leukemia associated with pre-B-cell leuke cause abnormal signal transduction associated with agamma mia (Leukemia 13: 2007-11 (1999)). Alternative form of globulinemia (J Exp Med 180: 461-70 (1994)). Decreased BCR mutant protein correlates with decreased isotype expression of BTK in B-lymphocytes correlates with agam Switching associated with chronic myeloid leukemia (Leuke maglobulinemia (Cell 72: 279-90 (1993)). Insertion mutation mia 13: 2007-11 (1999)). Translocation of the BCR gene in the BTK gene causes agammaglobulinemia (Blood 96: correlates with decreased isotype Switching associated with 610-7 (2000)). Deletion mutation in the BTK gene causes chronic myeloid leukemia (Leukemia 13: 2007-11 (1999)). agammaglobulinemia (Blood 96: 610-7 (2000)). Absence of (PhosphoSite(R), Cell Signaling Technology (Danvers, the protein kinase activity of BTK causes agammaglobuline Mass.), Human PSDTM, Biobase Corporation, (Beverly, mia (Blood 88: 561-73 (1996)). Decreased phosphatidyli Mass.)). nositol-3,4,5-triphosphate binding of BTK may cause agam 0079 Btk (Q06187), phosphorylated at Y39, Y333, is maglobulinemia (EMBO 16: 3396–404 (1997)). Missense among the proteins listed in this patent. Btk, Bruton agam mutation in the protein kinase domain of BTK causes agam maglobulinemia tyrosine kinase, functions in pre-B cell maglobulinemia (PNAS 91: 12803-7 (1994)). Missense receptor signaling and B cell development; gene mutation is mutation in the PH domain of BTK causes agammaglobu associated with X-linked agammaglobulinemia (XLA), linemia (EMBO J. 16: 3396–404 (1997)). Mutation in the mouse Btk gene mutation is associated with X-linked immu BTK gene causes abnormal B cell differentiation associated nodeficiency (Xid). This protein has potential diagnostic and/ with agammaglobulinemia (Hum Mol Genet 3: 161-6 or therapeutic implications based on the following findings. (1994)). Frameshift mutation in the BTK gene causes agam Mutation in the BTK gene causes immunologic deficiency maglobulinemia (FEBS Lett 346: 165-70 (1994)). Mutation syndromes associated with agammaglobulinemia (Proc Natl in the BTK gene causes agammaglobulinemia (Nucleic Acids AcadSci USA'91:9062-6 (1994)). Mutation in the BTK gene Res 26: 242-7 (1998)). Missense mutation in the PH domain causes abnormal B cell differentiation associated with agam of BTK causes agammaglobulinemia (EMBO 16: 3396–404 maglobulinemia (Nature 361: 226-33 (1993)). Mutation in (1997)). Missense mutation in the BTK gene causes agam the BTK gene causes agammaglobulinemia (Nucleic Acids maglobulinemia (Hum Mol Genet 3: 1751-6 (1994)). Dele Res 24:160-5 (1996)). Splice site mutation in the BTK gene tion mutation in the BTK gene causes agammaglobulinemia causes agammaglobulinemia (FEBS Lett 346: 165-70 (Proc Natl AcadSci USA'91:9062-6 (1994)). Deletion muta (1994)). Splice site mutation in the BTK gene causes agam tion in the BTK gene causes agammaglobulinemia (Proc Natl maglobulinemia (Hum Mol Genet 4: 693-700 (1995)). Muta AcadSci USA 91:9062-6 (1994)). Insertion mutation in the tion in the BTK gene causes immunologic deficiency syn BTK gene causes agammaglobulinemia (Hum Mol Genet 3: dromes associated with agammaglobulinemia (Proc Natl 79-83 (1994)). Decreased expression of BTK mRNA corre AcadSci USA 91:9062-6 (1994)). Insertion mutation in the lates with agammaglobulinemia (Blood 88: 561-73 (1996)). BTK gene causes agammaglobulinemia (Hum Mol Genet 3: Nonsense mutation in the BTK gene causes agammaglobu 161-6 (1994)). Missense mutation in the protein kinase linemia (Proc Natl Acad Sci USA 91:9062-6 (1994)). Point domain of BTK causes agammaglobulinemia (Hum Mol mutation in the BTK gene causes agammaglobulinemia Genet 4: 693-700 (1995)). Missense mutation in the BTK (Hum Mol Genet 4: 693-700 (1995)). Deletion mutation in gene causes agammaglobulinemia (PNAS 91: 9062-6 the SH3 domain of BTK causes abnormal B cell differentia (1994)). Splice site mutation in the BTK gene causes agam tion associated with agammaglobulinemia (J Exp Med 180: maglobulinemia (Am J Hum Genet 60: 798-807 (1997)). 461-70 (1994)). Missense mutation in the BTK gene causes Missense mutation in the BTK gene causes agammaglobu agammaglobulinemia (FEBS Lett 413: 205-10 (1997)). linemia (Proc Natl Acad Sci USA 91: 9062-6 (1994)). Non Frameshift mutation in the BTK gene causes agammaglobu sense mutation in the BTK gene causes immunologic defi linemia (PNAS 91:9062-6 (1994)). Nonsense mutation in the ciency syndromes associated with agammaglobulinemia BTK gene causes agammaglobulinemia (Proc Natl Acad Sci (Mol Med 2: 619-23 (1996)). Nonsense mutation in the BTK USA 91: 9062-6 (1994)). Splice site mutation in the BTK gene causes agammaglobulinemia (Hum Mol Genet 3: gene causes agammaglobulinemia (Hum Mol Genet 4: 51-8 1751-6 (1994)). Deletion mutation in the BTK gene causes (1995)). Nonsense mutation in the BTK gene causes agam less severe form of agammaglobulinemia (Clin Exp Immunol maglobulinemia (Hum Mol Genet 3:161-6 (1994)). Mutation 107: 235-40 (1997)). Mutation in the BTK gene causes agam in the BTK gene causes immunologic deficiency syndromes maglobulinemia (Am J Hum Genet 62: 1034-43 (1998)). associated with agammaglobulinemia (Clin Exp Immunol Mutation in the BTK gene causes immunologic deficiency 120: 512-7 (2000)). Missense mutation in the BTK gene US 2010/015 1483 A1 Jun. 17, 2010
causes agammaglobulinemia (Proc Natl Acad Sci USA 91: BUB1 may cause colorectal neoplasms (Cancer Res 60: 9062-6 (1994)). Missense mutation in the SH2 domain of 4349-52 (2000)). Deletion mutation in the BUB1 gene may BTK causes agammaglobulinemia (J Immunol 164: 4170-7 cause chromosome aberrations associated with acute HTLV (2000)). Missense mutation in the protein kinase domain of 1-associated leukemia (Cancer Lett 158: 141-50 (2000)). BTK causes agammaglobulinemia (Clin Exp Immunol 120: Splice site mutation in the BUB1 gene may cause abnormal 346-50 (2000)). Missense mutation in the PH domain of BTK mitotic sister chromatid segregation associated with colorec causes agammaglobulinemia (Blood 88: 561-73 (1996)). tal neoplasms (Nature 392: 300-303 (1998)). Missense muta Missense mutation in the BTK gene causes agammaglobu tion in the BUB1 gene may cause increased occurrence of linemia (J Immunol 161: 3925-9 (1998)). Mutation in the neoplasm metastasis associated with colorectal neoplasms BTK gene causes agammaglobulinemia (J Immunol 167: (Cancer Res 62: 13-7 (2002)). Point mutation in the BUB1 4038-45 (2001)). Decreased protein kinase activity of BTK gene may cause chromosome aberrations associated with causes agammaglobulinemia (Clin Exp Immunol 120: 346 acute HTLV-1-associated leukemia (Cancer Lett 158: 141-50 50 (2000)). Missense mutation in the BTK gene causes agam (2000)). Missense mutation in the BUB1 gene may cause maglobulinemia (Hum Mol Genet 4: 51-8 (1995)). Mutation recurrence associated with colorectal neoplasms (Cancer Res in the BTK gene causes agammaglobulinemia (Nucleic Acids 62: 13-7 (2002)). Decreased expression of BUB1 mRNA may Res 25: 166-71 (1997)). Deletion mutation in the BTK gene cause increased occurrence of malignant form of colorectal causes late onset form of agammaglobulinemia (Clin Exp neoplasms (Cancer Res 62: 13-7 (2002)). Splice site mutation Immunol 107: 235-40 (1997)). Frameshift mutation in the in the BUB1 gene may cause abnormal mitotic checkpoint BTK gene causes agammaglobulinemia (Hum Mol Genet 4: associated with colorectal neoplasms (Nature 392: 300-303 51-8 (1995)). Frameshift mutation in the BTK gene causes (1998)). Missense mutation in the BUB1 gene may cause agammaglobulinemia (Proc Natl Acad Sci USA 91: 9062-6 abnormal mitotic checkpoint associated with colorectal neo (1994)). Missense mutation in the PH domain of BTK causes plasms (Nature 392: 300-303 (1998)). Decreased expression agammaglobulinemia (EMBOJ 16:3396–404 (1997)). Muta of BUB1 mRNA may cause recurrence associated with col tion in the BTK gene causes agammaglobulinemia (Hum Mol orectal neoplasms (Cancer Res 62: 13-7 (2002)). (Phospho Genet 3: 79-83 (1994)). Missense mutation in the BTK gene Site(R), Cell Signaling Technology (Danvers, Mass.), Human causes agammaglobulinemia (Blood 96: 610-7 (2000)). PSDTM, Biobase Corporation, (Beverly, Mass.)). Deletion mutation in the BTK gene causes agammaglobu I0081 CD34 (P28906), phosphorylated at Y330, is among linemia (PNAS 91: 9062-6 (1994)). Frameshift mutation in the proteins listed in this patent. CD34. CD34 antigen, a the BTK gene causes agammaglobulinemia (Proc Natl Acad transmembrane sialomucin associated with hematopoietic Sci USA 91: 9062-6 (1994)). Missense mutation in the pro stem cells and an L-selectin ligand on high endothelial tein kinase domain of BTK causes agammaglobulinemia Venules, transduces signals that regulate cytoadhesion of (Proc Natl Acad Sci USA 91: 12803-7 (1994)). Deletion hematopoietic cells, may play a role in early stages of mutation in the BTK gene causes agammaglobulinemia hematopoiesis. This protein has potential diagnostic and/or (Hum Mol Genet 4: 693-700 (1995)). Splice site mutation in therapeutic implications based on the following findings. the BTK gene causes agammaglobulinemia (Hum Mol Genet Abnormal expression of CD34 protein may correlate with 3: 1751-6 (1994)). Nonsense mutation in the BTK gene acute myelocytic leukemia (Blood 86: 60-5 (1995)). causes agammaglobulinemia (Hum Mol Genet 4: 51-8 Increased expression of CD34 in hematopoietic stem cells (1995)). Decreased protein-tyrosine kinase activity of BTK may correlate with less severe form of HIV infections (Blood causes agammaglobulinemia (Clin Exp Immunol 107: 235 86: 1749-56 (1995)). Abnormal expression of CD34 protein 40 (1997)). Missense mutation in the protein kinase domain may correlate with chronic myeloid leukemia (Blood 86: of BTK causes agammaglobulinemia (Mol Med 6: 104-13 60-5 (1995)). (PhosphoSite(R), Cell Signaling Technology (2000)). Frameshift mutation in the BTK gene causes agam (Danvers, Mass.), Human PSDTM, Biobase Corporation, maglobulinemia (Hum Mol Genet 3: 1751-6 (1994)). Mis (Beverly, Mass.)). sense mutation in the SH2 domain of BTK causes agamma I0082 CLH-17 (Q00610), phosphorylated at Y1095, globulinemia (Hum Mol Genet 3: 161-6 (1994)). Y1205, Y1210, is among the proteins listed in this patent. (PhosphoSite(R), Cell Signaling Technology (Danvers, CLH-17, Clathrin heavy polypeptide Hc, binds huntingtin Mass.), Human PSDTM, Biobase Corporation, (Beverly, interacting protein 1 (HIP1), involved in endocytosis, may Mass.)). bind to endocytic proteins; gene fusion to ALK is associated 0080 Bub1 (O43683), phosphorylated at Y219, is among with inflammatory myofibroblastic tumor and large B-cell the proteins listed in this patent. Bub1, Budding uninhibited lymphoma. This protein has potential diagnostic and/orthera by benzimidazoles 1 homolog, acts in spindle assembly peutic implications based on the following findings. Translo checkpoint and chromosome congression, may regulate cation of the CLTC gene correlates with B-cell lymphoma vesicular traffic; mutations are associated with lung cancer, T associated with diffuse large-cell lymphoma (Blood 102: cell leukemia and colorectal cancer cell chromosomal insta 2568-73 (2003)). Translocation of the CLTC gene correlates bility. This protein has potential diagnostic and/or therapeutic with B-cell lymphoma (Blood 102: 2638-41 (2003)). Trans implications based on the following findings. Missense muta location of the CLTC gene correlates with diffuse large-cell tion in the BUB1 gene may cause abnormal mitotic sister lymphoma associated with B-cell lymphoma (Blood 102: chromatid segregation associated with colorectal neoplasms 2568-73 (2003)). (PhosphoSite(R), Cell Signaling Technology (Nature 392: 300-303 (1998)). Decreased expression of (Danvers, Mass.), Human PSDTM, Biobase Corporation, BUB1 mRNA may cause increased occurrence of neoplasm (Beverly, Mass.)). metastasis associated with colorectal neoplasms (Cancer Res I0083) CR2 (P20023), phosphorylated at Y1029, is among 62: 13-7 (2002)). Missense mutation in the BUB1 gene may the proteins listed in this patent. CR2, Complement receptor cause increased occurrence of malignant form of colorectal 2, binds to the breakdown products of complement C3 and neoplasms (Cancer Res 62: 13-7 (2002)). Locus instability of interacts with CD23 (FCER2); altered expression is associ US 2010/015 1483 A1 Jun. 17, 2010 34 ated with various diseases; inhibition by blocking antibody myeloid leukemia (Blood 88: 4304-13 (1996)). Increased may be therapeutic for HIV infection. This protein has poten phosphorylation of CRKL correlates with Philadelphia-posi tial diagnostic and/or therapeutic implications based on the tive myeloid leukemia (JBC 269: 22925-8 (1994)). Increased following findings. Decreased expression of CR2 protein cor phosphorylation of CRKL correlates with Philadelphia-posi relates with chronic B-cell leukemia (Clin Exp Immunol 83: tive myeloid leukemia (J Biol Chem 269: 22925-8 (1994)). 423–9 (1991)). Increased expression of CR2 in T-lympho Abnormal SH3/SH2 adaptor activity of CRKL may cause cytes correlates with glomerulonephritis associated with sys abnormal intracellular signaling cascade associated with temic lupus erythematosus (Clin Exp Immunol 90: 235-44 Philadelphia-positive myeloid leukemia (JBC 270: 21468-71 (1992)). Increased expression of CR2 protein may cause (1995)). Decreased SH3/SH2 adaptor activity of CRKL may increased cell-cell adhesion associated with multiple prevent increased cell proliferation associated with chronic myeloma (Blood 85: 3704-12 (1995)). Increased presence of myeloid leukemia (FASEBJ 14: 1529-38 (2000)). (Phospho CR2 antibody may prevent increased entry of virus into host Site(R), Cell Signaling Technology (Danvers, Mass.), Human cell associated with HIV infections (EurJImmunol 33: 2098 PSDTM, Biobase Corporation, (Beverly, Mass.)). 107 (2003)). Increased expression of CR2 in B-lymphocytes I0085 CRMP-2 (Q16555), phosphorylated at Y499, is correlates with asthma (Clin Exp Immunol 94: 337-40 among the proteins listed in this patent. CRMP-2, Dihydro (1993)). Decreased expression of CR2 in B-lymphocytes cor pyrimidinase-like 2, binds tubulin and axon growth cone pro relates with systemic lupus erythematosus (Clin Exp Immu teins, regulates microtubule formation, acts in neuronal nol 101: 60-5 (1995)). Increased expression of CR2 protein growth cone collapse and axonal growth, abnormal expres correlates with interstitial nephritis associated with Epstein sion or modification is linked to neuroinflammatory and Barr virus infections (J Clin Invest 104: 1673-81 (1999)). Alzheimer diseases. This protein has potential diagnostic Increased expression of CR2 in B-lymphocytes may correlate and/or therapeutic implications based on the following find with abnormal immune response associated with asthma (Eur ings. Increased phosphorylation of DPYSL2 correlates with J Immunol 24: 1109-14 (1994)). Decreased expression of Alzheimer disease (Biochemistry 39: 4267-75 (2000)). CR2 protein correlates with chronic lymphocytic leukemia Increased oxidation of DPYSL2 correlates with Alzheimer (Clin Exp Immunol 102: 575-81 (1995)). Decreased expres disease (J Neurochem 82: 1524-32 (2002)). Increased phos sion of CR2 in T-lymphocytes correlates with HIV infections phorylation of DPYSL2 correlates with Alzheimer disease (Immunology 75: 59-65 (1992)). (PhosphoSite R, Cell Sig (Biochemistry Usa 39: 4267-75 (2000)). (PhosphoSite R, naling Technology (Danvers, Mass.), Human PSDTM, Bio Cell Signaling Technology (Danvers, Mass.), Human PSDTM, base Corporation, (Beverly, Mass.)). Biobase Corporation, (Beverly, Mass.)). I0084) CrkL (P46109), phosphorylated at Y48,Y92, Y198, I0086 CSK (P41240), phosphorylated at Y416, is among is among the proteins listed in this patent. CrkL, V-crk sar the proteins listed in this patent. CSK, C-src tyrosine kinase, coma virus CT10 oncogene homolog (avian)-like, SH2-SH3 a protein tyrosine kinase with SH2 and SH3 domains, inacti adaptor protein and transcription cofactor, activates RAS and Vates the c-Src (SRC) oncoprotein, regulates receptor signal JUN kinase pathways, associated with chronic myelogenous ing pathways and possibly T-cell activation, and acts as a leukemia; mutations in mouse Crkl mimic DiGeorge Syn tumor antigen in carcinomas. This protein has potential diag drome. This protein has potential diagnostic and/or therapeu nostic and/or therapeutic implications based on the following tic implications based on the following findings. Decreased findings. Decreased expression of CSK protein correlates expression of CRKL protein may prevent increased cell pro with carcinoma tumors associated with colorectal neoplasms liferation associated with Philadelphia-positive myeloid leu (Cancer 92: 61-70 (2001)). Increased presence of CSK kemia (Biochem Biophys Res Commun 235: 383-8 (1997)). autoimmune antibody correlates with adenocarcinoma (Can Increased phosphorylation of CRKL correlates with Philadel cer Res 61: 1415-20 (2001)). Abnormal expression of CSK phia-positive myeloid leukemia (Blood 84: 2912-8 (1994)). protein may cause abnormal cell-cell adhesion associated Increased phosphorylation of CRKL may correlate with with colonic neoplasms (Oncogene 23: 289-97 (2004)). decreased response to drug associated with chronic myeloid Abnormal expression of CSK protein may cause abnormal leukemia (Leukemia 18: 401-8 (2004)). Abnormal SH3/SH2 integrin-mediated signaling pathway associated with colonic adaptor activity of CRKL may cause abnormal cell adhesion neoplasms (Oncogene 23: 289-97 (2004)). Increased expres associated with Philadelphia-positive myeloid leukemia sion of CSK protein correlates with adenocarcinoma (Cancer (JBC 270: 29145-50 (1995)). Decreased SH3/SH2 adaptor Res 61: 1415-20 (2001)). Increased protein binding of CSK activity of CRKL may prevent increased cell proliferation correlates with increased intracellular signaling cascade asso associated with chronic myeloid leukemia (FASEB 14: 1529 ciated with prostatic neoplasms (Int J Cancer 68: 164-71 38 (2000)). CRKL map position correlates with DiGeorge (1996)). Increased protein binding of CSK correlates with syndrome (Nat Genet. 27: 293-8 (2001)). Decreased phos malignant form of prostatic neoplasms (IntJ Cancer 68: 164 phorylation of CRKL may correlate with increased response 71 (1996)). Abnormal expression of CSK protein may cause to drug associated with chronic myeloid leukemia (Blood abnormal cell migration associated with colonic neoplasms 104:509-18 (2004)). Abnormal SH3/SH2 adaptor activity of (Oncogene 23: 289-97 (2004)). Increased protein binding of CRKL may cause abnormal cell adhesion associated with CSK correlates with increased carcinoma associated with Philadelphia-positive myeloid leukemia (J Biol Chem 270: prostatic neoplasms (Int J Cancer 68: 164-71 (1996)). 29145-50 (1995)). Abnormal SH3/SH2 adaptor activity of Increased presence of CSK autoimmune antibody correlates CRKL may cause abnormal intracellular signaling cascade with carcinoma associated with neoplasms (Cancer Res 61: associated with Philadelphia-positive myeloid leukemia (J 1415-20 (2001)). (PhosphoSite(R), Cell Signaling Technology Biol Chem 270: 21468-71 (1995)). Increased phosphoryla (Danvers, Mass.), Human PSDTM, Biobase Corporation, tion of CRKL correlates with Philadelphia-positive myeloid (Beverly, Mass.)). leukemia (Blood 84: 1731-6 (1994)). Increased phosphory I0087 DNA-PK (P78527), phosphorylated atY779, Y883, lation of CRKL correlates with Philadelphia-positive Y2936, is among the proteins listed in this patent. DNA-PK, US 2010/015 1483 A1 Jun. 17, 2010
DNA-dependent protein kinase catalytic subunit, a DNA sion of ENO2 in serum correlates with small cell carcinoma binding protein kinase involved in DNA double-strand break (Eur J Cancer: 198-202 (1993)). Increased expression of repair, V(D)J recombination, and transcriptional regulation, ENO2 protein may correlate with melanoma (Eur J Cancer phosphorylates and activates AKT: mouse Prkdc deficiency is 31: 1898-902 (1995)). Decreased expression of ENO2 pro associated with SCID. This protein has potential diagnostic tein correlates with decreased occurrence of death associated and/or therapeutic implications based on the following find with non-Small-cell lung carcinoma (Anticancer Res 22: ings. Decreased expression of PRKDC mRNA may prevent 1083-9 (2002)). Increased expression of ENO2 in serum cor decreased response to radiation associated with prostatic neo relates with adenocarcinoma tumors associated with lung plasms (Cancer Res 63: 1550-4 (2003)). Mutation in the neoplasms (EurJCancer: 198-202 (1993)). Increased expres PRKDC gene correlates with colorectal neoplasms (Cancer sion of ENO2 protein correlates with non-small-cell lung Res 62: 1284-8 (2002)). Decreased expression of PRKDC carcinoma (Anticancer Res 23: 885-93 (2003)). Increased protein may correlate with increased response to radiation expression of ENO2 in serum correlates with increased associated with lung neoplasms (Eur J Cancer 35: 111-6 occurrence of death associated with Small cell carcinoma (Br (1999)). Decreased expression of PRKDC mRNA may pre JCancer 67: 760-6 (1993)). Increased expression of ENO2 in vent decreased response to radiation associated with non serum correlates with neoplasm metastasis associated with small-cell lung carcinoma (Cancer Res 62: 6621-4 (2002)). non-small-cell lung carcinoma (Br J Cancer 84: 903-9 Increased cleavage of PRKDC may prevent multiple (2001)). (PhosphoSite(R), Cell Signaling Technology (Dan myeloma (Blood 101: 1530-4 (2003)). Abnormal expression vers, Mass.), Human PSDTM, Biobase Corporation, (Beverly, of PRKDC mRNA may correlate with chronic lymphocytic Mass.)). leukemia (Anticancer Res 22: 1787-93 (2002)). Single nucle (0090 ERK2 (P28482), phosphorylated at Y35, is among otide polymorphism in the PRKDC gene correlates with the proteins listed in this patent. ERK2, Mitogen-activated increased occurrence of disease Susceptibility associated with protein kinase 1, a serine-threonine kinase effector of the breast neoplasms (Cancer Res 63: 2440-6 (2003)). Decreased RAS-MAP kinase pathway, translocates to the nucleus to proteolysis of PRKDC may correlate with drug-resistant mediate transcription when activated, involved in the regula form of Burkitt Lymphoma (IntJ Cancer 77: 755-62 (1998)). tion of cell growth, differentiation, migration and apoptosis. MRNA instability of PRKDC may cause decreased double This protein has potential diagnostic and/or therapeutic Strand break repair associated with glioma (Oncogene 18: implications based on the following findings. Decreased 1361-8 (1999)). Gene instability of PRKDC may correlate nucleus localization of MAPK1 may correlate with increased with colorectal neoplasms (Hum Mol Genet 10: 513-8 transforming growth factor beta receptor signaling pathway (2001)). (PhosphoSite(R), Cell Signaling Technology (Dan associated with pancreatic neoplasms (Oncogene 19: 4531 vers, Mass.), Human PSDTM, Biobase Corporation, (Beverly, 41 (2000)). Decreased expression of MAPK1 in hippocam Mass.)). pus correlates with depression (J Neurochem 77: 916-28 I0088 elF2B (P20042), phosphorylated at Y298, is among (2001)). Insertion mutation in the MAPK1 gene correlates the proteins listed in this patent. eIF2B. Eukaryotic transla with hepatitis B associated with hepatocellular carcinoma tion initiation factor 2 subunit 2 beta 38 kDa, beta subunit of (Oncogene 22: 3911-6 (2003)). Induced stimulation of the eIF2, which is a translation initiation factor involved in the protein kinase activity of MAPK1 may cause increased apo initiation of protein synthesis; mutations are linked to leu ptosis associated with lung neoplasms (Oncogene 22: 5427 koencephalopathy with Vanishing white matter. This protein 35 (2003)). Increased nucleus localization of MAPK1 may has potential diagnostic and/or therapeutic implications cause decreased induction of apoptosis in response to chemi based on the following findings (PhosphoSite(R), Cell Signal cal stimulus associated with leukemia (J Biol Chem 279: ing Technology (Danvers, Mass.), Human PSDTM, Biobase 32813-23 (2004)). Induced stimulation of the MAP kinase 1 Corporation, (Beverly, Mass.)). activity of MAPK1 may cause increased actin filament orga I0089 ENO2 (P09104), phosphorylated at Y24, is among nization associated with ovarian neoplasms (J Biol Chem the proteins listed in this patent. ENO2, Neuron-specific eno 280: 11961-72 (2005)). Increased phosphorylation of lase (gamma enolase), catalyzes conversion of 2-phospho-D- MAPK1 may cause increased signal transduction associated glycerate to phosphoenolpyruvate in glycolysis, may be with ovarian neoplasms (JBiol Chem280: 11961-72 (2005)). involved in neuronal differentiation; altered expression is Increased phosphorylation of MAPK1 may cause increased seen inbreast cancer, lung cancer, and multiple Sclerosis. This cell death associated with breast neoplasms (FEBS Lett 458: protein has potential diagnostic and/or therapeutic implica 137-40 (1999)). Increased MAP kinase activity of MAPK1 tions based on the following findings. Abnormal expression correlates with malignant form of non-small-cell lung carci of ENO2 in serum correlates with decreased response to drug noma (Br J Cancer 90: 1047-52 (2004)). Increased cytosol associated with small cell carcinoma (Cancer 82: 1049-55 localization of MAPK1 may cause decreased induction of (1998)). Increased expression of ENO2 protein correlates apoptosis associated with leukemia (JBC 279: 32813–23 with leiomyosarcoma associated with ovarian neoplasms (2004)). Decreased phosphorylation of MAPK1 may cause (Anticancer Res 23: 3433-6 (2003)). Increased expression of increased apoptosis associated with colonic neoplasms (Br J ENO2 protein may correlate with carcinoma tumors associ Cancer 82: 905-12 (2000)). Induced inhibition of the MAP ated with pancreatic neoplasms (Cancer 70: 1514-9 (1992)). kinase 1 activity of MAPK1 may prevent drug-resistant form Increased expression of ENO2 in serum correlates with small of multiple myeloma (Blood 101: 703-5 (2003)). Increased cell carcinoma (Anticancer Res 11: 2107-10 (1991)). MAP kinase activity of MAPK1 correlates with advanced Increased expression of ENO2 in serum correlates with dis stage or high grade form of non-Small-cell lung carcinoma ease progression associated with Small cell carcinoma (Can (Br J Cancer 90: 1047-52 (2004)). Induced stimulation of the cer 72: 418-25 (1993)). Increased expression of ENO2 pro MAP kinase 1 activity of MAPK1 may cause increased signal tein correlates with carcinoma tumors associated with breast transduction associated with ovarian neoplasms (JBiol Chem neoplasms (Br J Cancer 82: 20-7 (2000)). Increased expres 280: 11961-72 (2005)). Induced inhibition of the MAP kinase US 2010/015 1483 A1 Jun. 17, 2010 36
1 activity of MAPK1 may correlate with increased transform (0091 EWS (Q01844), phosphorylated at Y417, is among ing growth factor beta receptor signaling pathway associated the proteins listed in this patent. EWS, Ewing sarcoma break with pancreatic neoplasms (Oncogene 19:4531-41 (2000)). point region 1, a transcriptional regulator that binds RNA and Increased cytosol localization of MAPK1 may cause may function in mRNA processing, signal transduction, or decreased induction of apoptosis associated with leukemia (J brain development, involved in many cancer-related translo Biol Chem 279: 32813-23 (2004)). Increased nucleus local cation-fusion events with transcription factors. This protein ization of MAPK1 may cause decreased induction of apop has potential diagnostic and/or therapeutic implications tosis in response to chemical stimulus associated with leuke based on the following findings. EWSR1 mutant protein mia (JBC 279: 32813-23 (2004)). Increased phosphorylation causes increased transcription initiation associated with of MAPK1 may cause increased signal transduction associ Ewing's sarcoma (Oncogene 24: 2715-22 (2005)). Abnormal ated with ovarian neoplasms (JBC 280: 11961-72 (2005)). expression of EWSR1 protein may cause increased cell pro Increased expression of MAPK1 protein correlates with liferation associated with chondrosarcoma (Cancer Res 63: breast neoplasms (Anticancer Res 19: 731-40 (1999)). Inser 449-54 (2003)). EWSR1 mutant protein may cause increased tion mutation in the MAPK1 gene correlates with hepatitis B transcription initiation associated with Ewing's sarcoma associated with liver neoplasms (Oncogene 22: 3911-6 (Cancer Res 63: 8338-44 (2003)). EWSR1 mutant protein (2003)). Decreased expression of MAPK1 in frontal cortex causes abnormal regulation of transcription associated with correlates with depression (JNeurochem 77: 916-28 (2001)). Ewing's sarcoma (Oncogene 20: 626-33 (2001)). Transloca Abnormal expression of MAPK1 mRNA may correlate with tion of the EWSR1 gene correlates with giant cell tumor of acute myelocytic leukemia (Oncogene 23:9381-91 (2004)). bone associated with bone neoplasms (IntJ Cancer 87: 328 Increased expression of MAPK1 protein may correlate with 35 (2000)). Translocation of the EWSR1 gene may cause hepatocellular carcinoma associated with liver neoplasms malignant form of melanoma (Oncogene 10: 1749-56 (1995)). Translocation of the EWSR1 gene correlates with (Biochem Biophys Res Commun 236: 54-8 (1997)). melanoma tumors associated with Soft tissue neoplasms (Nat Increased MAP kinase 1 activity of MAPK1 may correlate Genet 4:341-5 (1993)). Translocation of the EWSR1 gene with hepatocellular carcinoma associated with liver neo may cause Ewing's sarcoma associated with bone neoplasms plasms (Biochem Biophy's Res Commun 236: 54-8 (1997)). (Cancer Res 60: 1536-40 (2000)). EWSR1 mutant protein Increased phosphorylation of MAPK1 may cause increased may cause abnormal regulation of transcription associated actin filament organization associated with ovarian neo with Ewing's sarcoma (Oncogene 22: 1-9 (2003)). EWSR1 plasms (JBC 280: 11961-72 (2005)). Decreased phosphory mutant protein may cause abnormal regulation of transcrip lation of MAPK1 may cause decreased cell proliferation tion associated with Ewing's sarcoma (Oncogene 21:8302-9 associated with colonic neoplasms (Br J Cancer 82: 905-12 (2002)). Translocation of the EWSR1 gene correlates with (2000)). Decreased phosphorylation of MAPK1 may corre chondrosarcoma (Cancer Res 63:449-54 (2003)). Transloca late with drug-resistant form of prostatic neoplasms (Cancer tion of the EWSR1 gene correlates with clear cell sarcoma Res 61: 6060-3 (2001)). Decreased protein kinase activity of associated with soft tissue neoplasms (Oncogene 20: 6653-9 MAPK1 correlates with carcinoma associated with colorectal (2001)). Decreased expression of EWSR1 protein may pre neoplasms (Gut 44: 834-8 (1999)). Induced stimulation of the vent increased cell proliferation associated with primitive MAP kinase 1 activity of MAPK1 may cause increased signal neuroectodermal tumors (J Clin Invest 99: 239-47 (1997)). transduction associated with ovarian neoplasms (JBC 280: Translocation of the EWSR1 gene may cause abnormal tran 11961-72 (2005)). Increased MAP kinase activity of MAPK1 Scription, DNA-dependent associated with melanoma (Onco correlates with increased severity of non-Small-cell lung car gene 10: 1749-56 (1995)). Translocation of the EWSR1 gene cinoma associated with lung neoplasms (Br J Cancer 90: correlates with neuroblastoma (PNAS 93: 1038-43 (1996)). 1047-52 (2004)). Increased phosphorylation of MAPK1 may Translocation of the EWSR1 gene correlates with acute form cause increased actin filament organization associated with of leukemia (Cancer Res 62: 5408-12 (2002)). Translocation ovarian neoplasms (J Biol Chem 280: 11961-72 (2005)). of the EWSR1 gene correlates with small cell carcinoma Increased tyrosine phosphorylation of MAPK1 may correlate (Oncogene 19: 3799-804 (2000)). Induced inhibition of with increased cytokine and chemokine mediated signaling EWSR1 protein may cause increased apoptosis associated pathway associated with multiple myeloma (Blood 89: 261 with clear cell sarcoma (J. Biol Chem 274:34811-8 (1999)). 71 (1997)). Decreased protein kinase activity of MAPK1 Translocation of the EWSR1 gene correlates with peripheral correlates with adenoma associated with colorectal neo primitive neuroectodermal tumors (Cancer Res 58:24.69-76 plasms (Gut 44: 834-8 (1999)). Induced stimulation of the (1998)). Translocation of the EWSR1 gene correlates with MAP kinase 1 activity of MAPK1 may cause increased actin neuroblastoma (Proc Natl Acad Sci USA 93: 1038-43 filament organization associated with ovarian neoplasms (1996)). EWSR1 mutant protein may cause abnormal regu (JBC 280: 11961-72 (2005)). Induced inhibition of the inte lation of transcription associated with Ewing's sarcoma (On grin binding of MAPK1 may prevent disease progression cogene 20: 3258-65 (2001)). Translocation of the EWSR1 associated with colonic neoplasms (Br J Cancer 87: 348-51 gene causes abnormal regulation of transcription associated (2002)). Increased expression of MAPK1 protein correlates with Ewing's sarcoma (Oncogene 20: 626-33 (2001)). Trans with increased activation of MAPK activity associated with location of the EWSR1 gene correlates with Ewing's sarcoma hepatocellular carcinoma (Biochem Biophy's Res Commun (Cytogenet Cell Genet 82: 278-83 (1998)). Translocation of 236: 54-8 (1997)). Induced inhibition of the MAP kinase 1 the EWSR1 gene may correlate with chondrosarcoma (Can activity of MAPK1 may prevent abnormal cell proliferation cer 83: 1504-21 (1998)). Translocation of the EWSR1 gene associated with multiple myeloma (Blood 101: 703-5 may cause increased transcription, DNA-dependent associ (2003)). (PhosphoSite(R), Cell Signaling Technology (Dan ated with melanoma (Oncogene 12: 159-67 (1996)). Trans vers, Mass.), Human PSDTM, Biobase Corporation, (Beverly, location of the EWSR1 gene correlates with clear cell adeno Mass.)). carcinoma associated with Soft tissue neoplasms (Cancer Res US 2010/015 1483 A1 Jun. 17, 2010 37
64: 3395-405 (2004)). EWSR1 mutant protein may cause breast neoplasms (Cancer Res 60: 213-8 (2000)). Induced abnormal regulation of transcription associated with Ewing's inhibition of the fatty-acid synthase activity of FASN may sarcoma (Oncogene 24: 2512-24 (2005)). Translocation of prevent increased cell proliferation associated with breast the EWSR1 gene may cause abnormal regulation of tran neoplasms (PNAS 91: 6379-83 (1994)). Increased expression Scription associated with Ewing's sarcoma (Oncogene 22: of FASN protein correlates with increased occurrence of 1-9 (2003)). Translocation of the EWSR1 gene correlates more severe form of breast neoplasms (Cancer 77: 474-82 with neuroblastoma tumors associated with nose neoplasms (1996)). Increased expression of FASN in serum correlates (Proc Natl AcadSci USA93: 1038-43 (1996)). Translocation with breast neoplasms (Cancer Lett 167: 99-104 (2001)). of the EWSR1 gene may cause abnormal regulation of tran Increased expression of FASN protein correlates with Scription associated with Ewing's sarcoma (Oncogene 20: increased occurrence of invasive form of prostatic neoplasms 3258-65 (2001)). Induced inhibition of EWSR1 protein may (IntJ Cancer 98:19-22 (2002)). Induced inhibition of FASN cause increased apoptosis associated with clear cell sarcoma protein may cause increased apoptosis associated with breast (JBC 274: 34811-8 (1999)). Translocation of the EWSR1 neoplasms (Cancer Res 56: 2745-7 (1996)). Induced inhibi gene correlates with soft tissue neoplasms (Oncogene 20: tion of the fatty-acid synthase activity of FASN may prevent 6653-9 (2001)). Abnormal expression of EWSR1 protein increased cell proliferation associated with breast neoplasms may correlate with abnormal fibroblast growth factor recep (Proc Natl Acad Sci USA 91: 6379-83 (1994)). Increased tor signaling pathway associated with Ewing's sarcoma (On expression of FASN mRNA may correlate with breast neo cogene 19: 4298-301 (2000)). Translocation of the EWSR1 plasms (Cancer Lett 149: 43-51 (2000)). Induced inhibition gene correlates with neuroblastoma tumors associated with of the fatty-acid synthase activity of FASN may prevent nose neoplasms (PNAS 93: 1038-43 (1996)). Translocation increased cell proliferation associated with breast neoplasms of the EWSR1 gene correlates with small cell carcinoma (Proc Natl Acad Sci USA 91: 6379-83 (1994)). (Phospho (Proc Natl AcadSci USA92: 1028-32 (1995)). Translocation Site(R), Cell Signaling Technology (Danvers, Mass.), Human of the EWSR1 gene correlates with small cell carcinoma PSDTM, Biobase Corporation, (Beverly, Mass.)). (Proc Natl AcadSci USA92: 1028-32 (1995)). Translocation 0093. FGFR3 (P22607), phosphorylated at Y552, Y577, of the EWSR1 gene correlates with small cell carcinoma Y647, Y648, is among the proteins listed in this patent. (PNAS 92: 1028-32 (1995)). Translocation of the EWSR1 FGFR3, Fibroblast growth factor receptor 3, inhibits bone gene correlates with Ewing's sarcoma associated with bone formation, involved in cell proliferation, upregulated in uri neoplasms (Biochem Biophys Res Commun 293: 61-71 nary tract carcinoma; mutations in corresponding gene cause (2002)). Translocation of the EWSR1 gene correlates with achondroplasia, thanatophoric dwarfism, skeletal dysplasia clear cell sarcoma (Int J Cancer 99: 560-7 (2002)). Translo and multiple neoplasms. This protein has potential diagnostic cation of the EWSR1 gene correlates with neuroblastoma and/or therapeutic implications based on the following find tumors associated with nose neoplasms (Proc Natl Acad Sci ings. FGFR3 map position correlates with cherubism (Am J USA93: 1038-43 (1996)). Translocation of the EWSR1 gene Hum Genet 65: 151-7 (1999)). Missense mutation in the correlates with neuroblastoma (Proc Natl AcadSci USA 93: protein kinase domain of FGFR3 causes increased severity of 1038-43 (1996)). Translocation of the EWSR1 gene corre skeleton defects associated with thanatophoric dysplasia lates with clear cell sarcoma (Oncogene 20: 6653-9 (2001)). (Mol. Cell. Biol 16:4081-7 (1996)). Missense mutation in the Decreased expression of EWSR1 protein may prevent FGFR3 gene causes craniosynostoses (Lancet 349: 1059-62 increased cell proliferation associated with Ewing's sarcoma (1997)). Increased protein dimerization activity of FGFR3 (J Clin Invest 99: 239-47 (1997)). Translocation of the may cause abnormal chondrocytes differentiation associated EWSR1 gene may cause melanoma tumors associated with with thanatophoric dysplasia (Hum Mol Genet 6: 1899-906 soft tissue neoplasms (Oncogene 10: 1749-56 (1995)). Trans (1997)). Increased protein-tyrosine kinase activity of FGFR3 location of the EWSR1 gene correlates with Ewing's sarcoma may cause increased cell cycle arrest associated with thanato (Oncogene 19: 3799-804 (2000)). Translocation of the phoric dysplasia (Nature 386: 288-92 (1997)). Increased EWSR1 gene may cause abnormal regulation of transcription transmembrane receptor protein tyrosine kinase activity of associated with bone neoplasms (Cancer Res 60: 1536-40 FGFR3 causes increased severity of skeleton defects associ (2000)). Translocation of the EWSR1 gene correlates with ated with thanatophoric dysplasia (Mol Cell Biol 16: 4081-7 Ewing's sarcoma (Oncogene 10: 1229-34 (1995)). Translo (1996)). Increased fibroblast growth factor receptor activity cation of the EWSR1 gene may cause increased telomere of FGFR3 causes abnormal fibroblast growth factor receptor maintenanceviatelomerase associated with Ewing's sarcoma signaling pathway associated with achondroplasia (EMBOJ. (Cancer Res 63: 8338-44 (2003)). (PhosphoSite R, Cell Sig 15:520-7 (1996)). Mutation in the FGFR3 gene causes abnor naling Technology (Danvers, Mass.), Human PSDTM, Bio mal JAK-STAT cascade associated with thanatophoric dys base Corporation, (Beverly, Mass.)). plasia (JBC 273: 13007-14 (1998)). Missense mutation in the 0092 FASN (P49327), phosphorylated at Y222, is among FGFR3 gene causes achondroplasia associated with acantho the proteins listed in this patent. FASN, Fatty acid synthase, sis nigricans (Am J Hum Genet 64: 722-31 (1999)). Mutation multifunctional enzyme that synthesizes fatty acids from in the FGFR3 gene causes decreased calcium-mediated sig dietary proteins and carbohydrates, increased expression is naling associated with thanatophoric dysplasia (Hum Mol associated with various cancers and inhibition may be thera Genet 6: 681-8 (1997)). Missense mutation in the FGFR3 peutic for breast and prostate cancer. This protein has poten gene causes acanthosis nigricans associated with achondro tial diagnostic and/or therapeutic implications based on the plasia (Am J Hum Genet 64: 722-31 (1999)). Increased fibro following findings. Induced inhibition of the fatty-acid syn blast growth factor receptor activity of FGFR3 may cause thase activity of FASN may prevent increased fatty acid bio abnormal MAPKKK cascade associated with multiple synthetic process associated with ovarian neoplasms (Cancer myeloma (Blood 97: 729-736 (2001)). Mutation in the Immu Res 56: 1189-93 (1996)). Induced inhibition of FASN protein noglobulin domain of FGFR3 may cause carcinoma tumors may prevent increased cell proliferation associated with associated with colorectal neoplasms (Cancer Res 61:3541-3 US 2010/015 1483 A1 Jun. 17, 2010
(2001)). Missense mutation in the FGFR3 gene may cause droplasia (Am J Hum Genet 56: 368-73 (1995)). Missense abnormal fibroblast growth factor receptor signaling pathway mutation in the FGFR3 gene may cause abnormal skeletal associated with achondroplasia (J Clin Invest 104: 1517-25 development associated with achondroplasia (J Clin Invest (1999)). Increased expression of FGFR3 protein correlates 104: 1517-25 (1999)). Mutation in the FGFR3 gene causes with genetic translocation associated with multiple myeloma abnormal chondrocytes differentiation associated with tha (Blood 100: 1417-24 (2002)). Abnormal phosphorylation of natophoric dysplasia (JBC 273: 13007-14 (1998)). Missense FGFR3 may cause abnormal skeletal development associated mutation in the FGFR3 gene causes achondroplasia (Cell 78: with achondroplasia (J Clin Invest 104: 1517-25 (1999)). 335-42 (1994)). Mutation in the FGFR3 gene causes abnor Abnormal glycosylation of FGFR3 may cause abnormal mal chondrocytes differentiation associated with thanato tyrosine phosphorylation of Stat1 protein associated with phoric dysplasia (J. Biol Chem 273: 13007-14 (1998)). Mis thanatophoric dysplasia (JBC 278: 17344-9 (2003)). sense mutation in the FGFR3 gene causes non-familial form Increased stability of FGFR3 may cause thanatophoric dys of achondroplasia (Am J Hum Genet 63: 711-6 (1998)). Mis plasia associated with fetal diseases (Hum Mol Genet 6: sense mutation in the FGFR3 gene may cause transitional cell 1899-906 (1997)). Increased expression of FGFR3 in carti carcinoma associated with bladder neoplasms (Oncogene 20: lage may cause thanatophoric dysplasia associated with fetal 686-91 (2001)). Mutation in the FGFR3 gene correlates with diseases (Hum Mol Genet 6: 1899-906 (1997)). Increased carcinoma tumors associated with bladder neoplasms (Onco expression of FGFR3 mutant protein correlates with genetic gene 20: 5059-61 (2001)). Missense mutation in the FGFR3 translocation associated with multiple myeloma (Blood 92: gene may correlate with acrocephaloSyndactylia (Hum Mol 3025-34 (1998)). Increased fibroblast growth factor receptor Genet 6: 1369-73 (1997)). Mutation in the FGFR3 gene activity of FGFR3 causes abnormal fibroblast growth factor causes achondroplasia (Nature 371: 252-4 (1994)). Point receptor signaling pathway associated with achondroplasia mutation in the FGFR3 gene causes craniosynostoses (Lancet (EMBO 15:520-7 (1996)). Abnormal expression of FGFR3 349: 1059-62 (1997)). Increased phosphorylation of FGFR3 mRNA correlates with genetic translocation associated with may cause abnormal cell Surface receptor linked signal trans multiple myeloma (Cancer Res 60: 4058-61 (2000)). Mis duction associated with multiple myeloma (Oncogene 20: sense mutation in the protein kinase domain of FGFR3 causes 3553-62 (2001)). Increased transmembrane receptor protein increased severity of skeleton defects associated with tha tyrosine kinase activity of FGFR3 causes developmental natophoric dysplasia (Mol. Cell. Biol. 16: 4081-7 (1996)). bone diseases (Am J Hum Genet 67: 1411-21 (2000)). Mis Increased fibroblast growth factor receptor activity of FGFR3 sense mutation in the protein kinase domain of FGFR3 causes may cause defective bone development associated with increased severity of skeleton defects associated with tha achondroplasia (EMBOJ 15:520-7 (1996)). Increased trans natophoric dysplasia (MCB 16: 4081-7 (1996)). Mutation in membrane receptor protein tyrosine kinase activity of FGFR3 the FGFR3 gene causes decreased chondrocytes survival causes increased severity of skeleton defects associated with associated with thanatophoric dysplasia (JBC 273: 13007-14 thanatophoric dysplasia (Mol Cell Biol. 16: 4081-7 (1996)). (1998)). Abnormal glycosylation of FGFR3 may cause Translocation of the FGFR3 gene correlates with multiple abnormal tyrosine phosphorylation of Stat1 protein associ myeloma (Cancer Res 58:5640-5 (1998)). Increased expres ated with thanatophoric dysplasia (JBiol Chem 278: 17344-9 sion of FGFR3 mRNA may cause abnormal MAPKKK cas (2003)). Increased fibroblast growth factor receptor activity cade associated with multiple myeloma (Blood 97: 729-736 of FGFR3 may cause defective cartilage development asso (2001)). Mutation in the FGFR3 gene causes abnormal fibro ciated with achondroplasia (EMBO J 15: 520-7 (1996)). blast growth factor receptor signaling pathway associated Mutation in the FGFR3 gene correlates with early stage or with thanatophoric dysplasia (Hum Mol Genet 6: 681-8 low grade form of bladder neoplasms (Cancer Res 61: 1265-8 (1997)). Point mutation in the FGFR3 gene causes achondro (2001)). Missense mutation in the FGFR3 gene causes acro plasia (Cell 78: 335-42 (1994)). Increased transmembrane cephalosyndactylia (Am J Hum Genet 62: 1370-80 (1998)). receptor protein tyrosine kinase activity of FGFR3 causes Alternative form of FGFR3 mRNA may cause carcinoma increased severity of skeleton defects associated with tha tumors associated with colorectal neoplasms (Cancer Res 60: natophoric dysplasia (Mol. Cell Biol 16: 4081-7 (1996)). 4049-52 (2000)). Point mutation in the FGFR3 gene causes Mutation in the FGFR3 gene causes abnormal JAK-STAT defective skeleton development associated with craniosynos cascade associated with thanatophoric dysplasia (J Biol toses (Am J Hum Genet 60:555-64 (1997)). Missense muta Chem 273: 13007-14 (1998)). Mutation in the FGFR3 gene tion in the protein kinase domain of FGFR3 causes develop causes increased induction of apoptosis associated with tha mental bone diseases (Am J Hum Genet 67: 1411-21 (2000)). natophoric dysplasia (JBC 273: 13007-14 (1998)). Increased Translocation of the FGFR3 gene correlates with plasmacytic fibroblast growth factor receptor activity of FGFR3 may leukemia (Cancer Res 58: 5640-5 (1998)). Translocation of cause defective cartilage development associated with achon the FGFR3 locus correlates with multiple myeloma (Blood droplasia (EMBO 15:520-7 (1996)). Increased expression of 90: 4062-70 (1997)). Increased fibroblast growth factor FGFR3 mRNA correlates with genetic translocation associ receptor activity of FGFR3 may cause defective bone devel ated with multiple myeloma (Blood 90: 4062-70 (1997)). opment associated with achondroplasia (EMBO 15: 520-7 Increased fibroblast growth factor receptor activity of FGFR3 (1996)). Abnormal phosphorylation of FGFR3 may cause may cause thanatophoric dysplasia associated with fetal dis abnormal fibroblast growth factor receptor signaling pathway eases (Hum Mol Genet 6: 1899-906 (1997)). Increased associated with achondroplasia (J Clin Invest 104: 1517-25 expression of FGFR3 mutant protein may cause increased (1999)). Increased fibroblast growth factor receptor activity cell proliferation associated with multiple myeloma (Blood of FGFR3 may cause abnormal chondrocytes differentiation 95: 992-8 (2000)). Increased protein dimerization activity of associated with thanatophoric dysplasia (Hum Mol Genet 6: FGFR3 may cause thanatophoric dysplasia associated with 1899-906 (1997)). Increased transmembrane receptor protein fetal diseases (Hum Mol Genet 6: 1899-906 (1997)). Single tyrosine kinase activity of FGFR3 causes achondroplasia nucleotide polymorphism in the FGFR3 gene causes achon associated with acanthosis nigricans (Am J Hum Genet 64: US 2010/015 1483 A1 Jun. 17, 2010 39
722-31 (1999)). Mutation in the FGFR3 gene correlates with 288-92 (1997)). Mutation in the FGFR3 gene causes carcinoma tumors associated with bladder neoplasms (Onco decreased chondrocytes Survival associated with thanato gene 20: 4416-8 (2001)). Increased protein-tyrosine kinase phoric dysplasia (J. Biol Chem 273: 13007-14 (1998)). activity of FGFR3 may cause increased STAT protein nuclear Increased fibroblast growth factor receptor activity of FGFR3 translocation associated with thanatophoric dysplasia (Na may cause abnormal cell Surface receptor linked signal trans ture 386: 288-92 (1997)). Missense mutation in the protein duction associated with multiple myeloma (Oncogene 20: kinase domain of FGFR3 causes increased severity of skel 3553-62 (2001)). Increased nucleus localization of FGFR3 eton defects associated with thanatophoric dysplasia (Mol may cause abnormal chondrocytes differentiation associated Cell Biol 16: 4081-7 (1996)). Increased transmembrane with thanatophoric dysplasia (Hum Mol Genet 6: 1899-906 receptor protein tyrosine kinase activity of FGFR3 causes (1997)). Missense mutation in the protein kinase domain of FGFR3 causes increased severity of skeleton defects associ acanthosis nigricans associated with achondroplasia (Am J ated with thanatophoric dysplasia (Mol Cell Biol. 16: 4081-7 Hum Genet 64: 722-31 (1999)). Missense mutation in the (1996)). (PhosphoSite(R), Cell Signaling Technology (Dan FGFR3 gene causes thanatophoric dysplasia (Hum Mol vers, Mass.), Human PSDTM, Biobase Corporation, (Beverly, Genet 5:509-12 (1996)). Increased fibroblast growth factor receptor activity of FGFR3 may cause defective bone devel Mass.)). opment associated with achondroplasia (EMBO.J. 15:520-7 (0094) Fgr (P09769), phosphorylated at Y208, Y209, is (1996)). Increased expression of FGFR3 mutant protein may among the proteins listed in this patent. Fgr, Gardner cause increased anti-apoptosis associated with multiple Rasheed feline sarcoma viral oncogene homolog, acts in inte myeloma (Blood 95: 992-8 (2000)). Increased expression of grin signaling, neutrophil degranulation, and antiapoptosis, FGFR3 in cartilage may cause abnormal chondrocytes differ may be upregulated in Epstein-Barr-infected cells; gene entiation associated with thanatophoric dysplasia (Hum Mol amplification correlates with hormone-resistance in prostate Genet 6: 1899-906 (1997)). Mutation in the FGFR3 gene cancer. This protein has potential diagnostic and/or therapeu causes increased induction of apoptosis associated with tha tic implications based on the following findings. Induced natophoric dysplasia (J. Biol Chem 273: 13007-14 (1998)). inhibition of FGR protein may prevent increased cell prolif Mutation in the FGFR3 gene causes decreased fibroblast eration associated with acute B-cell leukemia (Nat Genet 36: growth factor receptor signaling pathway associated with 453-61 (2004)). Alternative form of FGR mRNA correlates achondroplasia (Hum Mol Genet 6: 681-8 (1997)). Increased with Epstein-Barr virus infections (Mol Cell Biol 11: 1500-7 fibroblast growth factor receptor activity of FGFR3 may (1991)). Alternative form of FGR mRNA correlates with cause defective cartilage development associated with achon Epstein-Barr virus infections (Mol. Cell Biol 11: 1500-7 droplasia (EMBO J. 15:520-7 (1996)). Increased fibroblast (1991)). Increased expression of FGR mRNA may correlate growth factor receptor activity of FGFR3 causes abnormal with Epstein-Barr virus infections (Nature 319: 238-40 fibroblast growth factor receptor signaling pathway associ (1986)). Alternative form of FGR mRNA correlates with ated with achondroplasia (EMBO J. 15: 520-7 (1996)). Epstein-Barr virus infections (Mol. Cell. Biol. 11: 1500-7 Increased stability of FGFR3 may cause abnormal chondro (1991)). Alternative form of FGR mRNA correlates with cytes differentiation associated with thanatophoric dysplasia Epstein-Barr virus infections (Mol Cell Biol. 11: 1500-7 (Hum Mol Genet 6: 1899-906 (1997)). Increased nucleus (1991)). Alternative form of FGR mRNA correlates with localization of FGFR3 may cause thanatophoric dysplasia Epstein-Barr virus infections (MCB 11: 1500-7 (1991)). associated with fetal diseases (Hum Mol Genet 6: 1899-906 (PhosphoSite(R), Cell Signaling Technology (Danvers, (1997)). Missense mutation in the FGFR3 gene causes defec Mass.), Human PSDTM, Biobase Corporation, (Beverly, tive skeleton development associated with craniosynostoses Mass.)). (Am J Hum Genet 60: 555-6 (1997)). Increased transmem 0095. In the specification and the appended claims, the brane receptor protein tyrosine kinase activity of FGFR3 singular forms include plural referents unless the context causes increased severity of skeleton defects associated with clearly dictates otherwise. As used in this specification, the thanatophoric dysplasia (MCB 16:4081-7 (1996)). Increased singular forms “a,” “an and “the specifically also encom transmembrane receptor protein tyrosine kinase activity of pass the plural forms of the terms to which they refer, unless FGFR3 causes increased severity of skeleton defects associ the content clearly dictates otherwise. As used herein, unless ated with thanatophoric dysplasia (Mol. Cell. Biol. 16: specifically indicated otherwise, the word 'or' is used in the 4081-7 (1996)). Increased expression of FGFR3 mRNA cor “inclusive' sense of “and/or” and not the “exclusive' sense of relates with genetic translocation associated with multiple “eitherfor myeloma (Blood 99: 1745-57 (2002)). Increased expression 0096. The term “about is used herein to mean approxi of FGFR3 mRNA may cause drug-resistant form of multiple mately, in the region of roughly, or around. When the term myeloma (Blood 100: 3819-21 (2002)). Mutation in the "about is used in conjunction with a numerical range, it FGFR3 gene causes decreased calcium-mediated signaling modifies that range by extending the boundaries above and associated with achondroplasia (Hum Mol Genet 6: 681-8 below the numerical values set forth. In general, the term (1997)). Abnormal mRNA splicing of FGFR3 may cause “about is used herein to modify a numerical value above and carcinoma tumors associated with colorectal neoplasms below the stated value by a variance of 20%. (Cancer Res 60: 4049-52 (2000)). Increased protein-tyrosine 0097. As used herein, the recitation of a numerical range kinase activity of FGFR3 may cause abnormal regulation of for a variable is intended to convey that the invention may be ossification associated with thanatophoric dysplasia (Nature practiced with the variable equal to any of the values within 386: 288-92 (1997)). Missense mutation in the FGFR3 gene that range. Thus, for a variable that is inherently discrete, the may cause transitional cell carcinoma (Oncogene 20: 686-91 variable can be equal to any integer value of the numerical (2001)). Increased protein-tyrosine kinase activity of FGFR3 range, including the end-points of the range. Similarly, for a may cause increased tyrosine phosphorylation of Stat1 pro variable that is inherently continuous, the variable can be tein associated with thanatophoric dysplasia (Nature 386: equal to any real value of the numerical range, including the US 2010/015 1483 A1 Jun. 17, 2010 40 end-points of the range. As an example, a variable that is (0103) The term “antibody” or “antibodies” refers to all described as having values between 0 and 2, can be 0, 1 or 2 classes of polyclonal or monoclonal immunoglobulins, for variables which are inherently discrete, and can be 0.0, including IgG, IgM, IgA, Ig), and IgE, including whole 0.1, 0.01, 0.001, or any other real value for variables which antibodies and any antigen binding fragment thereof. This are inherently continuous. includes any combination of immunoglobulin domains or 0098. As used in this specification, whether in a transi chains that contains a variable region (V(H) or V(L)) that tional phrase or in the body of the claim, the terms “comprise retains the ability to bind the immunogen. Such fragments (s)' and "comprising are to be interpreted as having an include F(ab) fragments (V(H)—C(H1), V(L)-C(L)); open-ended meaning. That is, the terms are to be interpreted monovalent Fab fragments (V(H)—C(H1), V(L)-C(L)). Fv synonymously with the phrases “having at least” or “includ fragment (V(H)-V(L); single-chain Fv fragments (Kobayashi ing at least'. When used in the context of a process, the term et al., Steroids July; 67(8):733-42 (2002). “comprising means that the process includes at least the 0.104 Monoclonal antibodies refer to clonal antibodies recited steps, but may include additional steps. When used in produced from fusions between immunized murine, rabbit, the context of a compound or composition, the term "com human, or other vertebrate species, and produced by classical prising means that the compound or composition includes at fusion technology Kohler G. Milstein C. Continuous cultures least the recited features or components, but may also include of fused cells secreting antibody of predefined specificity. additional features or components. Nature 1975 Aug. 7:256(55.17):495-7 or by alternative meth 0099. “Antibody” or “antibodies” refers to all classes of ods which may isolate clones of immunoglobulin secreting immunoglobulins, including cells from transformed plasma cells. 0100 IgG, IgM, IgA, Ig), and IgE, including whole anti 0105. When used with respect to an antibody's binding to bodies and any antigen biding fragment thereof (e.g., F.) or one phospho-form of a sequence, the expression “does not single chains thereof, including chimeric, polyclonal, and bind' means that a phospho-specific antibody either does not monoclonal antibodies. Antibodies are antigen-specific pro apparently bind to the non-phospho form of the antigen as tein molecules produced by lymphocytes of the B cell lin ascertained in commonly used experimental detection sys eage. Following antigenic stimulation, B cells that have Sur tems (Western blotting, IHC. Immunofluorescence, etc.). face immunoglobulin receptors that bind the antigen clonally One of skill in the art will appreciate that the expression may expand, and the binding affinity for the antigen increases be applicable in those instances when (1) a phospho-specific through a process called affinity maturation. The B cells antibody either does not apparently bind to the non-phospho further differentiate into plasma cells, which secrete large form of the antigen as ascertained in commonly used experi quantities of antibodies into the serum. While the physiologi mental detection systems (Western blotting, IHC. Immunof cal role of antibodies is to protect the host animal by specifi luorescence, etc.); (2) where there is some reactivity with the cally binding and eliminating microbes and microbial patho Surrounding amino acid sequence, but that the phosphory gens from the body, large amounts of antibodies are also lated residue is an immunodominant feature of the reaction. induced by intentional immunization to produce specific anti In cases Such as these, there is an apparent difference in bodies that are used extensively in many biomedical and affinities for the two sequences. Dilutional analyses of Such therapeutic applications. antibodies indicates that the antibodies apparent affinity for 0101 Antibody molecules are shaped somewhat like the the phosphorylated form is at least 10-100 fold higher than for letter “Y”, and consist of 4 protein chains, two heavy (H) and the non-phosphorylated form; or where (3) the phospho-spe two light (L) chains. Antibodies possess two distinct and cific antibody reacts no more than an appropriate control spatially separate functional features. The ends of each of the antibody would react under identical experimental condi two arms of the “Y” contain the variable regions (variable tions. A control antibody preparation might be, for instance, heavy (V(H)) and variable light (V(L)) regions), which form purified immunoglobulin from a pre-immune animal of the two identical antigen-binding sites. The variable regions same species, an isotype- and species-matched monoclonal undergo a process of “affinity maturation” during the immune antibody. Tests using control antibodies to demonstrate speci response, leading to a rapid divergence of amino acids within ficity are recognized by one of skill in the art as appropriate these variable regions. The other end of the antibody mol and definitive. ecule, the stem of the “Y”, contains only the two heavy 0106 “Target signaling protein/polypeptide' means any constant (CH) regions, interacts with effector cells to deter protein (or polypeptide derived therefrom) enumerated in mine the effector functions of the antibody. There are five Column A of Table 1/FIG. 2, which is disclosed herein as different CH region genes that encode the five different being phosphorylated in one or more cell line(s). Target sig classes of immunoglobulins: IgM, Ig), IgG, IgA and IgE. naling protein(s)/polypeptide(s) may be tyrosine kinases, These constant regions, by interacting with different effector such as TTN or BCR, or serine/threonine kinases, or direct cells and molecules, determine the immunoglobulin mol Substrates of Such kinases, or may be indirect Substrates ecule's biological function and biological response. downstream of Such kinases in signaling pathways. Target 0102) Each V(H) and V(L) region contains three subre signaling protein/polypeptide where elucidated in leukemia gions called complementarity determining regions. These cell lines, however one of skill in the art will appreciate that a include CDR1-3 of the V(H) domain and CDR1-3 of the V(L) target signaling protein/polypeptide may also be phosphory domain. These six CDRS generally form the antigen binding lated in other cell lines (non-leukemic) harboring activated Surface, and include those residues that hypermutate during kinase activity. the affinity maturation phase of the immune response. The 0107 “Heavy-isotope labeled peptide' (used interchange CDR3 of the V(H) domain seems to play a dominant role in ably with AQUA peptide) means a peptide comprising at least generating diversity of both the B cellantigen receptor (BCR) one heavy-isotope label, which is suitable for absolute quan and the T cell antigen receptor systems (Xu et al., Immunity tification or detection of a protein as described in 13:37-45 (2000)). WO/03016861, “Absolute Quantification of Proteins and US 2010/015 1483 A1 Jun. 17, 2010
Modified Forms Thereof by Multistage Mass Spectrometry” WSU-NHL, XG2, Z-55, cs001, cs015, cs025, cs041, cs042, (Gygiet al.), further discussed below. gZ21, gz68, gz73, gz74, gZB1, hl 144b, hl 152b, lung tumor 0108) “Protein' is used interchangeably with peptide and T26, lung tumor T57, normal human lung, pancreatic polypeptide, and includes protein fragments and domains as Xenograft, patient 1, rat brain, Swa80. The isolation and iden well as whole protein. tification of phosphopeptides from these cell lines, using an 0109 "Phosphorylatable amino acid' means any amino immobilized general phosphotyrosine-specific antibody, or acid that is capable of being modified by addition of a phos an antibody recognizing the phosphorylated motif PXpSP is phate group, and includes both forms of Such amino acid. described in detail in Example 1 below. In addition to the 0110) “Phosphorylatable peptide sequence” means a pep protein phosphorylation sites (tyrosine) described herein, tide sequence comprising a phosphorylatable amino acid. many known phosphorylation sites were also identified (not 0111 "Phosphorylation site-specific antibody' means an described herein). The immunoaffinity/mass spectrometric antibody that specifically binds a phosphorylatable peptide technique described in the 896 Patent (the “IAP' method)– sequence/epitope only when phosphorylated, or only when and employed as described in detail in the Examples—is not phosphorylated, respectively. The term is used inter briefly summarized below. changeably with “phospho-specific' antibody. 0114. The IAP method employed generally comprises the 0112 Technical and scientific terms used herein have the following steps: (a) a proteinaceous preparation (e.g. a meaning commonly understood by one of skill in the art to digested cell extract) comprising phosphopeptides from two which the present invention pertains, unless otherwise or more different proteins is obtained from an organism; (b) defined. Reference is made herein to various methodologies the preparation is contacted with at least one immobilized and materials known to those of skill in the art. Standard general phosphotyrosine-specific antibody; (c) at least one reference works setting forth the general principles of recom phosphopeptide specifically bound by the immobilized anti binant DNA technology include Sambrook et al., Molecular body in step (b) is isolated; and (d) the modified peptide Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor isolated in step (c) is characterized by mass spectrometry Laboratory Press, New York (1989); Kaufman et al., Eds. (MS) and/or tandem mass spectrometry (MS-MS). Subse Handbook of Molecular and Cellular Methods in Biology in quently, (e) a search program (e.g., Sequest) may be utilized Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., to substantially match the spectra obtained for the isolated, Directed Mutagenesis: A Practical Approach, IRL Press, modified peptide during the characterization of step (d) with Oxford (1991). Standard reference works setting forth the the spectra for a known peptide sequence. A quantification general principles of pharmacology include Goodman and step employing, e.g., SILAC or AQUA, may also be Gilman's The Pharmacological Basis of Therapeutics, 11th employed to quantify isolated peptides in order to compare Ed., McGraw Hill Companies Inc., New York (2006). peptide levels in a sample to a baseline. 0113 A. Identification of Phosphorylation Sites. The tar 0.115. In the IAP method as employed herein, a general get signaling protein/polypeptide phosphorylation sites dis phosphotyrosine-specific monoclonal antibody (commer closed herein and listed in Table 1/FIG. 2 were discovered by cially available from Cell Signaling Technology, Inc., Bev employing the modified peptide isolation and characteriza erly, Mass., Cat. #9411 (p-Tyr-100)) was used in the immu tion techniques described in U.S. Pat. No. 7,198.896 using noaffinity step to isolate the widest possible number of cellular extracts from the following human cancer cell lines, phospho-tyrosine containing peptides from the cell extracts. tissues and patient samples: 0 1364548-cll, 223-CLL, 293T. 0116 Extracts from the following human cancer cell lines, 3T3 TrkB, 3T3-Src, 3T3-TrkA, 3T3-wit, 577, A172, AML tissues and patient samples were employed: 013.64548-cll, 4833, AML-6246, AML-6735, AML-7592, BaF3-10ZF, 223-CLL, 293T, 3T3 TrkB, 3T3-Src, 3T3-Trk A, 3T3-wt, BaF3-4ZF, BaF3-APR, BaF3-FLT3(D842V), BaF3-FLT3 577, A172, AML-4833, AML-6246, AML-6735, AML-7592, (D842Y), BaF3-FLT3(K663Q), BaF3-FLT3(WT), BaF3 BaF3-10ZF, BaF3-4ZF, BaF3-APR, BaF3-FLT3(D842V), FLT3/ITD, BaF3-PRTK, BaF3-TDII, BaF3-Tel/FGFR3, BaF3-FLT3(D842Y), BaF3-FLT3(K663Q), BaF3-FLT3 Baf3, Baf3-V617F-jak2, Baf3/E255K, Baf3/H396P. Baf3/ (WT), BaF3-FLT3/ITD, BaF3-PRTK, BaF3-TDII, BaF3-Tel/ Jak2(IL-3 dep), Baf3/M351T, Baf3/T315I, Baf3/TpoR, Baf3/ FGFR3, Baf3, Baf3-V617F-jak2, Baf3/E255K, Baf3/H396P. TpoR-Y98F, Baf3/Tyk2, Baf3/V617F-jak2 (IL-3), Baf3/ Baf3/Jak2(IL-3 dep), Baf3/M351T, Baf3/T315I, Baf3/TpoR, Y253F, Baf3/cc-TpoR-IV, Baf3/p210wt, CHRF, CI-1, CMK, Baf3/TpoR-Y98F, Baf3/Tyk2, Baf3N617F-jak2 (IL-3), CTV-1, DMS 53, DND41, DU-528, DU145, ELF-153, EOL Baf3/Y253F, Baf3/cc-TpoR-IV, Baf3/p210wt, CHRF, CI-1, 1, GDM-1, H1703, H1734, H1793, H1869, H1944, H1993, CMK, CTV-1, DMS 53, DND41, DU-528, DU145, ELF-153, H2023, H226, H3255, H358, H520, H82, H838, HCC1428, EOL-1, GDM-1, H1703, H1734, H1793, H1869, H1944, HCC1435, HCC1806, HCC1937, HCC366, HCC827, H1993, H2023, H226, H3255, H358, H520, H82, H838, HCT116, HEL, HL107B, HL117B, HL131A, HL131B, HCC1428, HCC1435, HCC1806, HCC1937, HCC366, HL133A, HL53B, HL59b, HL60, HL61a, HL61b, HL66B, HCC827, HCT116, HEL, HL107B, HL117B, HL131A, HL68A, HL75A, HL84A, HL97B, HL98A, HT29, HU-3, HL131B, HL133A, HL53B, HL59b, HL60, HL61a, HL61b, HUVEC, Jurkat, K562, KG-1, KG 1-A, KMS 11, KMS 18, HL66B, HL68A, HL75A, HL84A, HL97B, HL98A, HT29, KMS27, KOPT-K1, KY821, Karpas 299, Karpas-1 106p. HU-3, HUVEC, Jurkat, K562, KG-1, KG 1-A, KMS 11, M-07e, M01043, MO59K, MC-1 16, MCF-10A (Y561F), KMS18, KMS27, KOPT-K1, KY821, Karpas 299, Karpas MCF-10A (Y969F), MDA-MB-453, MDA-MB-468, MEC 1106p, M-07e, M01043, MO59K, MC-1 16, MCF-10A 2, MKPL-1, ML-1, MO-91, MOLT15, MV4-11, Me-F2, (Y561F), MCF-10A (Y969F), MDA-MB-453, MDA-MB Molm 14, Monomac 6, NCI-N87, Nomo-1, OCI-M1, OCI 468, MEC-2, MKPL-1, ML-1, MO-91, MOLT15, MV4-11, ly4, OCI-ly8, OCI/AML2, OPM-1, PL21, Pfeiffer, RC-K8, Me-F2, Molm 14, Monomac 6, NCI-N87, Nomo-1, OCI-M1, RI-1, SCLC T1, SEM, SK-N-AS, SK-N-MC, SKBR3, OCI-ly4, OCI-ly8, OCI/AML2, OPM-1, PL21, Pfeiffer, RC SR-786, SU-DHL1, SUP-M2, SUPT-13, SuDHL5, T17, K8, RI-1, SCLC T1, SEM, SK-N-AS, SK-N-MC, SKBR3, TRE-cll patient, TS, UT-7, VAL, Verona, Verona 1, Verona 4, SR-786, SU-DHL1, SUP-M2, SUPT-13, SuDHL5, T17, US 2010/015 1483 A1 Jun. 17, 2010 42
TRE-cll patient, TS, UT-7, VAL, Verona, Verona 1, Verona 4, site sequence information provided in Column E of Table 1. WSU-NHL, XG2, Z-55, cs001, cs015, cs025, cs041, cs042, The ANK1 adaptor/scaffold protein phosphorylation site (ty gZ21, gz68, gz73, gz74, gzB1, hl 144b, hl 152b, lung tumor rosine 1258) (see Row #16 of Table 1/FIG. 2) is presently T26, lung tumor T57, normal human lung, pancreatic disclosed. Thus, an antibody that specifically binds this novel Xenograft, patient 1, rat brain and SWA80. ANK1 adaptor/scaffold site can now be produced, e.g. by 0117. As described in more detail in the Examples, lysates immunizing an animal with a peptide antigen comprising all were prepared from these cells and digested with trypsin after or part of the amino acid sequence encompassing the respec treatment with DTT and iodoacetamide to redue and alkylate tive phosphorylated residue (e.g., a peptide antigen compris cysteine residues. Before the immunoaffinity step, peptides ing the sequence set forth in Row #16, Column E, of Table were pre-fractionated by reversed-phase solid phase extrac 1, SEQ ID NO: 15, respectively) (which encompasses the tion using Sep-Pak Cs columns to separate peptides from phosphorylated tyrosine at position 1258 in ANK1, to pro other cellular components. The solid phase extraction car duce an antibody that only binds ANK1 adaptor/scaffold tridges were eluted with varying steps of acetonitrile. Each when phosphorylated at that site. lyophilized peptide fraction was redissolved in MOPS IP 0.122 Polyclonal antibodies of the invention may be pro buffer and treated with phosphotyrosine (P-Tyr-100, CST duced according to standard techniques by immunizing a #9411) immobilized on protein G-Sepharose. Immunoaffin Suitable animal (e.g., rabbit, goat, etc.) with a peptide antigen ity-purified peptides were eluted with 0.1%TFA and a portion corresponding to the phosphorylation site of interest (i.e., a of this fraction was concentrated with Stage or Zip tips and phosphorylation site enumerated in Column E of Table 1, analyzed by LC-MS/MS, using either a LCQ or Ther which comprises the corresponding phosphorylatable amino moFinnigan LTQ ion trap mass spectrometer. Peptides were acid listed in Column D of Table 1), collecting immune serum eluted from a 10 cmx75 um reversed-phase column with a from the animal, and separating the polyclonal antibodies 45-min linear gradient of acetonitrile. MS/MS spectra were from the immune serum, in accordance with known proce evaluated using the program Sequest with the NCBI human dures. For example, a peptide antigen corresponding to all or protein database. part of the novel Crkl adaptor/scaffold phosphorylation site 0118. This revealed the tyrosine phosphorylation sites in disclosed herein (SEQ ID NO: 37=IHyLDTTTLIEPAPR, signaling pathways affected by kinase activation or active in encompassing phosphorylated tyrosine 92 (see Row 38 of leukemia cells. The identified phosphorylation sites and their Table 1)) may be employed to produce antibodies that only parent proteins are enumerated in Table 1/FIG. 2. The bind Crkl when phosphorylated at Tyr 92. Similarly, a peptide tyrosine at which phosphorylation occurs is provided in Col comprising all or part of any one of the phosphorylation site umnD, and the peptide sequence encompassing the phospho sequences provided in Column E of Table 1 may employed as rylatable tyrosine residue at the site is provided in Column E. an antigen to produce an antibody that only binds the corre Ifa phosphorylated tyrosine was found in mouse, the ortholo sponding protein listed in Column A of Table 1 when phos gous site in human was identified using either Homologene or phorylated (or when not phosphorylated) at the correspond BLAST at NCBI; the sequence reported in column E is the ing residue listed in Column D. If an antibody that only binds phosphorylation site flanked by 7 amino acids on each side. the protein when phosphorylated at the disclosed site is FIG. 2 also shows the particular type of leukemic disease (see desired, the peptide antigen includes the phosphorylated form Column G) and cell line(s) (see Column F) in which a par of the amino acid. Conversely, if an antibody that only binds ticular phosphorylation site was discovered. the protein when not phosphorylated at the disclosed site is 0119. As a result of the discovery of these phosphorylation desired, the peptide antigen includes the non-phosphorylated sites, phospho-specific antibodies and AQUA peptides for the form of the amino acid. detection of and quantification of these sites and their parent I0123 Peptide antigens suitable for producing antibodies proteins may now be produced by standard methods, as of the invention may be designed, constructed and employed described below. These new reagents will prove highly useful in accordance with well-known techniques. See, e.g., Anti in, e.g., studying the signaling pathways and events underly bodies: A Laboratory Manual, Chapter 5, p. 75-76, Harlow & ing the progression of leukemias and the identification of new Lane Eds. Cold Spring Harbor Laboratory (1988); Czernik, biomarkers and targets for diagnosis and treatment of Such Methods In Enzymology, 201: 264-283 (1991); Merrifield, J. diseases in a mammal. Am. Chem. Soc. 85: 21-49 (1962)). 0120. The methods of the present invention are intended 0.124. It will be appreciated by those of skill in the art that for use with any mammal that may experience the benefits of longer or shorter phosphopeptide antigens may be employed. the methods of the invention. Foremost among Such mam See Id. For example, a peptide antigen may comprise the full mals are humans, although the invention is not intended to be sequence disclosed in Column E of Table 1/FIG. 2, or it may so limited, and is applicable to veterinary uses. Thus, in comprise additional amino acids flanking Such disclosed accordance with the invention, “mammals' or “mammal in sequence, or may comprise of only a portion of the disclosed need” include humans as well as non-human mammals, par sequence immediately flanking the phosphorylatable amino ticularly domesticated animals including, without limitation, acid (indicated in Column E by lowercase “y”). Typically, a cats, dogs, and horses. desirable peptide antigen will comprise four or more amino 0121 B. Antibodies and Cell Lines. Isolated phosphory acids flanking each side of the phosphorylatable amino acid lation site-specific antibodies that specifically bind a target and encompassing it. Polyclonal antibodies produced as signaling protein/polypeptide disclosed in Column A of Table described herein may be screened as further described below. 1 only when phosphorylated (or only when not phosphory 0.125 Monoclonal antibodies of the invention may be pro lated) at the corresponding amino acid and phosphorylation duced in a hybridoma cell line according to the well-known site listed in Columns D and E of Table 1/FIG. 2 may be technique of Kohler and Milstein. See Nature 265: 495-97 produced by Standard antibody production methods, such as (1975); Kohler and Milstein, Eur: J. Immunol. 6: 511 (1976): anti-peptide antibody methods, using the phosphorylation see also, Current Protocols in Molecular Biology, Ausubel et US 2010/015 1483 A1 Jun. 17, 2010
al. Eds (1989); Harlow E. D Lane. Antibodies, A Laboratory may be recombinant monoclonal antibodies produced Manual. CSHP 1988. Monoclonal antibodies so produced are according to the methods disclosed in U.S. Pat. No. 4,474,893 highly specific, and improve the selectivity and specificity of or U.S. Pat. No. 4,816,567. The antibodies may also be diagnostic assay methods provided by the invention. For chemically constructed by specific antibodies made accord example, a solution containing the appropriate antigen may ing to the method disclosed in U.S. Pat. No. 4,676.980. be injected into a mouse or other species and, after a sufficient time (in keeping with conventional techniques), the animal is I0131 The invention also provides immortalized cell lines sacrificed and spleen cells obtained. The spleen cells are then that produce an antibody of the invention. For example, hybri immortalized by fusing them with myeloma cells, typically in doma clones, constructed as described above, that produce the presence of polyethylene glycol, to produce hybridoma monoclonal antibodies to the protein phosphorylation sites cells. Rabbit fusion hybridomas, for example, may be pro disclosed herein are also provided. Similarly, the invention duced as described in U.S. Pat. No. 5.675,063. The hybri includes recombinant cells producing an antibody of the doma cells are then grown in a suitable selection media, Such invention, which cells may be constructed by well known as hypoxanthine-aminopterin-thymidine (HAT), and the techniques; for example the antigen combining site of the Supernatant Screened for monoclonal antibodies having the monoclonal antibody can be cloned by PCR and single-chain desired specificity, as described below. The secreted antibody antibodies produced as phage-displayed recombinant anti may be recovered from tissue culture Supernatant by conven bodies or soluble antibodies in E. coli (see, e.g., ANTIBODY tional methods such as precipitation, ion exchange or affinity ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul chromatography, or the like. editor.) 012.6 Monoclonal F, fragments may also be produced in I0132) Phosphorylation site-specific antibodies of the Escherichia coli by recombinant techniques known to those invention, whether polyclonal or monoclonal, may be skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 screened for epitope and phospho-specificity according to (1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87: 8095 standard techniques. See, e.g., Czernik et al., Methods in (1990). If monoclonal antibodies of one isotype are prefer Enzymology, 201: 264-283 (1991). For example, the antibod able for a particular application, particular isotypes can be ies may be screened against the phospho and non-phospho prepared directly, by selecting from the initial fusion, or pre peptide library by ELISA to ensure specificity for both the pared secondarily, from a parental hybridoma Secreting a desired antigen (i.e. that epitope including a phosphorylation monoclonal antibody of different isotype by using the sib site sequence enumerated in Column E of Table 1) and for selection technique to isolate class-switch variants reactivity only with the phosphorylated (or non-phosphory (Steplewski, et al., Proc. Natl. Acad. Sci., 82: 8653 (1985); lated) form of the antigen. Peptide competition assays may be Spira et al., J. Immunol. Methods, 74: 307 (1984)). carried out to confirm lack of reactivity with other phospho 0127. Additional methods available include vaccination epitopes on the given target Signal Protein/Polypepetide. The of the animal with DNA or virus encoding the protein of antibodies may also be tested by Western blotting against cell interest (Bates et al., Biotechniques. February:40(2): 199-208 preparations containing the signaling protein, e.g. cell lines (2006)). over-expressing the target protein, to confirm reactivity with 0128. An epitope of a phosphorylation-site specific anti the desired phosphorylated epitope/target. body of the invention is a peptide fragment consisting essen I0133. In an exemplary embodiment, phage display librar tially of about 8 to 17 amino acids including the phosphory ies containing more than 10" phage clones are used for high latable tyrosine, wherein about 3 to 8 amino acids are throughput production of monoclonal antibodies that target positioned on each side of the phosphorylatable tyrosine (for post-translational modification sites (e.g., phosphorylation example, the CGN tyrosine 55 phosphorylation site sequence sites) and, for validation and quality control, high-throughput disclosed in Row 35, Column E of Table 1), and antibodies of immunohistochemistry is utilized to screen the efficacy of the invention thus specifically bind a target Signal Protein/ these antibodies. Western blots, protein microarrays and flow Polypeptide comprising Such epitopic sequence. Epitopes cytometry can also be used in high-throughput screening of bound by the antibodies of the invention comprise all or part phosphorylation site-specific polyclonal or monoclonal anti of a phosphorylatable site sequence listed in Column E of bodies of the present invention. See, e.g., Blow N. Nature, Table 1, including the phosphorylatable amino acid. 447: 741-743 (2007). 0129. Included in the scope of the invention are equivalent 0.134 Specificity against the desired phosphorylated non-antibody molecules, such as protein binding domains or epitope may also be examined by constructing mutants lack nucleic acid aptamers, which bind, in a phospho-specific ing phosphorylatable residues at positions outside the desired manner, to essentially the same phosphorylatable epitope to epitope that are known to be phosphorylated, or by mutating which the phospho-specific antibodies of the invention bind. the desired phospho-epitope and confirming lack of reactiv See, e.g., Neuberger et al., Nature 312: 604 (1984). Such ity. Phosphorylation-site specific antibodies of the invention equivalent non-antibody reagents may be suitably employed may exhibit some limited cross-reactivity to related epitopes in the methods of the invention further described below. in non-target proteins. This is not unexpected as most anti 0130 Antibodies provided by the invention may be any bodies exhibit some degree of cross-reactivity, and anti-pep type of immunoglobulins, including IgG, IgM, IgA, Ig|D, and tide antibodies will often cross-react with epitopes having IgE, including F, or antigen-recognition fragments thereof. high homology to the immunizing peptide. See, e.g., Czernik, The antibodies may be monoclonal or polyclonal and may be Supra. Cross-reactivity with non-target proteins is readily of any species of origin, including (for example) mouse, rat, characterized by Western blotting alongside markers of rabbit, horse, or human, or may be chimeric antibodies. See, known molecular weight. Amino acid sequences of cross e.g., M. Walker et al., Molec. Immunol. 26: 403-11 (1989); reacting proteins may be examined to identify sites highly Morrision et al., Proc. Natl. Acad. Sci. 81: 6851 (1984): homologous to the target signaling protein/polypeptide Neuberger et al., Nature 312: 604 (1984)). The antibodies epitope for which the antibody of the invention is specific. US 2010/015 1483 A1 Jun. 17, 2010 44
0135) In certain cases, polyclonal antisera may exhibit the production of corresponding heavy-isotope labeled pep Some undesirable general cross-reactivity to phosphotyrosine tides for the absolute quantification of such signaling proteins or phosphoserine itself, which may be removed by further (both phosphorylated and not phosphorylated at a disclosed purification of antisera, e.g., over a phosphotyramine column. site) in biological samples. The production and use of AQUA Antibodies of the invention specifically bind their target pro peptides for the absolute quantification of proteins (AQUA) tein (i.e., a protein listed in Column A of Table 1) only when in complex mixtures has been described. See WO/03016861, phosphorylated (or only when not phosphorylated, as the case Gerber et al., Proc. Natl. Acad. Sci. U.S.A. 100: 6940-5 may be) at the site disclosed in corresponding Columns D/E, (2003). and do not (substantially) bind to the other form (as compared 0.141. The AQUA methodology employs the introduction to the form for which the antibody is specific). of a known quantity of at least one heavy-isotope labeled 0.136 Antibodies may be further characterized via immu peptide standard (which has a unique signature detectable by nohistochemical (IHC) staining using normal and diseased LC-SRM chromatography) into a digested biological sample tissues to evaluate phosphorylation and activation status in in order to determine, by comparison to the peptide standard, diseased tissue. IHC may be carried out according to well the absolute quantity of a peptide with the same sequence and known techniques. See, e.g., ANTIBODIES: A LABORATORY protein modification in the biological sample. Briefly, the MANUAL, Chapter 10, Harlow & Lane Eds. Cold Spring AQUA methodology has two stages: peptide internal stan Harbor Laboratory (1988). Briefly, paraffin-embedded tissue dard selection and validation and method development; and (e.g., tumor tissue) is prepared for immunohistochemical implementation using validated peptide internal standards to staining by deparaffinizing tissue sections with Xylene fol detect and quantify a target protein in Sample. The method is lowed by ethanol; hydrating in water then PBS; unmasking a powerful technique for detecting and quantifying a given antigen by heating slide in Sodium citrate buffer, incubating peptide?protein within a complex biological mixture, such as sections in hydrogen peroxide; blocking in blocking solution; a cell lysate, and may be employed, e.g., to quantify change in incubating slide in primary antibody and secondary antibody; protein phosphorylation as a result of drug treatment, or to and finally detecting using ABC avidin/biotin method accord quantify differences in the level of a protein in different ing to manufacturer's instructions. biological states. 0.137 Antibodies may be further characterized by flow 0.142 Generally, to develop a suitable internal standard, a cytometry carried out according to standard methods. See particular peptide (or modified peptide) within a target pro Chow et al., Cytometry (Communications in Clinical Cytom tein sequence is chosen based on its amino acid sequence and etry) 46: 72-78 (2001). Briefly and by way of example, the the particular protease to be used to digest. The peptide is then following protocol for cytometric analysis may be employed: generated by Solid-phase peptide synthesis such that one resi samples may be centrifuged on Ficoll gradients to remove due is replaced with that same residue containing stable iso erythrocytes, and cells may then be fixed with 2% paraform topes ('C, 'N). The result is a peptide that is chemically aldehyde for 10 minutes at 37°C. followed by permeabiliza identical to its native counterpartformed by proteolysis, but is tion in 90% methanol for 30 minutes on ice. Cells may then be easily distinguishable by MS via a 7-Damass shift. A newly stained with the primary phosphorylation-site specific anti synthesized AQUA internal standard peptide is then evaluated body of the invention (which detects a target Signal Protein/ by LC-MS/MS. This process provides qualitative informa Polypepetide enumerated in Table 1), washed and labeled tion about peptide retention by reverse-phase chromatogra with a fluorescent-labeled secondary antibody. Additional phy, ionization efficiency, and fragmentation via collision fluorochrome-conjugated marker antibodies (e.g., CD45. induced dissociation. Informative and abundant fragment CD34) may also be added at this time to aid in the subsequent ions for sets of native and internal standard peptides are identification of specific hematopoietic cell types. The cells chosen and then specifically monitored in rapid succession as would then be analyzed on a flow cytometer (e.g., a Beckman a function of chromatographic retention to form a selected Coulter FC500) according to the specific protocols of the reaction monitoring (LC-SRM) method based on the unique instrument used. profile of the peptide standard. 0138 Antibodies of the invention may also be advanta 0143. The second stage of the AQUA strategy is its imple geously conjugated to fluorescent dyes (e.g., Alexa488, PE) mentation to measure the amount of a protein or modified for use in multi-parametric analyses along with other signal protein from complex mixtures. Whole cell lysates are typi transduction (phospho-CrkL, phospho-Erk 1/2) and/or cell cally fractionated by SDS-PAGE gel electrophoresis, and marker (CD34) antibodies. regions of the gel consistent with protein migration are 0139 Phosphorylation-site specific antibodies of the excised. This process is followed by in-gel proteolysis in the invention specifically bind to a target signaling protein/ presence of the AQUA peptides and LC-SRM analysis (See polypeptide only when phosphorylated at a disclosed site, but Gerber et al., supra.) AQUA peptides are spiked in to the are not limited only to binding the human species, perse. The complex peptide mixture obtained by digestion of the whole invention includes antibodies that also bind conserved and cell lysate with a proteolytic enzyme and Subjected to immu highly homologous or identical phosphorylation sites in noaffinity purification as described above. The retention time respective target signaling protein/polypeptide from other and fragmentation pattern of the native peptide formed by species (e.g., mouse, rat, monkey, yeast), in addition to bind digestion (e.g., trypsinization) is identical to that of the ing the human phosphorylation site. Highly homologous or AQUA internal standard peptide determined previously; thus, identical sites conserved in other species can readily be iden LC-MS/MS analysis using an SRM experiment results in the tified by Standard sequence comparisons, such as using highly specific and sensitive measurement of both internal BLAST, with the human target signaling protein/polypeptide standard and analyte directly from extremely complex pep phosphorylation sites disclosed herein. tide mixtures. Because an absolute amount of the AQUA 0140 C. Heavy-Isotope Labeled Peptides (AQUA Pep peptide is added (e.g., 250 fmol), the ratio of the areas under tides). The phosphorylation sites disclosed herein now enable the curve can be used to determine the precise expression US 2010/015 1483 A1 Jun. 17, 2010 levels of a protein orphosphorylated form of a protein in the than the fragments of all the possible amino acids. As a result, original cell lysate. In addition, the internal standard is the labeled amino acids and peptides are readily distinguished present during in-gel digestion as native peptides are formed, from unlabeled ones by the ion/mass pattern in the resulting Such that peptide extraction efficiency from gel pieces, abso mass spectrum. Preferably, the ion mass signature component lute losses during sample handling (including vacuum cen imparts a mass to a protein fragment that does not match the trifugation), and variability during introduction into the LC residue mass for any of the 20 natural amino acids. MS system do not affect the determined ratio of native and 014.9 The label should be robust under the fragmentation AQUA peptide abundances. conditions of MS and not undergo unfavorable fragmenta 0144. An AQUA peptide standard is developed for a tion. Labeling chemistry should be efficient under a range of known phosphorylation site sequence previously identified conditions, particularly denaturing conditions, and the by the IAP-LC-MS/MS method within a target protein. One labeled tag preferably remains soluble in the MS buffer sys AQUA peptide incorporating the phosphorylated form of the tem of choice. The label preferably does not suppress the particular residue within the site may be developed, and a ionization efficiency of the protein and is not chemically second AQUA peptide incorporating the non-phosphorylated reactive. The label may contain a mixture of two or more form of the residue developed. In this way, the two standards isotopically distinct species to generate a unique mass spec may be used to detect and quantify both the phosphorylated trometric pattern at each labeled fragment position. Stable and non-phosphorylated forms of the site in a biological isotopes, such as H, C, N, O, O, or 'S, are suitable sample. labels. Pairs of peptide internal standards that incorporate a 0145 Peptide internal standards may also be generated by different isotope label may also be prepared. Amino acid examining the primary amino acid sequence of a protein and residues into which a heavy isotope label may be incorporated determining the boundaries of peptides produced by protease include leucine, proline, Valine, and phenylalanine. cleavage. Alternatively, a protein may actually be digested 0150 Peptide internal standards are characterized accord with a protease and a particular peptide fragment produced ing to their mass-to-charge (m/z) ratio, and preferably, also can then sequenced. Suitable proteases include, but are not according to their retention time on a chromatographic col limited to, serine proteases (e.g., trypsin, hepsin), metallo umn (e.g. an HPLC column). Internal standards that co-elute proteases (e.g., PUMP1), chymotrypsin, cathepsin, pepsin, with unlabeled peptides of identical sequence are selected as thermolysin, carboxypeptidases, etc. optimal internal standards. The internal standard is then ana 0146 A peptide sequence within a target protein is lyzed by fragmenting the peptide by any suitable means, for selected according to one or more criteria to optimize the use example by collision-induced dissociation (CID) using, e.g., of the peptide as an internal standard. Preferably, the size of argon or helium as a collision gas. The fragments are then the peptide is selected to minimize the chances that the pep analyzed, for example by multi-stage mass spectrometry tide sequence will be repeated elsewhere in other non-target (MS) to obtain a fragmention spectrum, to obtain a peptide proteins. Thus, a peptide is preferably at least about 6 amino fragmentation signature. Preferably, peptide fragments have acids. The size of the peptide is also optimized to maximize significant differences in m/z ratios to enable peaks corre ionization frequency. A workable range is about 7 to 15 amino sponding to each fragment to be well separated, and a signa acids. A peptide sequence is also selected that is not likely to ture that is unique for the target peptide is obtained. If a be chemically reactive during mass spectrometry, thus Suitable fragment signature is not obtained at the first stage, sequences comprising cysteine, tryptophan, or methionine additional stages of MS are performed until a unique signa are avoided. ture is obtained. 0147 A peptide sequence that does not include a modified 0151 Fragment ions in the MS/MS and MS spectra are region of the target region may be selected so that the peptide typically highly specific for the peptide of interest, and, in internal standard can be used to determine the quantity of all conjunction with LC methods, allow a highly selective means forms of the protein. Alternatively, a peptide internal standard of detecting and quantifying a target peptide?protein in a encompassing a modified amino acid may be desirable to complex protein mixture, such as a cell lysate, containing detect and quantify only the modified form of the target many thousands or tens of thousands of proteins. Any bio protein. Peptide standards for both modified and unmodified logical sample potentially containing a target protein/peptide regions can be used together, to determine the extent of a of interest may be assayed. Crude or partially purified cell modification in a particular sample (i.e., to determine what extracts may be employed. Generally, the sample has at least fraction of the total amount of protein is represented by the 0.01 mg of protein, typically a concentration of 0.1-10 modified form). For example, peptide standards for both the mg/mL, and may be adjusted to a desired buffer concentration phosphorylated and unphosphorylated form of a protein and pH. known to be phosphorylated at a particular site can be used to 0152. A known amount of a labeled peptide internal stan quantify the amount of phosphorylated form in a sample. dard, preferably about 10 femtomoles, corresponding to a 0148. The peptide is labeled using one or more labeled target protein to be detected/quantified is then added to a amino acids (i.e. the label is an actual part of the peptide) or biological sample, such as a cell lysate. The spiked sample is less preferably, labels may be attached after synthesis accord then digested with one or more protease(s) for a Suitable time ing to standard methods. Preferably, the label is a mass period to allow digestion. A separation is then performed altering label selected based on the following considerations: (e.g., by HPLC, reverse-phase HPLC, capillary electrophore the mass should be unique to shift fragment masses produced sis, ion exchange chromatography, etc.) to isolate the labeled by MS analysis to regions of the spectrum with low back internal standard and its corresponding target peptide from ground; the ion mass signature component is the portion of other peptides in the sample. Microcapillary LC is a method the labeling moiety that preferably exhibits a unique ion mass contemplated. signature in MS analysis; the Sum of the masses of the con 0153. Each isolated peptide is then examined by monitor stituent atoms of the label is preferably uniquely different ing of a selected reaction in the MS. This involves using the US 2010/015 1483 A1 Jun. 17, 2010 46 prior knowledge gained by the characterization of the peptide site). It will be appreciated that a larger AQUA peptide com internal standard and then requiring the MS to continuously prising a disclosed phosphorylation site sequence (and addi monitor a specific ion in the MS/MS or MS" spectrum for both tional residues downstream or upstream of it) may also be the peptide of interest and the internal standard. After elution, constructed. Similarly, a smaller AQUA peptide comprising the area under the curve (AUC) for both peptide standard and less than all of the residues of a disclosed phosphorylation site target peptide peaks are calculated. The ratio of the two areas sequence (but still comprising the phosphorylatable residue provides the absolute quantification that can be normalized enumerated in ColumnD of Table 1/FIG.2) may alternatively for the number of cells used in the analysis and the protein's be constructed. Such larger or shorter AQUA peptides are molecular weight, to provide the precise number of copies of within the scope of the present invention, and the selection the protein per cell. Further details of the AQUA methodology and production of AQUA peptides may be carried out as are described in Gygi et al., and Gerber et al. Supra. described above (see Gygiet al., Gerber et al., Supra.). 0154) In accordance with the present invention, AQUA internal peptide standards (heavy-isotope labeled peptides) 0158 Certain subsets of AQUA peptides provided by the may now be produced, as described above, for any of the invention are described above (corresponding to particular phosphorylation sites disclosed herein. Peptide standards for protein types/groups in Table 1, for example, tyrosine protein a given phosphorylation site (e.g., the tyrosine 644 in CD93— kinases or adaptor/scaffold proteins). Example 4 is provided see Row 48 of Table 1) may be produced for both the phos to further illustrate the construction and use, by standard phorylated and non-phosphorylated forms of the site (e.g., see methods described above, of exemplary AQUA peptides pro FASN site sequence in Column E. Row 195 of Table 1 (SEQ vided by the invention. For example, the above-described ID NO: 196) and such standards employed in the AQUA AQUA peptides corresponding to both the phosphorylated methodology to detect and quantify both forms of such phos and non-phosphorylated forms of the disclosed claspin cell phorylation site in a biological sample. cycle regulation protein tyrosine 887 phosphorylation site 0155 AQUA peptides of the invention may comprise all, (see Row 80 of Table 1/FIG. 2) may be used to quantify the or part of a phosphorylation site peptide sequence disclosed amount of phosphorylated claspin (Tyr 887) in a biological herein (see Column E of Table 1/FIG. 2). In an embodiment, sample, e.g., a tumor cell sample (or a sample before or after an AQUA peptide of the invention comprises a phosphoryla treatment with a test drug). tion site sequence disclosed herein in Table 1/FIG. 2. For 0159 AQUA peptides of the invention may also be example, an AQUA peptide of the invention for detection/ employed within a kit that comprises one or multiple AQUA quantification of BAG4 Apoptosis protein when phosphory peptide(s) provided herein (for the quantification of a target lated at tyrosine Y 102 may comprise the sequence SGYGPS signaling protein/polypeptide disclosed in Table 1/FIG. 2), DGPSyGR (y-phosphotyrosine), which comprises and, optionally, a second detecting reagent conjugated to a phosphorylatable tyrosine 102 (see Row 54, Column E: (SEQ detectable group. For example, a kit may include AQUA ID NO. 53)). Heavy-isotope labeled equivalents of the pep peptides for both the phosphorylated and non-phosphory tides enumerated in Table 1/FIG. 2 (both in phosphorylated lated form of a phosphorylation site disclosed herein. The and unphosphorylated form) can be readily synthesized and reagents may also include ancillary agents such as buffering their unique MS and LC-SRM signature determined, so that agents and protein stabilizing agents, e.g., polysaccharides the peptides are validated as AQUA peptides and ready for use and the like. The kit may further include, where necessary, in quantification experiments. other members of the signal-producing system of which sys 0156 The phosphorylation site peptide sequences dis tem the detectable group is a member (e.g., enzyme Sub closed herein (see Column E of Table 1/FIG.2) are well suited strates), agents for reducing background interference in a test, for development of corresponding AQUA peptides, since the control reagents, apparatus for conducting a test, and the like. IAP method by which they were identified (see Part A above The test kit may be packaged in any suitable manner, typically and Example 1) inherently confirmed that Such peptides are in with all elements in a single container along with a sheet of fact produced by enzymatic digestion (trypsinization) and are printed instructions for carrying out the test. in fact suitably fractionated/ionized in MS/MS. Thus, heavy 0160 AQUA peptides provided by the invention will be isotope labeled equivalents of these peptides (both in phos useful in the further study of signal transduction anomalies phorylated and unphosphorylated form) can be readily Syn associated with diseases Such as for example cancer, includ thesized and their unique MS and LC-SRM signature ing leukemias, and in identifying diagnostic/bio-markers of determined, so that the peptides are validated as AQUA pep these diseases, new potential drug targets, and/or in monitor tides and ready for use in quantification experiments. ing the effects of test compounds on target Signaling Proteins/ 0157 Accordingly, the invention provides heavy-isotope Polypeptides and pathways. labeled peptides (AQUA peptides) for the detection and/or 0.161. D. Immunoassay Formats. Antibodies provided by quantification of any of the phosphorylation sites disclosed in the invention may be advantageously employed in a variety of Table 1/FIG. 2 (see Column E) and/or their corresponding standard immunological assays (the use of AQUA peptides parent proteins/polypeptides (see Column A). A phosphopep provided by the invention is described separately above). tide sequence comprising any of the phosphorylation Assays may be homogeneous assays or heterogeneous sequences listed in Table 1 may be considered an AQUA assays. In a homogeneous assay the immunological reaction peptide of the invention. For example, an AQUA peptide usually involves a phosphorylation-site specific antibody of comprising the sequence EMVSQYLyTSK (SEQID NO:38) the invention), a labeled analyte, and the sample of interest. (where y may be either phosphotyrosine or tyrosine, and The signal arising from the label is modified, directly or where V-labeled valine (e.g., ''C)) is provided for the quan indirectly, upon the binding of the antibody to the labeled tification of phosphorylated (or non-phosphorylated) diapha analyte. Both the immunological reaction and detection of the nous 1 (Tyr130) in a biological sample (see Row 39 of Table 1, extent thereof are carried out in a homogeneous solution. tyrosine 130 being the phosphorylatable residue within the Immunochemical labels that may be employed include free US 2010/015 1483 A1 Jun. 17, 2010 47 radicals, radioisotopes, fluorescent dyes, enzymes, bacte col for cytometric analysis may be employed: fixation of the riophages, coenzymes, and so forth. cells with 1% paraformaldehyde for 10 minutes at 37° C. 0162. In a heterogeneous assay approach, the reagents are followed by permeabilization in 90% methanol for 30 min usually the specimen, a phosphorylation-site specific anti utes on ice. Cells may then be stained with the primary anti body of the invention, and Suitable means for producing a body (a phospho-specific antibody of the invention), washed detectable signal. Similar specimens as described above may and labeled with a fluorescent-labeled secondary antibody. be used. The antibody is generally immobilized on a Support, Alternatively, the cells may be stained with a fluorescent Such as a bead, plate or slide, and contacted with the specimen labeled primary antibody. The cells would then be analyzed Suspected of containing the antigen in a liquid phase. The on a flow cytometer (e.g., a Beckman Coulter EPICS-XL) Support is then separated from the liquid phase and either the according to the specific protocols of the instrument used. Support phase or the liquid phase is examined for a detectable Such an analysis would identify the presence of activated signal employing means for producing Such signal. The sig target Signaling Protein(s)/Polypeptide(s) in the malignant nal is related to the presence of the analyte in the specimen. cells and reveal the drug response on the targeted protein. Means for producing a detectable signal include the use of 0166 Alternatively, antibodies of the invention may be radioactive labels, fluorescent labels, enzyme labels, and so employed in immunohistochemical (IHC) staining to detect forth. For example, if the antigen to be detected contains a differences in signal transduction or protein activity using second binding site, an antibody which binds to that site can normal and diseased tissues. IHC may be carried out accord be conjugated to a detectable group and added to the liquid ing to well-known techniques. See, e.g., ANTIBODIES: A phase reaction Solution before the separation step. The pres LABORATORY MANUAL, supra. Briefly, paraffin-embed ence of the detectable group on the solid Support indicates the ded tissue (e.g., tumor tissue) is prepared for immunohis presence of the antigen in the test sample. Examples of Suit tochemical staining by deparaffinizing tissue sections with able immunoassays are the radioimmunoassay, immunofluo xylene followed by ethanol; hydrating in water then PBS: rescence methods, enzyme-linked immunoassays, and the unmasking antigen by heating slide in Sodium citrate buffer; like. incubating sections in hydrogen peroxide; blocking in block 0163. Immunoassay formats and variations thereof that ing solution; incubating slide in primary antibody and sec may be useful for carrying out the methods disclosed herein ondary antibody; and finally detecting using ABC avidin/ are well known in the art. Seegenerally E. Maggio, Enzyme biotin method according to manufacturer's instructions. Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); 0.167 Antibodies of the invention may be also be opti see also, e.g., U.S. Pat. No. 4,727,022; U.S. Pat. No. 4,659, mized for use in other clinically-suitable applications, for 678; U.S. Pat. No. 4,376, 110. Conditions suitable for the example bead-based multiplex-type assays, such as IGEN. formation of reagent-antibody complexes are well described. LuminexTM and/or BioplexTM assay formats, or otherwise Seeid. Monoclonal antibodies of the invention may be used in optimized for antibody array formats, such as reversed-phase a “two-site' or “sandwich' assay, with a single cell line serv array applications (see, e.g., Paweletz et al., Oncogene ing as a source for both the labeled monoclonal antibody and 20(16): 1981-89 (2001)). Accordingly, in another embodi the bound monoclonal antibody. Such assays are described in ment, the invention provides a method for the multiplex U.S. Pat. No. 4,376,110. The concentration of detectable detection of phosphorylation in a biological sample, the reagent should be sufficient Such that the binding of a target method comprising utilizing two or more antibodies or signaling protein/polypeptide is detectable compared to AQUA peptides of the invention to detect the presence of two background. or more phosphorylated proteins enumerated in Column A of 0164 Phosphorylation site-specific antibodies disclosed Table 1/FIG. 2. In an embodiment, two to five antibodies or herein may be conjugated to a solid Support Suitable for a AQUA peptides of the invention are employed in the method. diagnostic assay (e.g., beads, plates, slides or wells formed In another embodiment, six to ten antibodies or AQUA pep from materials such as latex or polystyrene) in accordance tides of the invention are employed, while in another embodi with known techniques, such as precipitation. Antibodies, or ment eleven to twenty Such reagents are employed. other target protein or target site-binding reagents, may like 0168 Antibodies and/or AQUA peptides of the invention wise be conjugated to detectable groups such as radiolabels may also be employed within a kit that comprises at least one (e.g., S.I. ''I), enzyme labels (e.g., horseradish peroxi phosphorylation site-specific antibody or AQUA peptide of dase, alkaline phosphatase), and fluorescent labels (e.g., fluo the invention (which binds to or detects a target signaling rescein) in accordance with known techniques. protein/polypeptide disclosed in Table 1/FIG.2), and, option 0.165 Antibodies of the invention may also be optimized ally, a second antibody conjugated to a detectable group. In for use in a flow cytometry (FC) assay to determine the Some embodies, the kit is suitable for multiplex assays and activation/phosphorylation status of a target signaling pro comprises two or more antibodies or AQUA peptides of the tein/polypeptide in patients before, during, and after treat invention, and in Some embodiments, comprises two to five, ment with a drug targeted at inhibiting phosphorylation of six to ten, or eleven to twenty reagents of the invention. The Such a protein at the phosphorylation site disclosed herein. kit may also include ancillary agents such as buffering agents For example, bone marrow cells or peripheral blood cells and protein stabilizing agents, e.g., polysaccharides and the from patients may be analyzed by flow cytometry for target like. The kit may further include, where necessary, other signaling protein/polypeptide phosphorylation, as well as for members of the signal-producing system of which system the markers identifying various hematopoietic cell types. In this detectable group is a member (e.g., enzyme Substrates), manner, activation status of the malignant cells may be spe agents for reducing background interference in a test, control cifically characterized. Flow cytometry may be carried out reagents, apparatus for conducting a test, and the like. The test according to standard methods. See, e.g. Chow et al., Cytom kit may be packaged in any suitable manner, typically with all etry (Communications in Clinical Cytometry) 46: 72-78 elements in a single container along with a sheet of printed (2001). Briefly and by way of example, the following proto instructions for carrying out the test. US 2010/015 1483 A1 Jun. 17, 2010 48
0169. Reference is made hereinafter in detail to specific T26, lung tumor T57, normal human lung, pancreatic embodiments of the invention. While the invention will be Xenograft, patient 1, rat brain and SWA80. described in conjunction with these specific embodiments, it 0173 Tryptic phosphotyrosine containing peptides were will be understood that it is not intended to limit the invention purified and analyzed from extracts of each of the cell lines to Such specific embodiments. On the contrary, it is intended mentioned above, as follows. Cells were cultured in DMEM to cover alternatives, modifications, and equivalents as may medium or RPMI 1640 medium supplemented with 10% fetal be included within the spirit and scope of the invention as bovine serum and penicillin/streptomycin. defined by the appended claims. In the description, numerous 0.174 Suspension cells were harvested by low speed cen specific details are set forth in order to provide a thorough trifugation. After complete aspiration of medium, cells were understanding of the present invention. The present invention resuspended in 1 mL lysis buffer per 1.25x10 cells (20 mM may be practiced without some or all of these specific details. HEPES pH 8.0, 9 Murea, 1 mM sodium vanadate, supple In other instances, well known process operations have not mented or not with 2.5 mM sodium pyro-phosphate, 1 mM been described in detail, in order not to unnecessarily obscure B-glycerol-phosphate) and Sonicated. the present invention. 0.175 Sonicated cell lysates were cleared by centrifuga 0170 The following examples are intended to further tion at 20,000xg, and proteins were reduced with DTT at a illustrate certain embodiments of the invention and are not final concentration of 4.1 mM and alkylated with iodoaceta limiting in nature. Those skilled in the art will recognize, or be mide at 8.3 mM. For digestion with trypsin, protein extracts able to ascertain, using no more than routine experimentation, were diluted in 20 mMHEPES pH 8.0 to a final concentration numerous equivalents to the specific Substances and proce of 2 M urea and soluble TLCKR)-trypsin (Worthington(R) dures described herein. Biochemcial Corporation, Lakewood, N.J.) was added at 0171 Any suitable materials and/or methods known to 10-20 g/mL. Digestion was performed for 1-2 days at room those of skill can be utilized in carrying out the present inven temperature. tion. However, materials and methods are described. Materi 0176 Trifluoroacetic acid (TFA) was added to protein als, reagents and the like to which reference is made in the digests to a final concentration of 1%, precipitate was following description and examples are obtainable from com removed by centrifugation, and digests were loaded onto mercial Sources, unless otherwise noted. Sep-PakR) C columns (provided by Waters Corporation, Milford, Mass.) equilibrated with 0.1% TFA. A column vol Example 1 ume of 0.7-1.0 ml was used per 2x10 cells. Columns were Isolation of Phosphotyrosine-Containing Peptides washed with 15 volumes of 0.1%TFA, followed by 4 volumes of 5% acetonitrile (MeCN) in 0.1% TFA. Peptide fraction I from Extracts of Cancer Cell Lines and Identification was obtained by eluting columns with 2 volumes each of 8, of Phosphorylation Sites 12, and 15% MeCN in 0.1% TFA and combining the eluates. 0172 IAP isolation techniques were employed to identify Fractions II and III were a combination of eluates after eluting phosphotyrosine containing peptides in cell extracts from the columns with 18, 22, 25% MeCN in 0.1% TFA and with 30, following human cancer cell lines, tissues and patient cell 35, 40% MeCN in 0.1%TFA, respectively. All peptide frac lines: 0 1364548-cl1, 223-CLL, 293T, 3T3 TrkB, 3T3-Src, tions were lyophilized. 3T3-TrkA, 3T3-wit, 577, A172, AML-4833, AML-6246, (0177 Peptides from each fraction corresponding to 2x10 AML-6735, AML-7592, BaF3-10ZF, BaF3-4ZF, BaF3 cells were dissolved in 1 ml of IAP buffer (20 mM Tris/HC1 or APR, BaF3-FLT3(D842V), BaF3-FLT3(D842Y), BaF3 50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM FLT3(K663Q), BaF3-FLT3(WT), BaF3-FLT3/ITD, BaF3 NaCl) and insoluble material was removed by centrifugation. PRTK, BaF3-TDII, BaF3-Tel/FGFR3, Baf3, Baf3-V617F IAP was performed on each peptide fraction separately. The jak2, Baf3/E255K, Baf3/H396P. Baf3/Jak2 (IL-3 dep), Baf3/ phosphotyrosine monoclonal antibody P-Tyr-100 (Cell Sig M351T, Baf3/T3151, Baf3/TpoR, Baf3/TpoR-Y98F, Baf3/ naling Technology(R), Inc., Danvers, Mass. catalog number Tyk2, Baf3N617F-jak2 (IL-3), Baf3/Y253F, Baf3/cc-TpoR 9411) was coupled at 4 mg/ml beads to protein G or protein A IV, Baf3/p210wt, CHRF, CI-1, CMK, CTV-1, DMS 53, agarose (Roche?R), Basel, Switzerland), respectively. Immo DND41, DU-528, DU145, ELF-153, EOL-1, GDM-1, bilized antibody (15ul, 60 g) was added as 1:1 slurry in IAP H1703, H1734, H1793, H1869, H1944, H1993, H2023, buffer to 1.4 ml of each peptide fraction, and the mixture was H226, H3255, H358, H520, H82, H838, HCC1428, incubated overnight at 4°C. with gentle rotation. The immo HCC1435, HCC1806, HCC1937, HCC366, HCC827, bilized antibody beads were washed three times with 1 ml HCT116, HEL, HL107B, HL117B, HL131A, HL131B, IAP buffer and twice with 1 ml water, all at 4° C. Peptides HL133A, HL53B, HL59b, HL60, HL61a, HL61b, HL66B, were eluted from beads by incubation with 75ul of 0.1%TFA HL68A, HL75A, HL84A, HL97B, HL98A, HT29, HU-3, at room temperature for 10 minutes. HUVEC, Jurkat, K562, KG-1, KG 1-A, KMS 11, KMS 18, 0.178 Alternatively, one single peptide fraction was KMS27, KOPT-K1, KY821, Karpas 299, Karpas-1 106p. obtained from Sep-PakRCs columns (provided by Waters M-07e, M01043, MO59K, MC-1 16, MCF-10A (Y561F), Corporation, Milford, Mass.) by elution with 2 volumes each MCF-10A (Y969F), MDA-MB-453, MDA-MB-468, MEC of 10%, 15%, 20%, 25%, 30%, 35% and 40% acetonitirile in 2, MKPL-1, ML-1, MO-91, MOLT15, MV4-11, Me-F2, 0.1%TFA and combination of all eluates. IAP on this peptide Molm 14, Monomac 6, NCI-N87, Nomo-1, OCI-M1, OCI fraction was performed as follows: After lyophilization, pep ly4, OCI-ly8, OCI/AML2, OPM-1, PL21, Pfeiffer, RC-K8, tide was dissolved in 1.4 ml IAP buffer (MOPS pH 7.2, 10 RI-1, SCLC T1, SEM, SK-N-AS, SK-N-MC, SKBR3, mM sodium phosphate, 50 mM NaCl) and insoluble material SR-786, SU-DHL1, SUP-M2, SUPT-13, SuDHL5, T17, was removed by centrifugation. Immobilized antibody (40 ul, TRE-cll patient, TS, UT-7, VAL, Verona, Verona 1, Verona 4, 160 ug) was added as 1:1 slurry in IAP buffer, and the mixture WSU-NHL, XG2, Z-55, cs001, cs015, cs025, cs041, cs042, was incubated overnight at 4°C. with gentle shaking. The gZ21, gz68, gz73, gz74, gzB1, hl 144b, hl 152b, lung tumor immobilized antibody beads were washed three times with 1 US 2010/015 1483 A1 Jun. 17, 2010 49 ml IAP buffer and twice with 1 ml water, all at 4°C. Peptides assignments, which are not yet universally accepted, and were eluted from beads by incubation with 40 ul of 0.15% guidelines for the publication of protein and peptide identifi TFA at room temperature for 10 min (eluate 1), followed by a cation results (see Carr et al., Mol. Cell Proteomics 3: 531 533 (2004)), which were followed in this Example. However, wash of the beads (eluate 2) with 40 ul of 0.15% TFA. Both because the immunoaffinity strategy separates phosphory eluates were combined. lated peptides from unphosphorylated peptides, observing Analysis by LC-MS/MS Mass Spectrometry. 40 ul or more of just one phosphopeptide from a protein is a common result, IAP eluate were purified by 0.2 ul StageTips (Proxeon, Staer since many phosphorylated proteins have only one tyrosine mosegaardsvei 6, DK-5230 Odense M. Denmark) or Zip phosphorylated site. For this reason, it is appropriate to use Tips(R (produced by Millipore(R), Billerica Mass.). Peptides additional criteria to validate phosphopeptide assignments. were eluted from the microcolumns with 1 ul of 40% MeCN, Assignments are likely to be correct if any of these additional 0.1% TFA (fractions I and II) or 1 ul of 60% MeCN, 0.1% criteria are met: (i) the same sequence is assigned to co eluting ions with different charge states, since the MS/MS TFA (fraction III) into 7.6 ul of 0.4% acetic acid/0.005% spectrum changes markedly with charge state; (ii) the site is heptafluorobutyric acid. This sample was loaded onto a 10 found in more than one peptide sequence context due to cmx75 um PicoFrit(R) capillary column (produced by New sequence overlaps from incomplete proteolysis or use of pro Objective, Woburn, Mass.) packed with Michrom Magic Bul teases other than trypsin; (iii) the site is found in more than lets(R C18 AQ reversed-phase resin (Michrom Bioresources, one peptide sequence context due to homologous but not Auburn Calif.) using a FamosTM autosampler with an inert identical protein isoforms; (iv) the site is found in more than sample injection valve (Dionex(R), Sunnyvale, Calif.). The one peptide sequence context due to homologous but not column was then developed with a 45-min linear gradient of identical proteins among species; and (v) sites validated by acetonitrile delivered at 200 ml/min (using an Ultimate(R) MS/MS analysis of synthetic phosphopeptides correspond ing to assigned sequences, since the ion trap mass spectrom pump, DioneXOR, Sunnyvale, Calif.), and tandem mass spectra eter produces highly reproducible MS/MS spectra. The last were collected in a data-dependent manner with an LTQR criterion is routinely employed to confirm novel site assign (produced by Thermo R. Finnigan R. San, Jose, Calif.), ion trap ments of particular interest. mass spectrometer essentially as described by Gygi et al., 0181 All spectra and all sequence assignments made by Supra. Sequest were imported into a relational database. The follow Database Analysis & Assignments. MS/MS spectra were ing Sequest scoring thresholds were used to select phospho evaluated using TurboSequestTM in the Sequest (owned by peptide assignments that are likely to be correct: RSp-6, Thermo (R) Finnigan R. San Jose, Calif.) Browser package (v. XCorre2.2, and DeltaCN>0.099. Further, the assigned sequences could be accepted or rejected with respect to accu 27, rev. 12) supplied as part of BioWorksTM 3.0 (Thermo.R. racy by using the following conservative, two-step process. Finnigan.R., San Jose, Calif.). Individual MS/MS spectra were 0182. In the first step, a Subset of high-scoring sequence extracted from the raw data file using the Sequest(R) Browser assignments should be selected by filtering for XCorr values program CreateDtaTM (owned by Thermo (R) Finnigan R. San of at least 1.5 for a charge state of +1, 2.2 for +2, and 3.3 for Jose, Calif.), with the following settings: bottom MW, 700; +3, allowing a maximum RSp value of 10. Assignments in top MW, 4.500; minimum number of ions, 20; minimum TIC, this subset should be rejected if any of the following criteria 4x10; and precursor charge state, unspecified. Spectra were were satisfied: (i) the spectrum contains at least one major extracted from the beginning of the raw data file before peak (at least 10% as intense as the most intense ion in the sample injection to the end of the eluting gradient. The Ion spectrum) that can not be mapped to the assigned sequence as QuestTM and VulDtaTM (owned by Thermo(R) Finnigan R. San ana, b, ory ion, as anion arising from neutral-loss of water or ammonia from ab ory ion, or as a multiply protonatedion; (ii) Jose, Calif.) programs were not used to further select MS/MS the spectrum does not contain a series of bory ions equivalent spectra for Sequest(R) analysis. MS/MS spectra were evalu to at least six uninterrupted residues; or (iii) the sequence is ated with the following TurboSequestTM parameters: peptide not observed at least five times in all the studies conducted mass tolerance, 2.5; fragment ion tolerance, 0.0; maximum (except for overlapping sequences due to incomplete pro number of differential amino acids per modification, 4, mass teolysis or use of proteases other than trypsin). type parent, average; mass type fragment, average; maximum 0183 In the second step, assignments with below-thresh number of internal cleavage sites, 10; neutral losses of water old scores should be accepted if the low-scoring spectrum and ammonia from b and y ions were considered in the cor shows a high degree of similarity to a high-scoring spectrum relation analysis. Proteolytic enzyme was specified except for collected in another study, which simulates a true reference spectra collected from elastase digests. library-searching strategy. 0179 Searches were performed against the NCBI human Example 2 protein database (as released on Aug. 24, 2004 and containing Production of Phospho-Specific Polyclonal Antibod 27, 960 protein sequences). Cysteine carboxamidomethyla ies for the Detection of Target Signal Protein/ tion was specified as a static modification, and phosphoryla Polypepetide Phosphorylation tion was allowed as a variable modification on serine, threo 0.184 Polyclonal antibodies that specifically bind a target nine, and tyrosine residues or on tyrosine residues alone. It signal protein/polypeptide only when phosphorylated at the was determined that restricting phosphorylation to tyrosine respective phosphorylation site disclosed herein (see Table residues had little effect on the number of phosphorylation 1/FIG. 2) are produced according to standard methods by first sites assigned. Furthermore, it should be noted that certain constructing a synthetic peptide antigen comprising the phos peptides were originally isolated in mouse and later normal phorylation site sequence and then immunizing an animal to ized to human sequences as shown by Table 1/FIG. 2. raise antibodies against the antigen, as further described 0180. In proteomics research, it is desirable to validate below. Production of exemplary polyclonal antibodies is pro protein identifications based solely on the observation of a vided below. single peptide in one experimental result, in order to indicate A. 14-3-3 Zeta (tyrosine 82) that the protein is, in fact, present in a sample. This has led to 0185. A 17 amino acid phospho-peptide antigen, the development of statistical methods for validating peptide KQQMAREy*REKIETELR (where y-phosphotyrosine) US 2010/015 1483 A1 Jun. 17, 2010 50 that corresponds to the sequence encompassing the tyrosine (i.e. phosphorylated 14-3-3 Zeta, Crkl or catalase), for 82 phosphorylation site in human 14-3-3 Zeta adaptor/scaf example, SEM and Jurkat cells, respectively. Cells are cul fold protein (see Row 2 of Table 1: SEQ ID NO: 1), plus tured in DMEM or RPMI supplemented with 10% FCS. Cell cysteine on the C-terminal for coupling, is constructed are collected, washed with PBS and directly lysed in cell lysis according to standard synthesis techniques using, e.g., a Rai buffer. The protein concentration of cell lysates is then mea nin/Protein Technologies, Inc., Symphony peptide synthe sured. The loading buffer is added into cell lysate and the sizer. See ANTIBODIES: A LABORATORY MANUAL, supra.: mixture is boiled at 100° C. for 5 minutes. 20 ul (10 ug Merrifield, supra. This peptide is then coupled to KLH and protein) of sample is then added onto 7.5% SDS-PAGE gel. used to immunize animals to produce (and Subsequently 0190. A standard Western blot may be performed accord screen) phospho-specific 14-3-3 Zeta (tyr82) polyclonal anti ing to the Immunoblotting Protocol set out in the CELL SIG bodies as described in Immunization/Screening below. NALING TECHNOLOGY, INC. 2003-04 Catalogue, p. 390. The B. Crkl (tyrosine 48) isolated phospho-specific antibody is used at dilution 1:1000. 0186. An 18 amino acid phospho-peptide antigen, Phosphorylation-site specificity of the antibody will be DSSTCPGDy*VLSVSENSR (where y*-phosphotyrosine) shown by binding of only the phosphorylated form of the that corresponds to the sequence encompassing the tyrosine target protein. Isolated phospho-specific polyclonal antibody 48 phosphorylation site in human Crkl adaptor/scaffold pro does not (Substantially) recognize the target protein when not tein (see Row 37 of Table 1 (SEQID NO:36)), plus cysteine phosphorylated at the appropriate phosphorylation site in the on the C-terminal for coupling, is constructed according to non-stimulated cells (e.g. Crkl is not bound when not phos standard synthesis techniques using, e.g., a Rainin/Protein phorylated at tyrosine 48). Technologies, Inc., Symphony peptide synthesizer. See ANTI 0191 In order to confirm the specificity of the isolated BODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. antibody, different cell lysates containing various phospho This peptide is then coupled to KLH and used to immunize rylated signal transduction proteins other than the target pro animals to produce (and Subsequently screen) phospho-spe tein are prepared. The Western blot assay is performed again cific Crkl (tyr 48) polyclonal antibodies as described in using these cell lysates. The phospho-specific polyclonal Immunization/Screening below. antibody isolated as described above is used (1:1000 dilution) C. Catalase (tyrosine 84) to test reactivity with the different phosphorylated non-target 0187. A 16 amino acid phospho-peptide antigen, proteins on Western blot membrane. The phospho-specific GAGAFGy*FEVTHDITK(wherey phosphotyrosine) that antibody does not significantly cross-react with other phos corresponds to the sequence encompassing the tyrosine 84 phorylated signal transduction proteins, although occasion phosphorylation site in human catalase apoptosis protein (see ally slight binding with a highly homologous phosphoryla Row 59 of Table 1 (SEQ ID NO. 58), plus cysteine on the tion-site on another protein may be observed. In Such case the C-terminal for coupling, is constructed according to standard antibody may be further purified using affinity chromatogra synthesis techniques using, e.g., a Rainin/Protein Technolo phy, or the specific immunoreactivity cloned by rabbit hybri gies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A doma technology. LABORATORY MANUAL, supra.; Merrifield, supra. This pep tide is then coupled to KLH and used to immunize animals to produce (and Subsequently screen) phospho-specific catalase Example 3 (tyr 84) antibodies as described in Immunization/Screening Production of Phospho-Specific Monoclonal Anti below. bodies for the Detection of Target Signal Protein/ Polypepetide Phosphorylation Immunization/Screening. 0188 A synthetic phospho-peptide antigenas described in 0.192 Monoclonal antibodies that specifically bind a tar A-C above is coupled to KLH, and rabbits are injected intra get signal protein/polypepetide only when phosphorylated at dermally (ID) on the back with antigen in complete Freunds the respective phosphorylation site disclosed herein (see adjuvant (500 ug antigen per rabbit). The rabbits are boosted Table 1/FIG. 2) are produced according to standard methods with same antigen in incomplete Freund adjuvant (250 ug by first constructing a synthetic peptide antigen comprising antigen per rabbit) every three weeks. After the fifth boost, the phosphorylation site sequence and then immunizing an bleeds are collected. The sera are purified by Protein A-affin animal to raise antibodies against the antigen, and harvesting ity chromatography by standard methods (see Antibodies. A spleen cells from Such animals to produce fusion hybridomas, Laboratory Manual, Cold Spring Harbor, supra.). The eluted as further described below. Production of exemplary mono immunoglobulins are further loaded onto a non-phosphory clonal antibodies is provided below. lated synthetic peptide antigen-resin Knotes column to pull A. ANXA11 (tyrosine 365) out antibodies that bind the non-phosphorylated form of the 0193 A 12 amino acid phospho-peptide antigen, phosphorylation site. The flow through fraction is collected DAQELy*AAGENR (where y-phosphotyrosine) that cor and applied onto a phospho-synthetic peptide antigen-resin responds to the sequence encompassing the tyrosine 365 column to isolate antibodies that bind the phosphorylated phosphorylation site in human ANXA11 calcium binding form of the site. After washing the column extensively, the protein (see Row 62 of Table 1 (SEQ ID NO: 61)), plus bound antibodies (i.e. antibodies that bind a phosphorylated cysteine on the C-terminal for coupling, is constructed peptide described in A-C above, but do not bind the non according to standard synthesis techniques using, e.g., a Rai phosphorylated form of the peptide) are eluted and kept in nin/Protein Technologies, Inc., Symphony peptide synthe antibody storage buffer. sizer. See ANTIBODIES: A LABORATORY MANUAL, supra.: 0189 The isolated antibody is then tested for phospho Merrifield, supra. This peptide is then coupled to KLH and specificity using Western blot assay using an appropriate cell used to immunize animals and harvest spleen cells for gen line that expresses (or overexpresses) target phospho-protein eration (and Subsequent screening) of phospho-specific US 2010/015 1483 A1 Jun. 17, 2010
monoclonal ANXA11 (tyr 365) antibodies as described in pho-specificity against the phosphorylated target (e.g. Immunization/Fusion/Screening below. ANXA5 phosphorylated at tyrosine 255). B. ANXA2 (tyrosine 199) 0194 An 9 amino acid phospho-peptide antigen, Example 4 DLy*DAGVKR (where y-phosphotyrosine) that corre Production and Use of AQUA Peptides for the Quan sponds to the sequence encompassing the tyrosine 199 phos tification of Target Signal Protein/Polypepetide phorylation site in human ANXA2 calcium binding protein Phosphorylation (see Row 63 of Table 1 (SEQID NO: 62)), plus cysteine on the C-terminal for coupling, is constructed according to standard 0199 Heavy-isotope labeled peptides (AQUA peptides synthesis techniques using, e.g., a Rainin/Protein Technolo (internal standards)) for the detection and quantification of a gies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A target signal protein/polypepetide only when phosphorylated LABORATORY MANUAL, supra.; Merrifield, supra. This pep at the respective phosphorylation site disclosed herein (see tide is then coupled to KLH and used to immunize animals Table 1/FIG. 2) are produced according to the standard and harvest spleen cells for generation (and Subsequent AQUA methodology (see Gygi et al., Gerber et al., Supra.) screening) of phospho-specific monoclonal ANXA2 (tyr199) methods by first constructing a synthetic peptide standard antibodies as described in Immunization/Fusion/Screening corresponding to the phosphorylation site sequence and below. incorporating a heavy-isotope label. Subsequently, the MS" C. ANXA5 (tyrosine 256) and LC-SRM signature of the peptide standard is validated, 0.195 A 15 amino acid phospho-peptide antigen, and the AQUA peptide is used to quantify native peptide in a SIPAYLAETLy*YAMK (where y-phosphotyrosine) that biological sample, Such as a digested cell extract. Production corresponds to the sequence encompassing the tyrosine 256 and use of exemplary AQUA peptides is provided below. phosphorylation site in human ANXA5 calcium binding pro A. ANXA11 (tyrosine 365) tein(see Row 64 of Table 1 (SEQID NO: 63), plus cysteine on 0200. An AQUA peptide comprising the sequence, the C-terminal for coupling, is constructed according to stan DAQELyAAGENR (y-phosphotyrosine; sequence incor dard synthesis techniques using, e.g., a Rainin/Protein Tech porating 'C/N-labeled leucine (indicated by bold L). nologies, Inc., Symphony peptide synthesizer. See ANTIBOD which corresponds to the tyrosine365 phosphorylation site in IES: A LABORATORY MANUAL, supra.; Merrifield, supra. This human ANXA11 chromatin or DNA binding/repair/replica peptide is then coupled to KLH and used to immunize animals tion protein (see Row 62 in Table 1 (SEQ ID NO: 61)), is and harvest spleen cells for generation (and subsequent constructed according to standard synthesis techniques using, screening) of phospho-specific monoclonal ANXA5 (tyr256) e.g., a Rainin/Protein Technologies, Inc., Symphony peptide antibodies as described in Immunization/Fusion/Screening synthesizer (see Merrifield, supra.) as further described below. below in Synthesis & MS/MS Signature. The ANXA11 (tyr 0196. Immunization/Fusion/Screening. A synthetic phos 365) AQUA peptide is then spiked into a biological sample to pho-peptide antigen as described in A-C above is coupled to quantify the amount of phosphorylated ANXA11 (tyr 365) in KLH, and BALB/C mice are injected intradermally (ID) on the sample, as further described below in Analysis & Quan the back with antigen in complete Freunds adjuvant (e.g. 50 tification. ug antigen per mouse). The mice are boosted with same B. Arp3 (tyrosine 16) antigen in incomplete Freund adjuvant (e.g. 25ug antigen per 0201 An AQUA peptide comprising the sequence mouse) every three weeks. After the fifth boost, the animals LPACVVDCGTGy*TK (y-phosphotyrosine; sequence are sacrificed and spleens are harvested. incorporating '"C/N-labeled leucine (indicated by bold L). (0197) Harvested spleen cells are fused to SP2/0 mouse which corresponds to the tyrosine 16 phosphorylation site in myeloma fusion partner cells according to the standard pro human Arp3 cytoskeletal protein (see Row 105 in Table 1 tocol of Kohler and Milstein (1975). Colonies originating (SEQ ID NO: 104)), is constructed according to standard from the fusion are screened by ELISA for reactivity to the synthesis techniques using, e.g., a Rainin/Protein Technolo phospho-peptide and non-phospho-peptide forms of the anti gies, Inc., Symphony peptide synthesizer (see Merrifield, gen and by Western blot analysis (as described in Example 1 supra.) as further described below in Synthesis & MS/MS above). Colonies found to be positive by ELISA to the phos Signature. The Arp3 (tyr16) AQUA peptide is then spiked into pho-peptide while negative to the non-phospho-peptide are a biological sample to quantify the amount of phosphorylated further characterized by Western blot analysis. Colonies Arp3 (tyr16) in the sample, as further described below in found to be positive by Western blot analysis are subcloned by Analysis & Quantification. limited dilution. Mouse ascites are produced from a single C. ADA (tyrosine 67) clone obtained from Subcloning, and tested for phospho 0202 An AQUA peptide comprising the sequence specificity (against the ANXA11, ANXA2 or ANXA5 phos FDy*YMPAIAGCR (y-phosphotyrosine; sequence incor pho-peptide antigen, as the case may be) on ELISA. Clones porating '"C/N-labeled phenylalanine (indicated by bold identified as positive on Western blot analysis using cell cul F), which corresponds to the tyrosine 67 phosphorylation site ture Supernatant as having phospho-specificity, as indicated in human ADA enzyme protein (see Row 146 in Table 1 (SEQ by a strong band in the induced lane and a weak band in the ID NO: 145)), is constructed according to standard synthesis uninduced lane of the blot, are isolated and subcloned as techniques using, e.g., a Rainin/Protein Technologies, Inc., clones producing monoclonal antibodies with the desired Symphony peptide synthesizer (see Merrifield, supra.) as fur specificity. ther described below in Synthesis & MS/MS Signature. The 0198 Ascites fluid from isolated clones may be further ADA (tyró7) AQUA peptide is then spiked into a biological tested by Western blot analysis. The ascites fluid should pro sample to quantify the amount of phosphorylated ADA duce similar results on Western blot analysis as observed (tyró7) in the sample, as further described below in Analysis previously with the cell culture Supernatant, indicating phos & Quantification. US 2010/015 1483 A1 Jun. 17, 2010 52
D. ASS (tyrosine 133) (0204 MS/MS spectra for each AQUA peptide should 0203. An AQUA peptide comprising the sequence, exhibit a strong Y-type ion peak as the most intense fragment ion that is suitable for use in an SRM monitoring/analysis. FELSCY*SLAPQIK (y-phosphotyrosine; sequence incor Reverse-phase microcapillary columns (0.1 A-150-220mm) porating '"C/N-labeled proline (indicated by bold P), are prepared according to standard methods. An Agilent 1100 which corresponds to the tyrosine 133 phosphorylation site in liquid chromatograph may be used to develop and deliver a human ASS enzyme protein (see Row 160 in Table 1 (SEQID solvent gradient 0.4% acetic acid/0.005% heptafluorobu NO: 159)), is constructed according to standard synthesis tyric acid (HFBA)/7% methanol and 0.4% acetic acid/0. techniques using, e.g., a Rainin/Protein Technologies, Inc., 0.05% HFBA/65% methanol/35% acetonitrile to the micro Symphony peptide synthesizer (see Merrifield, supra.) as fur capillary column by means of a flow splitter. Samples are then ther described below in Synthesis & MS/MS Signature. The directly loaded onto the microcapillary column by using a ASS (tyr133) AQUA peptide is then spiked into a biological FAMOS inert capillary autosampler (LC Packings, San Fran sample to quantify the amount of phosphorylated ASS cisco) after the flow split. Peptides are reconstituted in 6% acetic acid/0.01% TFA before injection. (tyr133) in the sample, as further described below in Analysis Analysis & Quantification. Target protein (e.g. a phosphory & Quantification. lated protein of A-D above) in a biological sample is quanti Synthesis & MS/MS Spectra. Fluorenylmethoxycarbonyl fied using a validated AQUA peptide (as described above). (Fmoc)-derivatized amino acid monomers may be obtained The IAP method is then applied to the complex mixture of from AnaSpec (San Jose, Calif.). Fmoc-derivatized stable peptides derived from proteolytic cleavage of crude cell isotope monomers containing one 'N and five to nine C extracts to which the AQUA peptides have been spiked in. atoms may be obtained from Cambridge Isotope Laboratories (0205 LC-SRM of the entire sample is then carried out. (Andover, Mass.). Preloaded Wang resins may be obtained MS/MS may be performed by using a ThermoFinnigan (San from Applied Biosystems. Synthesis scales may vary from 5 Jose, Calif.) mass spectrometer (LTQ ion trap or TSQ Quan to 25 umol. Amino acids are activated in situ with 1-H- tum triple quadrupole). On the LTQ, parentions are isolated benzotriazolium, 1-bis(dimethylamino)methylene at 1.6 m/z, width, the ion injection time being limited to 100 hexafluorophosphate (1-).3-oxide: 1-hydroxybenzotriazole ms per microScan, with one microscans per peptide, and with hydrate and coupled at a 5-fold molar excess over peptide. an AGC setting of 1x10; on the Quantum, Q1 is kept at 0.4 Each coupling cycle is followed by capping with acetic anhy and Q3 at 0.8 m/z, with a scan time of 200 ms per peptide. On dride to avoid accumulation of one-residue deletion peptide both instruments, analyte and internal standard are analyzed by-products. After synthesis peptide-resins are treated with a in alternation within a previously known reverse-phase reten standard Scavenger-containing trifluoroacetic acid (TFA)- tion window; well-resolved pairs of internal standard and water cleavage solution, and the peptides are precipitated by analyte are analyzed in separate retention segments to addition to cold ether. Peptides (i.e., a desired AQUA peptide improve duty cycle. Data are processed by integrating the described in A-D above) are purified by reversed-phase C18 appropriate peaks in an extracted ion chromatogram (60.15 HPLC using standard TFA/acetonitrile gradients and charac m/z from the fragment monitored) for the native and internal terized by matrix-assisted laser desorption ionization-time of standard, followed by calculation of the ratio of peak areas flight (Biflex III, Bruker Daltonics, Billerica, Mass.) and ion multiplied by the absolute amount of internal standard (e.g., trap (ThermoFinnigan, LCQ DecaXP) MS. 500 fmol).
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 499
<21 Os SEQ ID NO 1 &211s LENGTH: 17 212s. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 1 Lys Glin Gln Met Ala Arg Glu Tyr Arg Glu Lys Ile Glu Thr Glu Lieu. 1. 5 1O 15 Arg
<21 Os SEQ ID NO 2 &211s LENGTH: 12 212s. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs SEQUENCE: 2 Asn Asp Asp Gly Trp Tyr Glu Gly Val Cys Asn Arg 1. 5 1O US 2010/015 1483 A1 Jun. 17, 2010 53
- Continued <210s, SEQ ID NO 3 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 3 Cys Lieu Lys Asp Glu Asp Pro Tyr Val Arg 1. 5 1O
<210s, SEQ ID NO 4 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 4 Asn Val Glu Gly Glin Asp Met Lieu. Tyr Glin Ser Lieu Lys 1. 5 1O
<210s, SEQ ID NO 5 &211s LENGTH: 36 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 5 Val Ile Ser Ala Asn Pro Tyr Lieu. Gly Gly. Thir Ser Asn Gly Tyr Ala 1. 5 1O 15 His Pro Ser Gly Thr Ala Lieu. His Tyr Asp Asp Val Pro Cys Ile Asn 2O 25 3O Gly Ser Lieu Lys 35
<210s, SEQ ID NO 6 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 6 Trp Glu Ala Gly Ile Tyr Ala Asn Glin Glu Glu Glu Asp Asn. Glu 1. 5 1O 15
<210s, SEQ ID NO 7 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 7 Lieu. Phe Glu Asp Asp Glu. His Glu Lys Glu Glin Tyr Cys Ile Arg 1. 5 1O 15
<210s, SEQ ID NO 8 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 8 Ser Pro Gly Ala Lieu. Glu Thr Pro Ser Ala Ala Gly Ser Glin Gly Asn 1. 5 1O 15 Thr Ala Ser Glin Gly Lys Glu Gly Pro Tyr Ser Glu Pro Ser Lys Arg 2O 25 3O
<210s, SEQ ID NO 9 US 2010/015 1483 A1 Jun. 17, 2010 54
- Continued
&211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 9 Ser Asp Pro Phe Val Pro Pro Ala Ala Ser Ser Glu Pro Leu Ser Thr 1. 5 1O 15 Pro Trp Asn. Glu Lieu. Asn Tyr Val Gly Gly Arg 2O 25
<210s, SEQ ID NO 10 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 10 Cys Glu Val Asn Ala Glu Asp Llys Glu Asn. Ser Gly Asp Tyr Ile Ser 1. 5 1O 15 Glu Asn. Glu Asp Pro Glu Lieu. Glin Asp Tyr Arg 2O 25
<210s, SEQ ID NO 11 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens < 4 OO > SEQUENCE: 11 Ser Lieu. Glu Asn Gln Thr Tyr Phe Lys 1. 5
<210s, SEQ ID NO 12 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 12 Arg Ile Leu Tyr Glin Asn Lieu. Asn Glu Pro Thir Thr Trp Ser Lieu. Thr 1. 5 1O 15 Ser Asp Arg
<210s, SEQ ID NO 13 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 13 His Asp Val Ser Ala His His Asp Lieu. Asn. Ile Asp Glin Ser Glin Cys 1. 5 1O 15 Asn Glu Met Tyr Ile Asn Ser Ser Glin Arg 2O 25
<210s, SEQ ID NO 14 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 14 Gly Glin Glin Glin Glin Glin Glin Glin Glin Gly Ala Val Gly His Gly Tyr 1. 5 1O 15 Tyr Met Ala Gly Gly. Thir Ser Gln Lys US 2010/015 1483 A1 Jun. 17, 2010 55
- Continued
2O 25
<210s, SEQ ID NO 15 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 15 Lieu. Arg Cys Tyr Cys Met Thr Asp Asp Llys Val Asp Llys 1. 5 1O
<210s, SEQ ID NO 16 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 16 Pro Tyr Ser Val Gly Phe Arg 1. 5
<210s, SEQ ID NO 17 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 17 Glu Lieu Val ASn Tyr Gly Ala ASn Val Asn Ala Glin Ser Glin Lys 1. 5 1O 15
<210s, SEQ ID NO 18 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 18 Val Gly Tyr Lieu. Gly Ala Met Lieu. Lieu. Lieu. Asp Glu Arg 1. 5 1O
<210s, SEQ ID NO 19 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 19 Ala Lieu. Asp Lieu. Lieu. Tyr Gly Met Val Ser Lys 1. 5 1O
<210s, SEQ ID NO 2 O &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 2O Ser Val Val Ser Glin Ser Val Cys Asp Tyr Phe Phe Glu Ala Glin Glu 1. 5 1O 15 Lys
<210s, SEQ ID NO 21 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 2010/015 1483 A1 Jun. 17, 2010 56
- Continued <4 OOs, SEQUENCE: 21 Glu Gly Tyr Glin Asp Tyr Tyr Pro Glu Glu Ala Asn Gly Asn Thr Gly 1. 5 1O 15 Ala Ser Pro Tyr Arg 2O
<210s, SEQ ID NO 22 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 22 Thr His Cys Asp Cys Asn Val Asp Trp Cys Lieu. Tyr Glu Ile Tyr Pro 1. 5 1O 15 Glu Lieu. Glin Ile Glu Arg 2O
<210s, SEQ ID NO 23 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 23 Ala Ser Gly Ile Tyr Tyr Val Pro Llys 1. 5
<210s, SEQ ID NO 24 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 24 Ala Glu Phe Asn Tyr Ser Val Gly Phe Lys 1. 5 1O
<210s, SEQ ID NO 25 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 25 Ser Asp Asp His Pro Asp Val Val Tyr Glu Thr Met Arg 1. 5 1O
<210s, SEQ ID NO 26 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 26 Asp Phe Val Asp His Lieu. Asp Llys Val Asp Pro Val Asp Gly Val Val 1. 5 1O 15 Lieu Val Asp Pro Asp Tyr Lieu Lys Asp Arg 2O 25
<210s, SEQ ID NO 27 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 27 US 2010/015 1483 A1 Jun. 17, 2010 57
- Continued
Thir Arg Ser Glin Arg Llys Val Gly Gly Gly Ser Ala Glin Pro Cys Asp 1. 5 1O 15 Ser Ile Val Val Ala Tyr Tyr Phe Cys Gly Glu Pro Ile Pro Tyr Arg 2O 25 3O
<210s, SEQ ID NO 28 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 28 Thir Arg Ser Glin Arg Llys Val Gly Gly Gly Ser Ala Glin Pro Cys Asp 1. 5 1O 15 Ser Ile Val Val Ala Tyr Tyr Phe Cys Gly Glu Pro Ile Pro Tyr Arg 2O 25 3O
<210s, SEQ ID NO 29 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 29 Thir Arg Ser Glin Arg Llys Val Gly Gly Gly Ser Ala Glin Pro Cys Asp 1. 5 1O 15 Ser Ile Val Val Ala Tyr Tyr Phe Cys Gly Glu Pro Ile Pro Tyr Arg 2O 25 3O
<210s, SEQ ID NO 3 O &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 30 Val Glu Asn Glu Tyr Thr Ile Ser Val Lys 1. 5 1O
<210s, SEQ ID NO 31 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 31 Lieu. Asn His Asn Lieu. Tyr Glu Val Met Ser Lys 1. 5 1O
<210s, SEQ ID NO 32 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 32 Thr Gly His Gly Tyr Val Tyr Glu Tyr Pro Ser Arg 1. 5 1O
<210s, SEQ ID NO 33 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 33 US 2010/015 1483 A1 Jun. 17, 2010 58
- Continued Asp Thr Cys Tyr Ser Pro Llys Pro Ser Val Tyr Lieu Ser Thr Pro Ser 1. 5 1O 15 Ser Ala Ser Lys 2O
<210s, SEQ ID NO 34 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 34 Ala Ser Thr Tyr Gly Val Ala Val Arg 1. 5
<210s, SEQ ID NO 35 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 35 Gly Llys His Gly Asn Arg Asn. Ser Asn. Ser Tyr Gly Ile Pro Glu Pro 1. 5 1O 15 Ala His Ala Tyr 2O
<210 SEQ ID NO 36 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 36 Asp Ser Ser Thr Cys Pro Gly Asp Tyr Val Leu Ser Val Ser Glu Asn 1. 5 1O 15
Ser Arg
<210s, SEQ ID NO 37 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 37 Ile His Tyr Lieu. Asp Thir Thr Thr Lieu. Ile Glu Pro Ala Pro Arg 1. 5 1O 15
<210s, SEQ ID NO 38 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 38 Glu Met Val Ser Glin Tyr Lieu. Tyr Thr Ser Lys 1. 5 1O
<210s, SEQ ID NO 39 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 39 His His Thr Ser Ser Val Tyr Ser Ile Ser Glu Arg 1. 5 1O US 2010/015 1483 A1 Jun. 17, 2010 59
- Continued
<210s, SEQ ID NO 4 O &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 4 O Asn Llys Pro Val Pro Asp Glin Ile Ile Asin Phe Tyr Lys Ser Asn Tyr 1. 5 1O 15 Val Glin Arg
<210s, SEQ ID NO 41 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 41 Ser Lieu. Tyr Asp Ser Pro Glin Glu Pro Arg Gly Glu Ala Trp 1. 5 1O
<210s, SEQ ID NO 42 &211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 42 Glin Lieu Pro Asn. Ser Tyr Asn Lieu. Glu Lys Ile Thr Val Asn. Ser Val 1. 5 1O 15 Ser Arg Asp Asn. Ser Llys Tyr His Cys Thir Ala Tyr Arg 2O 25
<210s, SEQ ID NO 43 &211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 43 Glin Lieu Pro Asn. Ser Tyr Asn Lieu. Glu Lys Ile Thr Val Asn. Ser Val 1. 5 1O 15 Ser Arg Asp Asn. Ser Llys Tyr His Cys Thir Ala Tyr Arg 2O 25
<210s, SEQ ID NO 44 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 44 Asp Tyr Glu Pro Pro Ser Pro Ser Pro Ala Pro Gly Ala Pro Pro Pro 1. 5 1O 15 Pro Pro Glin Arg 2O
<210s, SEQ ID NO 45 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 45 Val Glu Glin Glin Pro Asp Tyr Arg US 2010/015 1483 A1 Jun. 17, 2010 60
- Continued
<210s, SEQ ID NO 46 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 46 Met Ser Glu Pro Pro Val Tyr Cys Asn Lieu 1. 5 1O
<210s, SEQ ID NO 47 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 47 Ala Met Glu Asn Glin Tyr Ser Pro Thr Pro Gly Thr Asp Cys 1. 5 1O
<210s, SEQ ID NO 48 &211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 48 Ser Pro Ile Ser Thr Gly Gln Pro Thr Asn Glin Ser Met Asp Asp Thr 1. 5 1O 15 Arg Glu Asp Ile Tyr Val Asn Tyr Pro Thr Phe Ser Arg 2O 25
<210s, SEQ ID NO 49 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 49 Asp Ser Ser Cys Gly Thr Gly Tyr Glu Lieu. Thr Glu Asp Asn Ser Cys 1. 5 1O 15 Lys
<210s, SEQ ID NO 50 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 50 Lieu. Glu Gly Glu Val Thr Pro Asn Ser Leu Ser Thr Ser Tyr Lys 1. 5 1O 15
<210s, SEQ ID NO 51 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 51 Ser Arg Ser Ala Pro Pro Asn Lieu. Trp Ala Ala Glin Arg Tyr Gly Arg 1. 5 1O 15 Glu Lieu. Arg Arg 2O US 2010/015 1483 A1 Jun. 17, 2010 61
- Continued
<210s, SEQ ID NO 52 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 52 Ser His Leu Met Ser Lieu. Tyr Ser Ala Cys Ser Ser Glu Val Pro His 1. 5 1O 15 Gly Pro Val Asp Glin Llys 2O
<210s, SEQ ID NO 53 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 53 Ala Gly Gly Ser His Glin Glu Gln Pro Pro Tyr Pro Ser Tyr Asn Ser 1. 5 1O 15 Asn Tyr Trp Asn. Ser Thr Ala Arg 2O
<210s, SEQ ID NO 54 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 54 Ser Gly Tyr Gly Pro Ser Asp Gly Pro Ser Tyr Gly Arg 1. 5 1O
<210s, SEQ ID NO 55 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 55 Ser Val Pro Glin Ser Gly Pro Thr Val Arg Pro Glin Glu Asp Ala Trp 1. 5 1O 15 Ala Ser Pro Gly Ala Tyr Gly Met Gly Gly Arg 2O 25
<210s, SEQ ID NO 56 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 56 Asp Ser Ser Tyr Pro Tyr Ser Glin Ser Asp Glin Ser Met Asn Arg 1. 5 1O 15
<210s, SEQ ID NO 57 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 57 Ser Gly Tyr Gly Pro Ser Asp Gly Pro Ser Tyr Gly Arg 1. 5 1O US 2010/015 1483 A1 Jun. 17, 2010 62
- Continued <210s, SEQ ID NO 58 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 58 Gly Ala Gly Ala Phe Gly Tyr Phe Glu Val Thr His Asp Ile Thr Lys 1. 5 1O 15
<210s, SEQ ID NO 59 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 59 Ala Lys Tyr Asp Thr Pro Tyr Ile Ile Trp Arg 1. 5 1O
<210s, SEQ ID NO 60 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 60 Thr Pro Val Lieu. Phe Asp Ile Tyr Glu Ile Lys Glu Ala Ile Llys 1. 5 1O 15
<210s, SEQ ID NO 61 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 61 Asp Ala Glin Glu Lieu. Tyr Ala Ala Gly Glu Asn Arg 1. 5 1O
<210s, SEQ ID NO 62 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 62 Asp Lieu. Tyr Asp Ala Gly Val Lys Arg 1. 5
<210s, SEQ ID NO 63 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 63 Ser Ile Pro Ala Tyr Lieu Ala Glu Thir Lieu. Tyr Tyr Ala Met Lys 1. 5 1O 15
<210s, SEQ ID NO 64 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 64 Asn Phe Ala Thr Ser Lieu. Tyr Ser Met Ile Llys 1. 5 1O US 2010/015 1483 A1 Jun. 17, 2010 63
- Continued
<210s, SEQ ID NO 65 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 65 Asn Llys Pro Lieu. Phe Phe Ala Asp Llys Lieu. Tyr Lys 1. 5 1O
<210s, SEQ ID NO 66 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 66 Lieu. Ile Val Gly Lieu Met Arg Pro Pro Ala Tyr Cys Asp Ala Lys 1. 5 1O 15
<210s, SEQ ID NO 67 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 67 Ser Gly Tyr Ile Asp Glu. His Glu Lieu. Asp Ala Lieu Lleu Lys Asp Lieu. 1. 5 1O 15 Tyr Glu Lys
<210s, SEQ ID NO 68 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 68 Phe Tyr Ala Leu Ser Ala Ser Phe Glu Pro Phe Ser Asn Lys 1. 5 1O
<210s, SEQ ID NO 69 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 69 Lieu. His Thr Lieu. Glu Glu Glu Lys Glu Glu Lieu Ala Glin Tyr Glin Lys 1. 5 1O 15 Trp Asp Llys
<210s, SEQ ID NO 70 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 7 O Lieu. Phe Tyr His Ile Val Asp Ser Asp Glu Val Ser Thr Lys 1. 5 1O
<210s, SEQ ID NO 71 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 2010/015 1483 A1 Jun. 17, 2010 64
- Continued <4 OOs, SEQUENCE: 71 Gly Ala Lieu. Thr Gly Gly Tyr Tyr Asp Thir Arg Llys 1. 5 1O
<210s, SEQ ID NO 72 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 72 Thr Pro Cys Asn Ala Gly Thr Phe Ser Glin Pro Glu Lys Val Tyr Thr 1. 5 1O 15 Lieu. Ser Val Ser Gly Asp Arg 2O
<210s, SEQ ID NO 73 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 73 Val Ala Val Glu Tyr Lieu. Asp Pro Ser Pro Glu Val Glin Lys 1. 5 1O
<210s, SEQ ID NO 74 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 74 Ala Ala Lieu. Tyr Phe Glin Arg 1. 5
<210s, SEQ ID NO 75 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 75 Lieu. Glu Tyr Phe Ser Cys Asp His Glin Glu Lieu. Lieu. Glin Arg 1. 5 1O
<210s, SEQ ID NO 76 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 76 Ile Ser His Ile Pro Glu Asn. Phe Asp Asp Tyr Val Asp Ile Asin Glu 1. 5 1O 15 Asp Glu Asp Cys Tyr Ser Asp Glu Arg 2O 25
<210s, SEQ ID NO 77 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 77 Ile Ser His Ile Pro Glu Asn. Phe Asp Asp Tyr Val Asp Ile Asin Glu 1. 5 1O 15 US 2010/015 1483 A1 Jun. 17, 2010 65
- Continued
Asp Glu Asp Cys Tyr Ser Asp Glu Arg 2O 25
<210s, SEQ ID NO 78 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 78 Glu Lys Asp Val Ser Glu Tyr Phe Tyr Glu Lys 1. 5 1O
<210s, SEQ ID NO 79 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 79 Asn Glin Tyr Glin Ala Lieu Lys Pro Arg 1. 5
<210s, SEQ ID NO 8O &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens < 4 OO > SEQUENCE: 8O Val Lieu. Thir Thr Gly Tyr Trp Pro Thr Glin Ser Ala Thr Pro Llys 1. 5 1O 15
<210s, SEQ ID NO 81 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 81 Val His Thr Val Glu Asp Tyr Glin Ala Ile Val Asp Ala Glu Trp Asn 1. 5 1O 15 Ile Lieu. Tyr Asp Llys Lieu. Glu Lys 2O
<210s, SEQ ID NO 82 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 82 Lys Arg Val Glu Asp Ala Tyr Ile Lieu. Thir Cys Asn Val Ser Lieu. Glu 1. 5 1O 15 Tyr Glu Lys
<210s, SEQ ID NO 83 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 83 Ser Lieu. Glu Tyr Glu Lys Thr Glu Val Asin Ser Gly 1. 5 1O US 2010/015 1483 A1 Jun. 17, 2010 66
- Continued <210s, SEQ ID NO 84 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 84 Glu Lys Llys Lieu. Tyr Ala Asn Met Phe Glu Arg 1. 5 1O
<210s, SEQ ID NO 85 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 85 Glu Ala Ala Gly Glu Gly Pro Ala Lieu. Tyr Glu Asp Pro Pro Asp Glin 1. 5 1O 15 Lys
<210s, SEQ ID NO 86 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 86 Ile Ser Met Glin Asp Pro Llys Met Glin Val Tyr Lys Asp Glu Glin Val 1. 5 1O 15 Val Val Ile Lys Asp Llys Tyr Pro Llys 2O 25
<210s, SEQ ID NO 87 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 87 Ala Arg Tyr His Trp Lieu Val Lieu Pro Trp Thir Ser Ile Ser Ser Leu 1. 5 1O 15 Lys
<210s, SEQ ID NO 88 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 88 Asn Asp Met Thr Tyr Asn Tyr Ala Asn Arg 1. 5 1O
<210s, SEQ ID NO 89 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 89 Asn Asp Met Thr Tyr Asn Tyr Ala Asn Arg 1. 5 1O
<210s, SEQ ID NO 90 &211s LENGTH: 22 212. TYPE: PRT US 2010/015 1483 A1 Jun. 17, 2010 67
- Continued <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 90 Thir Lieu Val Glin Asn. Asn. Cys Lieu. Thir Arg Pro Asn. Ile Tyr Lieu. Ile 1. 5 1O 15 Pro Asp Ile Asp Lieu Lys 2O
<210s, SEQ ID NO 91 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 91 Ser Ser Asn Ala Tyr Asp Pro Ser Glin Met Cys Ala Glu Lys 1. 5 1O
<210s, SEQ ID NO 92 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 92 Tyr Arg His Ile Llys Pro Val Ser Arg Asn. Ser Arg 1. 5 1O
<210s, SEQ ID NO 93 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 93 Thir Lys Llys Pro Asp Lieu. Glin Ile Tyr Glin Pro Gly Arg 1. 5 1O
<210s, SEQ ID NO 94 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 94 Phe Glin Asn Ser Asp Asn Pro Tyr Tyr Tyr Pro Arg 1. 5 1O
<210s, SEQ ID NO 95 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 95 Tyr Ala Ser Asn Lieu Pro Gly Ser Lieu. Lieu Lys Glu Gln Lys 1. 5 1O
<210s, SEQ ID NO 96 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 96 Val Asp Asn. Glu Ile Lieu. Asp Tyr Lys Asp Lieu Ala Ala Ile Pro Llys 1. 5 1O 15 US 2010/015 1483 A1 Jun. 17, 2010 68
- Continued
<210s, SEQ ID NO 97 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 97 Asn Gly Lieu. His Arg Pro Val Ser Thr Asp Phe Ala Glin Tyr Asn Ser 1. 5 1O 15 Tyr Gly Asp Val Ser Gly Gly Val Arg 2O 25
<210s, SEQ ID NO 98 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 98 Asp Tyr Glin Thr Lieu Pro Asp Gly His Met Pro Ala Met Arg 1. 5 1O
<210s, SEQ ID NO 99 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 99 Ile Val Glin Thr Tyr His Val Asn Met Ala Gly Thr Asn Pro Tyr Thr 1. 5 1O 15 Thir Ile Thr Pro Glin Glu Ile Asin Gly Lys 2O 25
<210s, SEQ ID NO 100 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 1.OO Ile Ser Ile Glu Met Asn Gly. Thir Lieu. Glu Asp Gln Lieu. Ser His Lieu. 1. 5 1O 15 Lys Glin Tyr Glu Arg 2O
<210s, SEQ ID NO 101 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 101 Ser Met Leu Glu Val Asn Tyr Pro Met Glu Asn Gly Ile Val Arg 1. 5 1O 15
<210s, SEQ ID NO 102 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 102 Asn Trp Asp Asp Met Lys His Lieu. Trp Asp Tyr Thr Phe Gly Pro Glu 1. 5 1O 15 Llys Lieu. Asn. Ile Asp Thr Arg US 2010/015 1483 A1 Jun. 17, 2010 69
- Continued
<210s, SEQ ID NO 103 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 103 Tyr Lieu. Arg Ala Glu Pro Glu Asp His Tyr Phe Leu Lleu. Thr Glu Pro 1. 5 1O 15 Pro Lieu. Asn Thr Pro Glu Asn Arg 2O
<210s, SEQ ID NO 104 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 104 Lieu Pro Ala Cys Val Val Asp Cys Gly Thr Gly Tyr Thr Lys 1. 5 1O
<210s, SEQ ID NO 105 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 105 Glu Thir Lys Asp Thr Asp Ile Val Asp Glu Ala Ile Tyr Tyr Phe Lys 1. 5 1O 15
<210s, SEQ ID NO 106 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 106 Ile Glin Val His Tyr Tyr Glu Asp Gly Asn Val Glin Lieu Val Ser His 1. 5 1O 15 Lys
<210s, SEQ ID NO 107 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 107 Pro Lieu. Asp His Ala Glin Pro Pro Ser Ser Lieu Val Ile Asp Llys Glu 1. 5 1O 15 Ser Glu Val Tyr Lys 2O
<210s, SEQ ID NO 108 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 108 Met Ile Tyr Ala Ser Ser Lys Asp Ala Ile Llys 1. 5 1O US 2010/015 1483 A1 Jun. 17, 2010 70
- Continued
<210s, SEQ ID NO 109 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 109 Tyr Phe Glu Ile Thr Asp Glu Ser Pro Tyr Val His Tyr Lieu. Asn Thr 1. 5 1O 15 Phe Ser Ser Lys Glu Pro Glin Arg 2O
<210s, SEQ ID NO 110 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 110 Arg Glu Glin Arg Tyr Glin Glu Glin Gly Gly Glu Ala Ser Pro Glin Arg 1. 5 1O 15
<210s, SEQ ID NO 111 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 111 Met Ile Tyr Ala Ser Ser Lys Asp Ala Ile Llys 1. 5 1O
<210s, SEQ ID NO 112 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 112 Thir Lieu. Asp Asp Ile Val Gly Arg Tyr Glu Asp Lieu. Ser Lys 1. 5 1O
<210s, SEQ ID NO 113 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 113 Ile Phe Glu Tyr Glu Thr Glin Arg 1. 5
<210s, SEQ ID NO 114 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 114 Lieu. Ser Pro Pro Ser Ser Ser Ala Ala Ser Ser Tyr Ser Phe Ser Asp 1. 5 1O 15 Lieu. Asn. Ser Thr Arg 2O
<210s, SEQ ID NO 115 &211s LENGTH: 15 212. TYPE: PRT US 2010/015 1483 A1 Jun. 17, 2010 71
- Continued <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 115 Val Ser Lieu. Lieu. Asp Asp Thr Val Tyr Glu. CyS Val Val Glu Lys 1. 5 1O 15
<210s, SEQ ID NO 116 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 116 Thir Ile Thr Tyr Glu Ala Ala Glin Thr Val Lys 1. 5 1O
<210s, SEQ ID NO 117 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 117 Tyr Ser Gly Lys Thr Glu Tyr Glin Thr Thr Lys 1. 5 1O
<210s, SEQ ID NO 118 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 118 Ala Ser Val Met Val Tyr Asp Asp Thir Ser Lys Llys 1. 5 1O
<210s, SEQ ID NO 119 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 119 Ile Asin Ile Tyr His Asn Thr Ala Ser Asn Thr Phe Arg 1. 5 1O
<210s, SEQ ID NO 120 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 120 Glu Gly Ile Leu Ser Asp Glu Ile Tyr Cys Pro Pro Glu Thir Ala Val 1. 5 1O 15 Lieu. Lieu. Gly Ser Tyr Ala Val Glin Ala Lys 2O 25
<210s, SEQ ID NO 121 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 121 Thr His Thr Gly Lys Tyr Trp Thr Lieu. Thir Ala Thr Gly Gly Val Glin 1. 5 1O 15 US 2010/015 1483 A1 Jun. 17, 2010 72
- Continued Ser Thr Ala Ser Ser Lys 2O
<210s, SEQ ID NO 122 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 122 Asn Ala Ser Cys Tyr Phe Asp Ile Glu Trp Arg 1. 5 1O
<210s, SEQ ID NO 123 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 123 Cys Ser Asn Glu Lys Gly Tyr Phe Ala Val Thr Glu Lys 1. 5 1O
<210s, SEQ ID NO 124 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 124 Val Gly Lieu. Gly Lieu. Gly Tyr Lieu. Glu Lieu Pro Glin Ile Asn Tyr Lys 1. 5 1O 15
<210s, SEQ ID NO 125 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 125 Asn Ser Glu Gly Trp Glu Glin Asn Gly Lieu. Tyr Glu Phe Phe Arg 1. 5 1O 15
<210s, SEQ ID NO 126 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 126 Gly Val Gly Val Ala Lieu. Asp Asp Pro Tyr Glu Asn Tyr Arg Arg 1. 5 1O 15
<210s, SEQ ID NO 127 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 127 Gly Val Gly Val Ala Lieu. Asp Asp Pro Tyr Glu Asn Tyr Arg Arg 1. 5 1O 15
<210s, SEQ ID NO 128 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 128 US 2010/015 1483 A1 Jun. 17, 2010 73
- Continued
His Tyr Lieu. Ser Thr Lys Asn Gly Ala Gly Lieu. Ser Lys 1. 5 1O
<210s, SEQ ID NO 129 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 129 Asp Asin Phe Trp Glu Met Gly Asp Thr Gly Pro Cys Gly Pro Cys Ser 1. 5 1O 15 Glu Ile His Tyr Asp Arg 2O
<210s, SEQ ID NO 130 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 130 Ala Val Tyr Thr Glin Asp Cys Pro Lieu Ala Ala Ala Lys 1. 5 1O
<210s, SEQ ID NO 131 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 131 Thir Arg Ala Asp Llys Glu Gly Asp Tyr Tyr Val Lieu. Asn Gly Ser Lys 1. 5 1O 15
<210s, SEQ ID NO 132 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 132 Glin Ala Ser Val Gly Ala Gly Ile Pro Tyr Ser Val Pro Ala Trp Ser 1. 5 1O 15 Cys Glin Met Ile Cys Gly Ser Gly Lieu Lys 2O 25
<210s, SEQ ID NO 133 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 133 Lieu Lys Glin Gly Lieu. Tyr Arg 1. 5
<210s, SEQ ID NO 134 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 134 Glu Ala Tyr Pro Glu Glu Ala Tyr Ile Ala Asp Lieu. Asp Ala Lys 1. 5 1O 15 US 2010/015 1483 A1 Jun. 17, 2010 74
- Continued
<210s, SEQ ID NO 135 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 135 Glu Ala Tyr Pro Glu Glu Ala Tyr Ile Ala Asp Lieu. Asp Ala Lys 1. 5 1O 15
<210s, SEQ ID NO 136 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 136 Arg Gly Gly Pro Asn Tyr Glin Glu Gly Lieu. Arg 1. 5 1O
<210s, SEQ ID NO 137 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 137 Lieu. Thir Tyr Gly Thr Met Val Phe Val Arg 1. 5 1O
<210s, SEQ ID NO 138 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 138 Tyr Asp Gly Asn Val Tyr Glu Asn Lieu. Phe Glu Trp Ala Lys 1. 5 1O
<210s, SEQ ID NO 139 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 139 Thr Glu Gly Gly Tyr Tyr Glin Ile Thr Gly Arg 1. 5 1O
<210s, SEQ ID NO 140 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 140 Asp Glin Asp Gly Tyr Tyr Trp Ile Thr Gly Arg 1. 5 1O
<210s, SEQ ID NO 141 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 141 Asp Glin Asp Gly Tyr Tyr Trp Ile Thr Gly Arg 1. 5 1O US 2010/015 1483 A1 Jun. 17, 2010 75
- Continued
<210s, SEQ ID NO 142 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 142 Val Glu Lieu. His Val His Lieu. Asp Gly Ser Ile Llys Pro Glu Thir Ile 1. 5 1O 15 Lieu. Tyr Tyr Gly Arg 2O
<210s, SEQ ID NO 143 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 143 Ser Thr Lieu. Asp Thr Asp Tyr Gln Met Thr Lys 1. 5 1O
<210s, SEQ ID NO 144 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens < 4 OO > SEQUENCE: 144 Glu Lieu. Lieu. Asp Lieu. Lieu. Tyr Lys Ala Tyr Gly Met Pro Pro Ser Ala 1. 5 1O 15 Ser Ala Gly Glin Asn Lieu. 2O
<210s, SEQ ID NO 145 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 145 Phe Asp Tyr Tyr Met Pro Ala Ile Ala Gly Cys Arg 1. 5 1O
<210s, SEQ ID NO 146 &211s LENGTH: 37 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 146 Ser His Met His Lieu Ala Ser Ala Phe Ala Gly Ile Gly Phe Gly Asn 1. 5 1O 15 Ala Gly Val His Lieu. Cys His Gly Met Ser Tyr Pro Ile Ser Gly Lieu. 2O 25 3O Val Lys Met Tyr Lys 35
<210s, SEQ ID NO 147 &211s LENGTH: 37 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 147 US 2010/015 1483 A1 Jun. 17, 2010 76
- Continued Ser His Met His Lieu Ala Ser Ala Phe Ala Gly Ile Gly Phe Gly Asn 1. 5 1O 15 Ala Gly Val His Lieu. Cys His Gly Met Ser Tyr Pro Ile Ser Gly Lieu. 2O 25 3O Val Lys Met Tyr Lys 35
<210s, SEQ ID NO 148 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 148 Lys Gly Ile Gly Pro Val Tyr Ser Ser Lys 1. 5 1O
<210s, SEQ ID NO 149 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 149 Asp Gly Val Tyr Phe Lieu. Tyr Glu Ala Lieu. His Gly Pro Pro Llys Llys 1. 5 1O 15
<210 SEQ ID NO 150 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 150 Asp Gly Val Tyr Phe Lieu. Tyr Glu Ala Lieu. His Gly Pro Pro Llys Llys 1. 5 1O 15
<210s, SEQ ID NO 151 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 151 Gly Lieu. Glu Val Thr Ala Tyr Ser Pro Lieu. Gly Ser Ser Asp Arg 1. 5 1O 15
<210s, SEQ ID NO 152 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 152 His Ile Asp Cys Ala Ala Ile Tyr Gly Asn. Glu Pro Glu Ile Gly Glu 1. 5 1O 15 Ala Lieu Lys Glu Asp Val Gly Pro Gly Lys 2O 25
<210s, SEQ ID NO 153 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 153 Ala Asn Asn Thr Phe Tyr Gly Lieu Ser Ala Gly Val Phe Thr Lys US 2010/015 1483 A1 Jun. 17, 2010 77
- Continued
1. 5 1O 15
<210s, SEQ ID NO 154 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 154 Glu Lieu. Gly Glu Tyr Gly Phe His Glu Tyr Thr Glu Val Lys 1. 5 1O
<210s, SEQ ID NO 155 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 155 Tyr Tyr Ala Gly Trp Ala Asp Lys Ile His Gly Met Thr Ile Pro Val 1. 5 1O 15 Asp Gly Asp Tyr Phe Thr Phe Thr Arg 2O 25
<210s, SEQ ID NO 156 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 156 Ser Ala Pro Tyr Glu Phe Pro Glu Glu Ser Pro Ile Glu Glin Leu Glu 1. 5 1O 15 Glu Arg
<210s, SEQ ID NO 157 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 157 Ile Asp Tyr Ile Ala Gly Lieu. Asp Ser Arg 1. 5 1O
<210s, SEQ ID NO 158 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 158 Tyr Tyr Ala Asp Gly Glu Asp Ala Tyr Ala Met Lys Arg 1. 5 1O
<210s, SEQ ID NO 159 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 159 Phe Glu Lieu Ser Cys Tyr Ser Leu Ala Pro Glin Ile Lys 1. 5 1O
<210s, SEQ ID NO 160 &211s LENGTH: 19