Quantitative profiling of signaling pathways using immunoaffinity purification and LC-MS/MS

Matthew P. Stokes, Jian Min Ren; Kimberly A. Lee; Xiaoying Jia; Jeffrey C. Silva Technology, Danvers MA USA 01923

MouSE TISSuE STuDy Introduction Multipathway Akt/PI3K Pathway 0 1 Monitoring more than 1,000 phosphorylation sites on over 400 critical signaling . In-depth analysis of PI3K/Akt and associated signaling pathways. Figure 5: Akt/PI3K Pathway mouse tissue profiling. Proteomic analysis of post-translationally modified peptides has traditionally employed methods that broadly sample the proteome but are unfocused with Hierarchical clustering of relative intensities for mouse liver,

www.cellsignal.com www.cellsignal.com Figure 4: Akt/PI3K Pathway - www.cellsignal.com www.cellsignal.com www.cellsignal.com SAPK/JNK Signaling Cascades Cell Cycle Control: G2/M DNA Damage Checkpoint MAPK/Erk in Growth and Differentiation respect to the sites identified. We have developed a novel immunoaffinity method for identification and quantitation of post-translationally modified pep- Regulation of Microtubule Dynamics Jak/Stat Signaling: IL-6 Receptor Family

IR UV ® RTKs Integrins FasL, UV IL-6 EGF Neurotrophins Cellular Stress, Inflammatory Cytokines brain, and embryo. Each row represents a different validated Wnt Ion Channels FANCD2 2+ RTKs γ Radiation Critically Short RTK [Ca ] Telomeres

P Growth Factors, UV Oxidative

Stress G12/13-coupled POT1 TRF2 DNA R IPA - Molecular Mechanisms of Cancer L Pax RTKs Receptors Repair SOS Ras SOS Tal PI3K Rap1 gp130 Rad50 IRS

α Shc

G q/o GRB2 CAS cdc42 FRS2 FAK PLCγ DNA Repair NBS1 Erk SHP-2 EGFR Spry Fyn C3G cdc42 Rho Mre11 GRB2 RACK1 CRK Rac SHP-1 PI3K Ras Rac cdc42 Akt PIP3 PTEN ROCK Ras SOCS3 Jak Sos RIP Shc EPAC TRADD ATM/ (MRN) Spred1 GRB2 2+ TAOK Daxx Dvl p120 TRAF2 Par6 aPKC [Ca ] GRB2 α ras-GAP Src G 12/13 ATR MAPKAPK Jak Shc

CrkL interaction map of validated targets. SOCS HIPK2 DNA-PK Crk βγ Par3 Actin Filaments PYK2 PAK Rac [cAMP] PI3K G Caffeine Spred BRCA1 tides from proteins that reside in the same signaling pathway or pathways, allowing a global view of pathway activation from a single LC-MS/MS run. TESK Par1 MARK2 PI3Kγ GSK-3β Stat3 Src PKA rpS6 TCPβ GCKs MARK E-Ras Raf PI3K c-Raf B-Raf Rac1 Stat3 PKC MEKK1/4 Cofilin Stat c-Raf B-Raf 14-3-3 eIF4B MLK2/3 BRCA1 MAPKKKs : DLK Tau CRMP2 MEK1 Heterodimer PD98059 c-Abl Rad51 Akt/PI3K pathway peptide. The maximum intensity across the PIP MAP1b Filamin A TpI-2 LIMK 3 Rho MEK Renewal P14 U0126 MDM4 Tpl2/Cot1 TAO1/2 PI3K of ES Cells MP1 PP1/ Nuclear Export, Ion Channels, IκBα HPK ASK1 Ubiquitination MDM2 p53 Chk2 Rad52 Receptors MEK1/2 PP2At TPPP LL5β Akt IMP Chk1 mDIA MAP1b PKA PAK Stat BI-D1870 Erk Mcl-1 Erk1 MAPKKKs cPLA2 C-TAK1 MLKs ICIS Cdk1 Akt KSR CaMK Erk eEF2K GSK-3 POSH Nucleolar Arf APC Plus End 1/2 p19 cdc25A 14-3-3 Tumor-like SOS Sequestration Proteins Crosstalk Bim or mDIA EB1 XMAP215 Properties Erk DAPK METTL1 MDM2 Aurora B mTOR Tumor Cells Late Endosome Control MNK1/2 JIP1,2,3 Apoptosis p53 Stabilization CLASP of ES Cells MKK4/7 Stathmin Apoptosis p90RSK p300/ cdc25A/C EB1 MCAK MKP-3 Erk1/2 BAD nNos TRIP12 p53 Nuclear Exclusion CLIP Erk1/2 p90RSK PCAF cdc25 Six different antibody reagents have been prepared that focus on diverse signaling areas: Ser/Thr Kinase activity, Tyr Kinase activity, PI3K/Akt signaling, CLIP TSC2 Ubiquitination + end Growth MT Stability PEA-15 Cytoplasmic Anchoring TAK1 JNK1/2/3 cdc25A/B Promoting Cytoplasm Cell Adhesion +/- end 14-3-3 MT IRS-1 AurA Polymerization Destabilizing sm Nucleus Cytopla c-Abl 14-3-3 14-3-3 Bax Bora γ CLIP Erk PIAS cdc25 PPAR M3/6 MKPs PLK1 Delivery to MT Catastrophe Erk Feedback σ Cip1 three tissues was set to 1 and the other two intensities normal- TopoII BRCA1 14-3-3 Reprimo GADD45 p21 CDK7 SCF MT Plus Ends Inhibition Nucleus Bax CLIP CLASP SUMO p90RSK β 14-3-3 Minus End C/EBP BUB1 p27 KIP1 Myt1 Plus End NF-κB p90RSK Mitochondrial Src EB1 TF Stat3 PTP MKP-1/2 Erk1/2 MSK1/2 m Translocation/ AP-1 as Apoptosis Nuclear etc. Cytopl NO JNK Exclusion c-Abl APC TF Stat3 SCF “Dynamic Accessory CIS, SOCS, Mcl-1, APPs, TIMP-1, JNK Tiam1 ISRE/GAS Nucleus c-Abl Microtubules” RhoGEF TF Motif Pim-1, c-Myc, cytokines, TFs, etc. ER Ets Elk-1 TIF1A Fox03 CREB ATF1 ER81 SRF c-Fos ERα Nur77 MITF C/EBPβ Wee1 Ubiquitination mDIA1 Rho c-Myc/ cdc2 Stat1/3 Pax6 c-Fos Histone HMGN1 ETV1 TIF1A ATF4 Myt1 Mad1 Ran BP3 Cell Cycle/DNA Damage signaling, Apoptotic/Autophagolytic pathways, and a Multipathway reagent for detection of critical signaling proteins across many Rac1 Trio Rho N-Myc H3 α M G Focal Adhesions Rho c-Jun ATF-2 Elk-1 Smad4 p53 NFAT4 NFATc1 Stat3 HSF1 ASE Cyclin B -PH UBF -PH AS G2 E Growth Transcription Differentiation Transcription ized to the max. Blue indicates higher intensity. Selected kinase Survival different pathways. Reagents were validated using human and mouse samples with a variety of treatments (inhibitors, growth factors, etc.). This technology Apoptosis © 2002 – 2010 Cell Signaling Technology, Inc. SAPK/JNK Signaling Cascades • created January 2002 • revised November 2010 © 2002 – 2010 Cell Signaling Technology, Inc. Cell Cycle Control: G2/M DNA Damage Checkpoint • created November 2002 • revised November 2010 © 2003 – 2010 Cell Signaling Technology, Inc. MAPK/Erk in Growth and Differentiation • created January 2003 • revised November 2010 © 2008 – 2010 Cell Signaling Technology, Inc. Regulation of Microtubule Dynamics • created September 2008 • revised November 2010 © 2002 – 2010 Cell Signaling Technology, Inc. Jak/Stat Signaling: IL-6 Receptor Family • created November 2002 • revised November 2010 www.cellsignal.com www.cellsignal.com www.cellsignal.com www.cellsignal.com peptides are shown in detail with accompanying western blots. www.cellsignal.com ErbB/HER Signaling NF-κB Signaling Toll-like Receptor Signaling Insulin Receptor Signaling PI3 Kinase Akt Signaling

NRG2 NRG3 HB-EGF TGF-α LPS Flagellin Triacyl Insulin Receptor NRG1 EGF Growth Factors: Lipopeptide NRG4 EGF EPG TNF MD-2

NRG4 AREG IL-1 BMP, EGF, HGH, Diacyl SNARE TNF

E

2/ R HE

E H

E H GF

HER4/ErbB E Lipopeptide Glucose HER2/ErbB GFR/ErbB1 β Complex H CEL. LPS, CpG, Insulin, NGF, TGF-α

HER2/ErbB

R R ER LT, CD40L, BCR

ssRNA, dsRNA CD14 GLUT4

3 bB

2/Er Ag-MHC E

R/ Cytokine

r /E

r /E 4 EGFR E IL-1R BAFF/BLys TNFR TLR5 TLR5 Ag / Ag Receptor rbB p85

HER4/Er Er TLR2 TLR1

1 B

rb TLR4 TLR4

3 B b Cav is broadly applicable to any experimental system in which quantitative profiling of specific critical signaling molecules is desirable. R1 TLRs Flotillin RTK CD19 b

TACE TCR GF-Rs PTEN PI3K Gab1 2 B 2 β 4 2 LT R, TLR2 TLR6 p110 SOCS3 4 MyD88

Synip CAP TNF 2 Cbl

MyD88 IRS TRIF bB1 BCR PIP Integrin TIRAP 3 mIg CD40, Shc α/β GPCR TIRAP ub MyD88 TRAM α β γ dsRNA or APS / b -Sec

MIG6 TRADD mIg

MyD88 Ras TRIAD3A B4 Dok GEP100 IRAK1/4 ubc5 BR3 SHIP SHP-2 PTP1B 5'-triphosphate RNA MyD88 Akt TIRAP TSAd For detailed signaling, TRAF2/5 GLUT4

Crkll unique sites on PSD95 Shc GAB2 GRB2 Translocation EHD1 Nck CXCR4 TNS3 TNS4 For detailed signaling, see TLR Pathway. Pellino SOCS1 TC10 C3G BCAP SHP-2 A20 RIP TRAF2/6 Crk Jak1

ATP RIG-I IRS-1 PI3K Calcineurin ST2L PI3K BDP1 GRB2 TRAF3 EHBP1 see BCR Pathway. CIP4/2 PKCλ/ζ Fyn Syk ITCH TAX1BP1 ub CYLD SOS IRS-1 Lyn Ubc13 PI3K PI3K Invasion PI3K FAK Gab1 ARF6 For detailed signaling, TRAF6 α 296 Metastasis PDK1 G Vav1 ASIP PI3K Gβγ PI3K see TCR Pathway. UEV1A PDK1 mTORC2 Gab2 GTP FAK c-Cbl ub TAB2/3 IRAK-4 θ Increase GRB2 PKC GRB10 FoxO1 Tumor TAB1/2 TAK1 Jnk ILK Gab1 Cell Motility Akt IKK Paxillin mTORC2 Rac1 Crk Yes Invasion MAVS GLUT4 PKCλ/ζ Akt Stress: ROIs, TOLLIP IRAK-M PIP PTEN PIP TAK1 γ/ Cot NIK vesicle Ras 3 3 PTEN PP2A PHLPP CTMP Shc Bad Akt p130 Cas Nck1 Spry UV, metals, IKK FFA SOS Syk NEMO PP2A S6 ischemia, shear P

Cell Survival c-Abl IRAK-1 IRAK-2 FADD Casp-8 NO r ub ub Tax LKB1 o

TRAF3 t PDK1 PDK1 α e Src ELKS IKKβ IKK ub β-TrCP ub RIP1 iNOS Membrane Membrane TRAF6 AS160 i Organization Muc1 Caveolin GLUT4 n Recruitment Recruitment PLC γ/ Akt

mTOR PAK1 IKK Exocytosis S LaminA of Nuclear Memo GSK Ras A20 and Activation and Activation Jak c-Raf y p27 NEMO α TBC1D1 n p70 S6K Proteins

ub α IKK t h ub IKK e PDK1 AMPK Tpl2 r

Apoptosis e β β-TrCP m NF-κB e

Paxillin o s

XIAP MDM2 -Cat s ROS h t o TRAF6 i

Calmodulin s Cell Cell d ε PDE3B Pathway IKK TSC2 α O n Anti-viral ECSIT MEKK-1 IKK Migration Cycle Raf MEK2/3 JNK E Ubc13 PDCD4 p70S6K MEKK1 ub ub Compounds, TSC1 Cardiovascular E-cadherin TBK1 UEV1A Bad TSC2 p53 cdc42 Proteasomal p65/ ssRNA 4E- eNOS Homeostasis WT1 Caspase CK2 RelA/cRel IκBα/β/ε IκBα RelA MEK1/2 mTORC1 TSC1 Cell Degradation RelB Inhibits BP1 Adhesion MEK MEK5 NF-κB2 NF-κB2 GSK-3 PRAS40 Rheb JNK p38 NF-κB1 ub IKKγ/ SGK Autophagy Synaptic α/β/ε p52 p100 [cAMP] GABAA

HER2/ICD p50 CaMK PKC UV IKK Genotoxic NEMO ATG13 Signaling Importin β1 Stress CpG dsRNA β α PRAS40 MAPK PKCζ IKK IKK TAK1 MKK 3/6 Akt e ERK5 β mTORC1 Huntingtin Blocks c Cell Cytoskeletal NAP1 NAK GSK-3 Apoptosis XIAP n e Nup358 Synthesis Survival Motility Regulation Stat TAB1/2 Aggregation i Apoptosis Nuclear-cytoplasmic eIF2B PKA Inhibits Apoptosis c Proliferation Promotes s shuttling of non- MSK1 Ataxin-1 o RSK1 Proteasomal 4E-BP1 r MEF2 phosphorylated forms p70 Erk MDM2 Neuroprotection u

Processing TLR9 TLR9 TLR7 TLR8 TLR3 TLR3 FoxO1 Cellular Proliferation, PP1 GS e p65/ S6K N lasm DNA Repair TRIF ATP-citrate PFKFB2 Aggregation Cytop RelA γ MKK 4/7 eIF4E Bad GSK-3

MyD88 MyD88 MyD88 HSL ub K63- PKA C CK2 IKK / lyase p53 Neurodegeneration ub CYLD κ NEMO 14-3-3 ucleus cdc2 PLNA DNA-PK NF- B IKKα ub p38 MAPK ENaC Glycogen Bim N ub K48-ubiquitin IKKα κ α Bcl-3 p50/52 I B Synthesis PIP5K AS160 RelB Fatty Acid Glycolysis IκBζ NF-κB2 p65/RelA Protein Lipolysis IRF-3 NF-κB Synthesis Synthesis p52 IRF-7 Proteasomal JNK Sodium Bcl-2

H Degradation Transport D

IC IRF-3 4 ER m IRF-7 Cytoplas S

H TAB2 lasm u

ICD r

4 R E PCAF SUMO Cytop v Bax Glycogen γ iv Wee1 Myt1 p27 Kip Glucose m Stat IKK / SGK Akt Erk a Synthase s N-CoR E2F Stat CREB Fos Jun Elk Stat MEF2 Nucleus NF-κB IKKα l li EGFR/ErbB-1 EGFR/ErbB-1 p65/ ac CBP/ NEMO IKKα m Transport o Cytoplas us b p50/52 RelA p300 Nucle Apoptosis ta IκBα/ε p21 Cip e RelA/cRel κ M NF- B NF-κB κ RelB Transcription Cytosolic e SUMO NF- B2 s Glycogen s NF-κB1 p50/52 p50/52 p52 Nucleu Factors FoxO3A FoxO4 FoxO1 Sequestration co Synthesis lu p50 Cyclin D1 G Cell Proliferation, , Cell Growth/Differentiation, Growth Differentiation H3 Genome Instability, Tumor Progression Cell Shape, Chemotaxis HDAC proteins γ PIAS ATM PARP1 Apoptosis Transcription Transcription Growth Survival, Proliferation, Inflammation, Immune Regulation Lymphogenesis, B Cell Maturation Cell Cycle Inflammation, Immune Regulation, Survival, Proliferation mTORC1 mTORC2 105 Proliferation ® GβL Raptor Sin1 PRR5 mTORC1 mTORC2 Rictor β mTOR G L Sin1 PRR5 © 2004 – 2010 Cell Signaling Technology, Inc. ErbB/HER Signaling Network • created October 2004 • revised November 2010 DEPTOR GβL Raptor mTOR DEPTOR mTOR Rictor GβL DEPTOR mTOR DEPTOR © 2003 – 2011 Cell Signaling Technology, Inc. NF-κB Signaling • created January 2003 • revised August 2011 © 2006 – 2010 Cell Signaling Technology, Inc. Toll-Like Receptor Signaling • created May 2006 • revised November 2010 © 2006 – 2011 Cell Signaling Technology, Inc. Insulin Receptor Signaling • created May 2006 • revised August 2011 PTMScan Direct Method © 2003 – 2010 Cell Signaling Technology, Inc. PI3K / Akt Signaling • created January 2003 • revised November 2010

www.cellsignal.com www.cellsignal.com Figure 1: PTMScan® Direct Reagent validation strategy. PTMScan® Direct is a published method (Stokes et al. 2012) adapted from the original PI3 Kinase Akt Signaling MAPK/Erk in Growth and Differentiation RTKs Integrins

BCR Ion Channels RTKs Cytokine [Ca2+] ® Receptor Ag RTK CD19 PhosphoScan method (Rush et al. 2005) developed at Cell Signaling Technology. PTMScan Direct Reagents are validated using mixtures of Pax SOS Ras SOS Tal Integrin mIg α/βα β GPCR Rap1 IRS

/ Shc

mIg GRB2 CAS PLCγ FRS2 FAK Spry Fyn C3G GRB2 RACK1 CRK PI3K BCAP EPAC Jak1 2+ PI3K PI3K [Ca ] Src Syk pervanadate-treated human cancer cell lines digested with trypsin. Peptides are desalted over C18 columns and immunoprecipitated with either IRS-1 Lyn PI3K FAK Gab1 PYK2 PAK Rac [cAMP] Gα Spred PI3K Gβγ PI3K Gab2 GTP rpS6 TCPβ ILK c-Raf PKA B-Raf Paxillin Akt mTORC2 PKC PIP3 PTEN PIP3 PTEN PP2A PHLPP CTMP c-Raf B-Raf 14-3-3 eIF4B S6 PD98059 the PTMScan Direct reagent or empty Protein G beads. Immunoprecipitated peptide mixtures are analyzed by LC-MS/MS and relative quantitation P MEK1 Heterodimer

r Filamin A o P14 U0126 t PDK1 PDK1

e Membrane Membrane Tpl2/Cot1 i Organization n Recruitment Recruitment MP1 PP1/

Ion Channels, κ α

S LaminA of Nuclear I B and Activation and Activation PP2At y Receptors MEK1/2

n p70 S6K Proteins IMP t

h BI-D1870

Tpl2 r e

NF-κB e Erk1 s

h cPLA2 t i C-TAK1 KSR s Pathway is performed. Validated peptides meet several strict criteria: they are targeted by the reagent or are homologous to a target, they must pass score IKKα O eEF2K GSK-3 HeLa -/+ uV DAMAgE PDCD4 1/2 Cardiovascular SOS TSC2 Translation Bim 4E- eNOS Homeostasis DAPK METTL1 mTORC1 TSC1 Late Endosome Control MNK1/2 Inhibits BP1 Autophagy Synaptic MKP-3 p90RSK BAD nNos GABAA Erk1/2 Signaling Erk1/2 p90RSK ATG13 Cytoplasmic Anchoring TSC2 PRAS40 PEA-15 Akt e filtering (Lundgren et al. 2009) and signal intensity thresholds, and they must be present in higher abundance in the PTMScan Direct reagent Huntingtin c Cell Adhesion Blocks DNA Damage/Cell Cycle XIAP n e Aggregation i Inhibits Apoptosis c Promotes s Cytoplasm Ataxin-1 o r PPARγ u cdc25 FoxO1 MDM2 Neuroprotection e PFKFB2 N Nucleus GSK-3 Aggregation p53 Bad Neurodegeneration immunoprecipitation than the empty Protein G beads immunoprecipitation (Stokes et al. 2012). 14-3-3 BUB1 Bim p90RSK p27 KIP1 AS160 MKP-1/2 Erk1/2 MSK1/2 PIP5K Glycolysis A comprehensive view of the DNA damage response. Bcl-2

S u ER Ets Elk-1 TIF1A Fox03 CREB ATF1 ER81 SRF c-Fos ERα Nur77 MITF C/EBPβ rv Bax Glycogen iv Wee1 Myt1 p27 Kip Glucose m a Synthase s l li Transport o c-Myc/ b Stat1/3 N-Myc Pax6 c-Fos Histone HMGN1 ETV1 TIF1A ATF4 Myt1 Mad1 Ran BP3 ta H3 Apoptosis p21 Cip e M Cytosolic e Glycogen s UBF Sequestration co Synthesis lu Cyclin D1 G Figure 6: DNA Damage/Cell Cycle - Transcription

Cell Cycle

Proliferation mTORC1 mTORC2 interaction map of validated targets.

GβL Raptor Sin1 PRR5 mTOR Rictor GβL DEPTOR mTOR DEPTOR

© 2003 – 2010 Cell Signaling Technology, Inc. MAPK/Erk in Growth and Differentiation • created January 2003 • revised November 2010 © 2003 – 2010 Cell Signaling Technology, Inc. PI3K / Akt Signaling • created January 2003 • revised November 2010 Figure 2: Multipathway coverage of selected signaling pathways. 263 unique sites on

Figure 7: DNA Damage/Cell Cycle profiling of response to Ser/Thr Kinases and Tyr Kinases 137 proteins uV damage. Hela cells were untreated or treated with 500 mJ/cm2 Profiling sites of activation and inhibition on protein kinases. UV light and harvested at 2 hr post treatment. Fold changes were calculated from chromatographic peak heights/areas. Green cells indicate peptides more abundant with UV damage; Red cells indicate peptides less abundant. Selected DNA damage response proteins are shown in detail with accompanying western blots. 1056 unique sites on 250 kinases Apoptosis/Autophagy HeLa -/+ uV DAMAgE Figure 9: Apoptosis/Autophagy profiling Monitoring activity of apoptotic proteins and autophagolytic pathways. Figure 8: Apoptosis/Autophagy - of response to uV damage. Hela cells were interaction map of validated targets. untreated or treated with 500 mJ/cm2 UV light and harvested at 2 hr post treatment. Fold changes ® were calculated from chromatographic peak PTMScan Direct heights/areas. Green cells indicate peptides more 1. Multipathway (updated) 175 unique sites on abundant with UV damage; Red cells indicate peptides less abundant. 2. Ser/Thr Kinases 100 proteins 3. Tyr Kinases 4. Akt/PI3K 5. DNA Damage/Cell Cycle 6. Apoptosis/Autophagy Conclusion References PTMScan® Direct is a recently published method that allows identification and quantification of hundreds of peptides 1. Stokes, M.P. et al. (2012) Mol. Cell Proteomics 11, 187–201. from selected protein types or signaling pathways. This approach allows focus on proteins of interest instead of the 2. Rush, J. et al. (2005) Nat. Biotechnol. 23, 94–101. Interaction maps were generated from IPA® pathway analysis or the STRING database (string.embl.de) using high confidence scores random sampling of peptides that occurs in traditional data-dependent proteomic analysis. PTMScan® Direct is widely 3. Lundgren, D.H. et al. (2009) Curr. Protoc. Bioinformatics 13, 13.3.1–13.3.21. (>0.700) from experimental, database, and text mining lines of evidence. Interactions were also defined from the substrate search page of applicable in drug development and discovery, as well as in any application where monitoring of known signaling 4. Stokes, M.P. et al. (2012) Int. J. Mol. Sci. 14, 286–307. ® PhosphoSitePlus (www.phosphosite.org). Node colors and shapes denote different protein classes. Edge color denotes interaction type. Figure 3: Ser/Thr Kinase and Tyr Kinase targets mapped onto the human kinome tree. Yellow highlighting indicates kinases for which pathways is desired. peptides are identified using the reagents. Tyr Kinase coverage is shown in the inset (“TK”). Contact Information Matthew P. Stokes, Cell Signaling Technology, Inc. Email: [email protected] © 2013 Cell Signaling Technology, Inc. / Cell Signaling Technology®, PhosphoScan®, PhosphoSitePlus®, and PTMScan® are trademarks of Cell Signaling Technology, Inc. / IPA® is a registered trademark of Ingenuity Systems, Inc. PTMScan® Services Department Email: [email protected] • web: www.cellsignal.com/services