Tyrosine Phosphorylation: from Discovery to the Kinome and Beyond

The 2007 Herbert Tabor - Journal of Biological Chemistry Lecture

Tony Hunter

Tyrosine phosphorylation: from discovery to the kinome and beyond

ASBMB Annual Meeting

April 28, 2007

So far 43 JBC papers and one submitted!

The History of Protein Phosphorylation

Protein kinase ATP ADP

Protein P.Protein

Protein phosphatase The History of Protein Phosphorylation

Phospho- Protein Phospho- Src serine in kinase tyrosine tyrosine proteins activity in fly eggs kinase 1932 1954 1964 1979

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

Phospho Phospho- Phosph- cAMP Gleevec -protein tyrosine orylase depen- approved discovery synthesis kinase dent PK for CML 1906 1933 1959 1968 2001 The History of Protein Phosphorylation J. Biol. Chem. 2:127 (1906)

Phospho- Protein Phospho- Src serine in kinase tyrosine tyrosine proteins activity in fly eggs kinase 1932 1954 1964 1979

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

Phospho Phospho- Phosph- cAMP Gleevec -protein tyrosine orylase depen- approved discovery synthesis kinase dent PK for CML 1906 1933 1959 1968 2001 The History of Protein Phosphorylation

J. Biol. Chem. 98:109 (1932)

Phospho- Protein Phospho- Src serine in kinase tyrosine tyrosine proteins activity in fly eggs kinase 1932 1954 1964 1979

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

Phospho Phospho- Phosph- cAMP Gleevec -protein tyrosine orylase depen- approved discovery synthesis kinase dent PK for CML 1906 1933 1959 1968 2001 The History of Protein Phosphorylation

J. Biol. Chem. 100:583 (1933)

Phospho- Protein Phospho- Src serine in kinase tyrosine tyrosine proteins activity in fly eggs kinase 1932 1954 1964 1979

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

Phospho- Protein Phospho- Src serine in kinase tyrosine tyrosine proteins activity in fly eggs kinase 1932 1954 1964 1979

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

Phospho Phospho- Phosph- cAMP Gleevec -protein tyrosine orylase depen- approved discovery synthesis kinase dent PK for CML 1906 1933 1959 1968 2001 The History of Protein Phosphorylation

Burnett and Kennedy, J. Biol. Chem. 211:969 (1954)

Phospho- Protein Phospho- Src serine in kinase tyrosine tyrosine proteins activity in fly eggs kinase 1932 1954 1964 1979

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

Phospho Phospho- Phosph- cAMP Gleevec -protein tyrosine orylase depen- approved discovery synthesis kinase dent PK for CML 1906 1933 1959 1968 2001 The History of Protein Phosphorylation

Phospho- Protein Phospho- Src serine in kinase tyrosine tyrosine proteins activity in fly eggs kinase 1932 1954 1964 1979

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

Phospho Phospho- Phosph- cAMP Gleevec -protein tyrosine orylase depen- approved discovery synthesis kinase dent PK for CML 1906 1933 1959 1968 2001 Historic moments in the discovery of phosphotyrosine

+ Pi Pi 18/9/79

P.SER P.SER pH “1.9” “X”

P.THR P.TYR P.THR

| mT 14/6/79 | | | | acid LT mT mT IgH/Src in vivo acid protease v-Src increases P.Tyr levels in transformed chick cells

uninfected v-Src-transformed

pH 1.9

+ pH 3.5 32P-labeled control and RSV-transformed chick fibroblasts

Hunter and Sefton, PNAS 77:1311 (1980) The History of Protein Phosphorylation

Phospho- Protein Phospho- Src serine in kinase tyrosine tyrosine proteins activity in fly eggs kinase 1932 1954 1964 1979

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

Phospho Phospho- Phosph- cAMP Gleevec -protein tyrosine orylase depen- approved discovery synthesis kinase dent PK for CML 1906 1933 1959 1968 2001 The History of Protein Phosphorylation

Phospho- Protein Phospho- Src serine in kinase tyrosine tyrosine proteins activity in fly eggs kinase 1932 1954 1964 1979

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

Phospho Phospho- Phosph- cAMP Gleevec -protein tyrosine orylase depen- approved discovery synthesis kinase dent PK for CML 1906 1933 1959 1968 2001

More than 29,000 papers on tyrosine kinases have been published since 1979! How many tyrosine kinases are there? 1. The finding that v-Src and c-Src phosphorylate tyrosine gave us the first tyrosine kinase in 1979 2. By the end of 1980 four tyrosine kinases were known (Src, Abl, EGF receptor, Fps/Fes) 3. By the end of 1990 over 50 tyrosine kinases had been identified in vertebrates and equal numbers of tyrosine kinases and serine kinases were known, leading to the prediction that there might be several 100 tyrosine kinases in a vertebrate genome and a total of over 1000 protein kinases 4. The complete human genome sequence reported in 2001 reveals that there are 90 tyrosine kinases (all the tyrosine kinases had been found by other means before the sequence was completed), out of a total of 518 protein kinases The History of Tyrosine Phosphorylation

SH2 PI3 kinase PLCγ STATs domain associates as RTK are PTK identified with MT substrate substrates 1986 1987 1989 1992

v-Src MT + Src v-Erb is PTP1B PTP1B PTB Human protein + tyrosine derived is first and IRK domain kinome has kinase kinase from EGFR PTPase structures binds pY 90 PTKs 1977/8 1979 1984 1988 1994 1995 2002

1975 1980 1985 1990 1995 2000 2005

c-Src Abl + Bcr-Abl SH2 SH2 Inactive Gleevec gene EGFR fusion domain domain c-Src approved discovery are PTKs in CML binds P.Tyr structure structure for CML 1976 1980 1985 1990 1992 1997 2001 v-Src v-Src ∆C Cdc2 is Yersinia Grb2 kinase activation inhibited encodes SH2/SH3 sequence mechanism by P.Tyr PTPase adaptor 1980 1987 1989 1990 1992 Insulin receptor tyrosine kinase catalytic domain

Hubbard et al. Nature 372:746 (1994) Receptor tyrosine kinases

EGFR INSR PDGFRα FLT1 FGFR1 CCK4 MET TRKA AXL TIE EphA1 RYK DDR1 RET ROS LTK ROR1 MuSK RTK106 HER2 IGF1R PDGFRβ FLK1 FGFR2 RON TRKB MER TEK EphA2 DDR2 ALK ROR2 HER3 IRR CSF1R FLT4 FGFR3 TRKC SKY EphA3 HER4 KIT FGFR4 EphA4 FLK2 EphA5 EphA6 LET-23 DAF-2 F59F3.1 EGL-15 KIN15 T01G5.1 EphA7 C16B8.1 F11D5.3 C16D9.2 CAM-1 F59F3.5 KIN16 T17A3.8 EphA8 C25F6.4 F40G9.13 TKR-1 W04G5.6N EphB1 T10H9.2 C08H9.8 W04G5.6C EphB2 EphB3 F59F5.3 Y50D4B-4 Human RTKs EphB4 LMR1 M01B2.1 ZK938.5 EphB5 LMR2 58 (20 classes) R09D1.12 B0198.3 EphB6 LMR3 R09D1.13 F54F7.5 VAB-1 Worm RTKs 29 (11 classes) (B0252.1, F11E6.8, F40A3.5, R151.4, T148.1, T22B11.3 11 Unclassified Y38H6C.20, C24G6.2A, F08F1.1, F09A5.2, F09G2.1) NonreceptorNonreceptor protein-tyrosine tyrosine kinase kinasess

(Myr) Y SH3 binding regio n DNA BD Actin BD Abl Y Fes/Fe r Y Syk/Zap70

JEF domai n Kinase-like domain Y Jak PH domain Y Tec

Integrin-binding/JEF domain Y Focal adhesion-binding Fak

Y Cdc42-binding Ack

Myr Y Y Src

Csk

Y Srm PTK catalytic domain

YY SH2 domain Rak/Fr k SH3 domain Y Y Brk/Si k What is tyrosine phosphorylation used for?

1. Growth factor signaling (and oncogenesis) 2. Cell adhesion, spreading, migration and shape 3. Cell differentiation in development 4. Cell cycle control 5. Gene regulation and transcription 6. Endocytosis and exocytosis 7. Insulin stimulation of glucose uptake 8. Angiogenesis (formation of new blood vessels) 9. Regulation of ion channels in nerve transmission TransmembraneTransmembrane signaling signaling by tyrosineby tyrosine phosphorylation phosphorylation

EGF receptor Interferon receptor PDGF receptor T cell receptor

NH 2NH 2 Ligand binding domain Out

Plasma membrane NH 2 In

Catalytic domain

Transmembrane receptor Bimolecular receptor protein-tyrosine kinase protein-tyrosine kinase Receptor tyrosine kinase (RTK) signaling through SH2 and PTB P.Tyr-binding domain proteins

Schlessinger, Cell 103:211 (2000) Stone age bioinformatics!

Manual alignment - March 1985 (BB) Eukaryotic protein kinases have related catalytic domains

Serine kinases PKA-C Cdc2

Tyrosine kinases c-Src EGFR Catalytic domain ~300 aa

Protein kinase catalytic domain subdomains

Hanks, Quinn and Hunter, Science 241:42 (1988) Structure of PKA catalytic subunit bound to PKI (5-24) and ATP

N-lobe

pT197 catalytic cleft

C-lobe

Knighton et al. Science 253:414 (1991) The birth of the kinome: a thousand and one protein kinases

Src

Phos K PKA

Hunter, Cell 50:823 (1987) The first kinome

Hunter and Plowman, TiBS 22:18 (1997) The first kinome tree

Hunter and Plowman, TiBS 22:18 (1997) How many protein kinases are there? • S. cerevisiae (6217 genes) 130 (116) protein kinases (2.1%) but no bona fide tyrosine kinases • S. pombe (4624 genes) 128 (114) protein kinases (2.8%) but no TKs • C. elegans (19100 genes) 454 (434) protein kinases (2.4%) including 90 tyrosine kinases (20%) • D. melanogaster (13600 genes) 239 (223) protein kinases (1.8%) including 32 tyrosine kinases (14%) • H. sapiens (23,000 genes) 518 (478) protein kinases (2.2%) including 90 tyrosine kinases (16%) (chimpanzee kinome is essentially identical) • A. thaliana (26,800 genes) 1055 protein kinases (3.8%) but no tyrosine kinases (>630 RLK) M. brevicolli (unicellular choanoflagellate) has bona fide tyrosine kinases, SH2 domains and protein-tyrosine phosphatases (PTPs); the yeasts have PTPs, but no tyrosine kinases or SH2 domains ~2% of all genes in eukaryotes encode protein kinases Some more recent kinomes • Tetrahymena (27424 genes) 1069 PKs (3.8%) - no true TKs, but has TKLs and 83 two-component HisK. Has 630 PKs not assignable to known families or subfamilies, with 37 novel classes and 100s of unique PKs • Dictyostelium (12500 genes) 285 PKs (2.3%) 246 ePKs, including TKLs but no true TKs, plus 26 aPKs and 14 HisKs • Sea urchin (24000 genes) 353 PKs (1.5%) 329 ePKs, 24 aPKs (Strongylocentrotus) and 53 TKs, but no HisK. Lacks only 4/187 human kinase families • Mouse kinome is ~99% identical to human kinome - 540 mouse protein kinase genes - 510 are orthologous to human protein kinases • One conclusion is that the tyrosine kinase-like kinases (TKLs) evolved in unicellular organisms, perhaps serving as tyrosine (TK) progenitors, and were secondarily eliminated from yeast. HisKs were lost during evolution of metazoans, apparently replaced by TKLs and TKs (Eisen et al. PLoS Biol 4:e286; Goldberg et al. PLoS Genet 2:e38; Bradham et al. Dev Biol 300:180) The evolution of the kinome

rostome e

Unikont OpisthokonMetazoan Coelomate Deut

+4 Vertebrates +9 182 +15 +3 Sea urchin 173 TKs +76/-3 Drosophila 158 -11 +6/-1 C. elegans 85 +15 / -7 +27 80 +6 / -28 S. cerevisiae Dictyostelium 53 +25/-4 TKLs Tetrahymena +37 Gerard Manning How many human protein kinases and phosphatases? Protein kinases (the kinome) 518 protein kinases including: (not quite a 1001!) 478 conventional protein kinases (ePKs) (16 have tandem catalytic domains) 388 protein-serine/threonine kinases 90 protein-tyrosine kinases 58 receptor protein-tyrosine kinases 32 non-receptor protein-tyrosine kinases (~50 may lack catalytic activity; ~106 pseudogenes) 40 atypical protein kinases in 7 families (e.g. alpha kinases) Manning et al. Science 208:1912 (2002) (http://www.kinase.com) Protein phosphatases (the phosphatome) Nature has invented several ways to remove phosphate from proteins

1. Ser/Thr - phosphatases Metal-containing enzymes DxH…DxxD…..N PP1, PP2A, PP2B (PPP); PP2C (PPM)

2. Protein-tyrosine phosphatases Protein-tyrosine phosphatases

C(X)5R Dual-specificity phosphatases Low molecular weight phosphatases

Cdc25

3. RNA pol CTD phosphatase family Haloacid dehalogenase-related enzymes DxDxT….GDxxxE Eyes absent

Many others How many human protein kinases and phosphatases? Protein kinases (the kinome) 518 protein kinases including: (not quite a 1001!) 478 conventional protein kinases (ePKs) (16 have tandem catalytic domains) 388 protein-serine/threonine kinases 90 protein-tyrosine kinases ~2.5% genes directly devoted to protein phosphorylation and 58 receptor protein-tyrosine kinases dephosphorylation, and possibly up to 5%, if regulatory subunits, 32 non-receptor protein-tyrosine kinases inhibitors and scaffolding/anchoring proteins are included. (~50 may lack catalytic activity; ~106 pseudogenes) 40 atypical protein kinases in 7 families (e.g. alpha kinases) There are as many tyrosine phosphatases as tyrosine kinases Manning et al. Science 208:1912 (2002) (http://www.kinase.com) Protein phosphatases (the phosphatome) ~140 protein phosphatases including: 38 protein-tyrosine phosphatases 38 serine/threonine phosphatases (18 PP1/2A (PPP); 20 PP2C (PPM)) 62 DSPs (e.g. MKPs, PTEN); 8 HADs (EyA/FCP) Manning, Whyte, Martinez, Hunter and Sudarsanam Science 208:1912 (2002) The beard kinome

A 1001 hairs? Marc Bitensky (March, 1990) Human tyrosine kinases (90) Alternative transcripts of mouse protein kinases and phosphatases

Products Number per locus Transcripts 6.7 Polypeptides 3.7 Domain combinations 1.6 5’ exons 1.8 3’ exons 1.6

Most of the alternative coding products differ outside the catalytic domain, but a significant number of kinases have variant catalytic domains

Forrest et al. Genome Biol 7:R5 (2006) (variant.imb.uq.edu.au) Global dynamics of protein phosphorylation Analysis of global phosphorylation events using enrichment of phosphopeptides by IMAC, TiO2, PAC or phosphoantibodies derived by proteolytic digestion of cellular fractions followed by tandem MS identification of phosphopeptides and phosphorylation sites • 2,002 phosphorylation sites identified on 967 HeLa nuclear proteins (Beausoleil, Gygi, PNAS 101:12130, 2004)

• 5,635 phosphorylation sites identified on 2,328 proteins from mouse liver (Villen, Gygi, PNAS 104:1488, 2007)

• 6,600 phosphorylation sites identified on 2,244 proteins in HeLa cells; ~14% of these change >2 fold within 20 min of EGF (Olsen, Mann, Cell 127:635, 2006)

• CST Phosphosite database has >48,400 phosphorylation sites, including 6,200 human proteins (N.B. Bodenmiller, Aebersold, Nat Meth 4:231, 2007) These data suggest that the majority of intracellular proteins are phosphorylated at one or more sites under the appropriate condition - occupancy of many of these sites can change in response to stimuli Functional categories of EGF-regulated phosphoproteins

QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.

Olsen, Mann, Cell 127:635 (2006) The human kinome

• The completion of the human genome sequence has permitted a complete cataloguing of all human protein kinases • There are 518 protein kinase genes, of which about 450 have kinase activity and 90 are tyrosine kinases. This represents about 2% of all human genes • There are more than 140 protein phosphatases, and a large number of additional types of protein that recognize proteins once they are phosphorylated through phosphobinding domains • Most of the proteins in a cell can be phosphorylated at one or often multiple sites under the right conditions • Protein phosphorylation is a major mechanism of signal transduction in eukaryotic cells and perturbation in phosphorylation-driven processes can lead to disease Protein kinases are implicated in diverse diseases

• >150/518 (~30%) of human protein kinases reportedly implicated in various diseases • Many more are likely to follow from expression, sequencing, and SNP analyses, genetics and functional genomics • Kinases are tractable drug targets with several approved drugs and huge development efforts Gerard Manning (CST “Kinase and Signaling Reference Guide”) (www.cellsignal.com/reference/kinase disease.asp) Human diseases caused by mutations in protein kinases DISEASE KINASE

• MYOTONIC MUSCULAR DYSTROPHY MYOTONIN PROTEIN KINASE • X-LINKED AGAMMAGLOBULINEMIA BRUTON TYROSINE KINASE (BTK) • HIRSCHSPRUNG DISEASE, MEN RET • CD8 DEFICIENCY FORM OF SCID ZAP70 • X-LINKED SCID JAK3 • CRANIOSYNOSTOSIS FGF RECEPTOR KINASES • PEUTZ-JEGHERS SYNDROME LKB1 • COFFIN-LOWRY SYNDROME RSK2 • ATAXIA-TELANGIELTASIA ATM • LI-FRAUMENI SYNDROME CHK2 • WILLIAMS SYNDROME LIMK1 • LEPRECHAUNISM, DIABETES INSULIN RECEPTOR • WOLFF-PARKINSON-WHITE SYNDROME AMP-ACTIVATED KINASE (AMPK) • GORDON HYPERTENSION SYNDROME WNK1 AND WNK4 • WOLCOTT-RALLISON SYNDROME eIF2AK3/PEK • HEREDITARY EARLY-ONSET PARKINSON’S DISEASE PINK1 • HEREDITARY EARLY-ONSET PARKINSON’S DISEASE LRRK2 • PULMONARY HYPERTENSION TGFβ FAMILY RECEPTOR BMPR-II • FAMILIAL ADVANCED SLEEP PHASE SYNDROME CKIδ • RETT SYNDROME (NEURODEVELOPMENT DISORDER) CDKL5 • HYPORESPONSIVENESS TO BACTERIAL INFECTION IRAK4

• POLYCYTHEMIA VERA JAK2 • MELANOMA & OTHER SPORADIC CANCERSB-RAF • PAPILLARY RENAL CANCER MET RECEPTOR KINASE • CHRONIC MYELOGENOUS LEUKEMIA TEL-PDGF RECEPTOR KINASE • CHRONIC MYELOGENOUS LEUKEMIA BCR-ABL • NON-HODGKINS LYMPHOMA ALK • 5-10% NONSMALL CELL LUNG CANCERS EGF RECEPTOR KINASE • COLON, BREAST & OTHER SPORADIC CANCERS PI-3 KINASE (P110α SUBUNIT) Protein kinases with disease connections

PKs associated with human diseases

Cell Signaling Technology, 2005 Human cancer genes • 355 cancer genes implicated by mutation - ~1% of all human genes • 90% cancer genes show somatic mutations in cancer, 20% show germ line mutations and 10% show both • The most common mutation class among cancer genes are chromosomal translocations that create chimeric genes or appose a gene next a regulatory element of another gene • More cancer genes have been found in leukemias, lymphomas and sarcomas than in other types of cancer despite the fact that they represent only 10% all cancers • The protein kinase catalytic domain is the commonest domain among cancer genes (30). Domains involved in DNA binding and transcriptional regulation are also common

Futreal et al. Nat Rev Cancer 4:177 (2004) Cancer and the kinome Kinases control cancer pathways • 120 kinases strongly implicated in cancer; more to come • Kinases control complex pathways and signal transduction, implicated in all major steps/pathways of transformation Somatic mutations and kinases • Kinases as the test case for large scale resequencing of tumor genes: efforts at Sanger Center, JHU, Venter, Broad, elsewhere • Results to date are a mixture of stunning (BRAF in melanoma), impressive (high frequency of PI3-kinase pathway gene mutations in colon cancer - RTKs, IRS2, PIK3CA, PTEN, PDK1, AKT2, PAK4 - as well as other tyrosine kinase and PTP genes), and underwhelming (breast cancer kinome) (Davies et al. Nature 417:949; Bardelli et al. Science 300:949; Wang et al. Science 304:1164; Davies et al. Cancer Res 65:7591; Stephens et al. Nature Genet 37:590; Parsons et al. Nature 436:792; Rand et al. PNAS 102:1434; Sjoblom et al. Science 314:268; Bignell et al Genes Chr Cancer 45:42; Human Cancer Genome Project) Protein Kinases/Phosphatases and Cancer

• Over half of the 90 tyrosine kinases are implicated in human cancer either through gain of function mutations (e.g. Bcr-Abl), gene amplification (e.g. EGF receptor) or overexpression (e.g. c-Src) or as tumor suppressors (e.g. Syk, c-Fes, Csk, EphB2, EphB3, EphB4) • Many serine kinases are also implicated in cancer through activating mutations (e.g. B-Raf), overexpression (e.g. Aurora A), or loss of function mutations (e.g. Lkb1) • 164 protein kinase genes map to amplicons found in tumors • 80 protein kinase genes map to chromosomal disease loci and these are candidate genes for the causative mutation in hereditary disease (e.g. activating mutations in the Ret and Met RTKs in predisposition to cancer) • Inactivating and activating mutations in tyrosine/lipid and serine phosphatases have also been implicated in cancer (e.g. PTEN, SHP-2, PRL-3; RPTPβ, PTP-BAS, PEZ, RPTPγ, LAR, PTPH; Pr65/PP2A) Protein kinases as drug targets in disease therapy

• The involvement of protein kinases and altered phosphorylation in cancer and other diseases has been well established • Protein kinases are often mutated or overexpressed in cancer. More than 25 protein kinase genes are known to be mutated in human cancer, and ~120 protein kinases are implicated in cancer. It seems likely that additional currently uncharacterized protein kinases will prove to play a role in cancer, and all these protein kinases make potential drug targets for cancer therapy • Enzymes (e.g. protein kinases) generally make good drug targets • Initial concerns that it would not be possible to make specific inhibitors because most known kinase inhibitors bind to the conserved ATP binding site and because high intracellular ATP concentrations would compete for binding have proved groundless • Selective protein kinase inhibitors have been developed and are proving effective in cancer therapy - GleevecTM, TarcevaTM,SutentTM Cancer drugs that act against tyrosine kinases

DRUG CANCER TARGET Small molecule drugs GleevecTM (imatinib) leukemia (CML) Bcr-Abl tyrosine kinase IressaTM (gefitinib) lung cancer EGF receptor TK TarcevaTM (erlotinib) lung cancer EGF receptor TK SutentTM (sunitinib) GI stromal tumor/RCC Kit receptor TK SprycelTM (dasatinib) leukemia (CML) Bcr-Abl tyrosine kinase TykerbTM (lapatinib) breast cancer ErbB2 RTK

Many in trials several types/AML angiogenesis/Flt3 RTKs Monoclonal antibody drugs HerceptinTM (trastuzumab) breast cancer ErbB2 RTK ErbituxTM (cetuximab) breast/renal cancer EGF receptor TK AvastinTM (cevacizumab) colon cancer VEGF

>70 protein kinase inhibitors are in cancer clinical trials, including several directed against serine/threonine kinases implicated in cancer (Mark Via, Cambridge Healthtech). The Raf inhibitor sorafenib (NexavarTM) has recently been approved for treatment of renal carcinoma. Rapamycin, an mTOR inhibitor, and analogues are also in clinical trials for several cancers The long road to the GLEEVECTM cancer drug

1845 1960 1973 1982 1984 1990 1996 CML Chr 22∆ t(9:22) Bcr-Abl Bcr-Abl Bcr-Abl causes STI571 inhibits Ph Chr translocation (22:9) PTK CML in mice CML cell growth activity and v-Abl tumors

1970 1978 1980 2000 Ab-MuLV v-Abl v-Abl STI571/Abl protein tyrosine structure kinase 1988 1992 1998 First PTK CGP57148 STI571 1911 1970 1975 1977 1978 1979 inhibitors PDGFR/ in CML RSV v-src c-src v-Src v-Src v-Src reported Kit/Abl TKI patients gene gene protein PK tyrosine (TKI) (Novartis) kinase 2001 NEJM 1952 1977 1979 1983 papers polyoma mT mT mT reporting virus antigen associated associated STI571 PTK activity c-Src efficacy 1927 1988 1996 1998 2000 in CML W mutant W is c-kit gof c-kit gof STI571 and GIST mouse c-kit leukemia GIST in GIST mutants mutants patients 1986 1986

HZ4-FeSV v-Kit TM PTK Gleevec approved by the FDA May 10, 2001 Some outstanding questions • How/when did tyrosine kinases and phosphatases evolve? • Are there additional functions for tyrosine phosphorylation? • Are there additional types of P.Tyr-binding domain? • How large is the P.Tyr phosphoproteome? • How are the actions of the tyrosine kinases and phosphatases coordinated and do they act in combinations? How fast do phosphates turn over at a particular site? • Where do critical tyrosine phosphorylation and dephosphorylation events occur in the cell? • Can we develop more specific inhibitors or inhibitors that target a desired set of tyrosine kinases or phosphatases, or block P.Tyr interactions for disease therapy? Kinomics Gerard Manning (Sugen/Salk) Science 208:1912 TiBS 27:514 Greg Plowman PNAS 101:11707 Sucha Sudarsanam Sean Caenepeel http://www.kinase.com THANKS GO TO

AND OF COURSE THE OLD BUFFER!

Bart Sefton Walter Eckhart Jon Cooper Mary Anne Hutchinson Karen Beemon