tyrosine phosphorylation: from discovery to the kinome …super7/30011-31001/30941.pdf · tyrosine...

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

Protein P.Protein

ATP ADP

P

Protein phosphatase


Phospho-proteindiscovery1906

Phospho-serine inproteins1932

Phospho-tyrosinesynthesis1933

Protein kinaseactivity1954

Phosph-orylasekinase1959

cAMPdepen-dent PK1968

Srctyrosine kinase1979

Phospho-tyrosinein fly eggs1964

Gleevecapprovedfor CML2001

1910 1920 1930 1940 1950 1970 1980 1990 200019601900











1910 1920 1930 1940 1950 1970 1980 1990 200019601900

J. Biol. Chem. 2:127 (1906)











1910 1920 1930 1940 1950 1970 1980 1990 200019601900

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











1910 1920 1930 1940 1950 1970 1980 1990 200019601900

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











1910 1920 1930 1940 1950 1970 1980 1990 200019601900

J. Biol. Chem. 211:969 (1954)











1910 1920 1930 1940 1950 1970 1980 1990 200019601900

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











1910 1920 1930 1940 1950 1970 1980 1990 200019601900

v-Src increases P.Tyr levels in transformed chick cells

uninfected v-Src-transformed

+

pH 1.9

Hunter and Sefton, PNAS 77:1311 (1980)

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

pH 3.5











1910 1920 1930 1940 1950 1970 1980 1990 200019601900





Protein kinase activity1954






1910 1920 1930 1940 1950 1970 1980 1990 200019601900

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 theprediction 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

c-Srcgene

discovery1976

v-Erb isderived

from EGFR1984

Abl + EGFR

are PTKs1980

PTP1Bis first

PTPase1988

Bcr-Abl fusion

in CML1985

Cdc2 isinhibited by P.Tyr

1989

PTP1Band IRKstructures

1994

Gleevecapprovedfor CML

2001

MT + Src tyrosine kinase1979

SH2domain

binds P.Tyr1990

YersiniaencodesPTPase1990

PI3 kinaseassociateswith MT

1987

SH2domain

structure1992

PLCγas RTKsubstrate

1989

STATsare PTK

substrates1992

Inactivec-Src

structure1997

PTBdomain

binds pY1995

Humankinome has

90 PTKs2002

SH2domain

identified1986

v-Src ∆C activation

mechanism1987

Grb2SH2/SH3adaptor1992

v-Srckinase

sequence1980

v-Src protein +

kinase1977/8

1975 1980 1990 20001985 1995 2005

Insulin receptor tyrosine kinase catalytic domain

Hubbard et al. Nature 372:746 (1994)

Receptor tyrosine kinases

F59F3.1F59F3.5F40G9.13

EGL-15DAF-2 CAM-1C16B8.1

VAB-1

F11D5.3C25F6.4

C16D9.2KIN15KIN16TKR-1C08H9.8F59F5.3M01B2.1R09D1.12R09D1.13

T01G5.1T17A3.8W04G5.6NW04G5.6CY50D4B-4ZK938.5B0198.3F54F7.5

LET-23

CCK4FLT1FLK1FLT4

PDGFRαPDGFRβCSF1RKITFLK2

FGFR1FGFR2FGFR3FGFR4

IGF1RINSR

IRR

EGFRHER2HER3HER4

METRON

TRKATRKBTRKC

AXLMERSKY

TIETEK

EphA1EphA2EphA3EphA4EphA5EphA6EphA7EphA8EphB1EphB2EphB3EphB4EphB5EphB6

RYK ROSRET LTKALK

DDR1DDR2

RTK106ROR1ROR2

MuSK

LMR1LMR2LMR3

?

Human RTKs58 (20 classes)

Worm RTKs29 (11 classes)11 Unclassified

T10H9.2

(B0252.1, F11E6.8, F40A3.5, R151.4, T148.1, T22B11.3Y38H6C.20, C24G6.2A, F08F1.1, F09A5.2, F09G2.1)

Y DNA BD Actin BDAbl

YFes/Fe r

YKinase-like domainJak

YSyk/Zap70

YPH domain

Tec

Y Cdc42-bindingAck

YIntegrin-binding/JEF domain Focal adhesion-binding

Fak

YYSrc

CskY

Srm

Y YRak/Fr k

YYBrk/Si k

Myr

(Myr)

SH3 domain

SH2 domain

PTK catalytic domain

Nonreceptor protein-tyrosine kinase s

JEF domai n

SH3 binding regio n

Nonreceptor tyrosine kinases

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

Transmembrane receptorprotein-tyrosine kinase

Plasma membrane

Ligand binding domain

Bimolecular receptor protein-tyrosine kinase

Out

In

Catalytic domain

NH 2 NH 2

NH 2

EGF receptor PDGF receptor

Interferon receptor T cell receptor

Transmembrane signaling by tyrosine phosphorylationTransmembrane signaling by tyrosine phosphorylation

Schlessinger, Cell 103:211 (2000)

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

Manual alignment - March 1985 (BB)

Stone age bioinformatics!

Eukaryotic protein kinases have related catalytic domains

Serine kinases PKA-CCdc2

Tyrosine kinasesc-SrcEGFR

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

pT197

N-lobe

C-lobe

catalyticcleft

Knighton et al. Science 253:414 (1991)

Hunter, Cell 50:823 (1987)

The birth of the kinome: a thousand and one protein kinases

Phos K PKA

Src

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%) including90 tyrosine kinases (16%) (chimpanzee kinomeis 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

• 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, includingTKLs 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 proteinkinase 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

Some more recent kinomes

(Eisen et al. PLoS Biol 4:e286; Goldberg et al. PLoS Genet 2:e38; Bradham et al. Dev Biol 300:180)

Vertebrates

Sea urchin

Drosophila

+4

-11

+15

+9

53

C. elegans+15 / -7

+3

S. cerevisiae

Dictyostelium

Tetrahymena

+6 / -28

+76/-3

+25/-4

+6/-1

+37

Unikon

t

Metazo

an

Opistho

kont

+27

Coelom

ateDeu

teros

tome

80

85

158

173

182

The evolution of the kinome

Gerard Manning

TKLs

TKs

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 kinases58 receptor protein-tyrosine kinases32 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?

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)

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 kinases58 receptor protein-tyrosine kinases32 non-receptor protein-tyrosine kinases

(~50 may lack catalytic activity; ~106 pseudogenes) 40 atypical protein kinases in 7 families (e.g. alpha kinases)

~2.5% genes directly devoted to protein phosphorylation and dephosphorylation, and possibly up to 5%, if regulatory subunits,

inhibitors and scaffolding/anchoring proteins are included.

There are as many tyrosine phosphatases as tyrosine kinases

Manning, Whyte, Martinez,Hunter and SudarsanamScience 208:1912 (2002)

A 1001 hairs?Marc Bitensky (March, 1990)

The beard kinome

Human tyrosinekinases

(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

• 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, Cell127:635, 2006)

• CST Phosphosite database has >48,400 phosphorylation sites, including 6,200 human proteins (N.B. Bodenmiller, Aebersold, Nat Meth 4:231, 2007)

Analysis of global phosphorylation events using enrichment ofphosphopeptides by IMAC, TiO2, PAC or phosphoantibodies derived by proteolytic digestion of cellular fractions followed by tandem MS identification of phosphopeptides and phosphorylation sites

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 aTIFF (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)

http://www.cellsignal.com/reference/kinasedisease.asp

Human diseases caused by mutations in proteinkinases

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)

Cell Signaling Technology, 2005

PKs associated with human diseases

Protein kinases with disease connections

• 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

Human cancer genes

Futreal et al. Nat Rev Cancer 4:177 (2004)

Cancer and the kinomeKinases 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. Science304:1164; 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 TARGETSmall molecule drugs

GleevecTM (imatinib) leukemia (CML) Bcr-Abl tyrosine kinaseIressaTM (gefitinib) lung cancer EGF receptor TKTarcevaTM (erlotinib) lung cancer EGF receptor TKSutentTM (sunitinib) GI stromal tumor/RCC Kit receptor TKSprycelTM (dasatinib) leukemia (CML) Bcr-Abl tyrosine kinaseTykerbTM (lapatinib) breast cancer ErbB2 RTK

Many in trials several types/AML angiogenesis/Flt3 RTKs

Monoclonal antibody drugsHerceptinTM (trastuzumab) breast cancer ErbB2 RTKErbituxTM (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

1845CML

1960Chr 22∆Ph Chr

1973t(9:22)translocation

1982Bcr-Abl(22:9)

1984Bcr-Abl

PTKactivity

1980v-Abltyrosinekinase

1988First PTKinhibitorsreported(TKI)

1992CGP57148PDGFR/Kit/Abl TKI(Novartis)

1998STI571in CMLpatients

1990Bcr-Abl causesCML in mice

1970Ab-MuLV

1978v-Ablprotein

2000STI571/Ablstructure

1911RSV

1970v-srcgene

1975c-srcgene

1977v-Srcprotein

1978v-SrcPK

1979v-Srctyrosinekinase

1927W mutantmouse

1988W isc-kit

1986HZ4-FeSV

1986v-KitPTK

1998c-kit gofGISTmutants

1996c-kit gofleukemiamutants

2001NEJMpapersreportingSTI571efficacyin CMLand GIST

2000STI571in GISTpatients

1996STI571 inhibitsCML cell growthand v-Abl tumors

1977mTantigen

1952polyomavirus

1979mTassociatedPTK activity

1983mTassociatedc-Src

GleevecTM 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?

KinomicsGerard Manning (Sugen/Salk)

Greg PlowmanSucha SudarsanamSean Caenepeel

Science 208:1912TiBS 27:514PNAS 101:11707

http://www.kinase.com

Walter EckhartMary Anne Hutchinson

Bart SeftonJon CooperKaren Beemon

THANKS GO TO

AND OF COURSE THE OLD BUFFER!

tyrosine phosphorylation: from discovery to the kinome …super7/30011-31001/30941.pdf · tyrosine...

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