COMMEMORATIVE LECTURE
Tyrosine phosphorylation in cell signaling and disease
Tony Hunter
The Salk Institute for Biological Studies, La Jolla, CA, USA(Received for publication on April 16, 2002)

Key words: phosphorylation, protein kinases, receptor tyrosine kinase, actin cytoskeleton, tyrosine
kinase inhibitor

But interestingly, although there are a few dedicatedtyrosine kinases, like Swe1p, which phosphorylates
Cdc28 (Cdc2) on tyrosine, bona ﬁde tyrosine kinases
like those that belong to the large family found inmetazoans are lacking. Indeed, it appears that the useof tyrosine phosphorylation as a signaling mechanism
evolved hand-in-hand with multicellularity as a mechanism for intercellular communication. Interestingly, it
has recently been shown that one species of choanoﬂagellate, which are unicellular organisms closely related to metazoa, has a tyrosine kinase receptor.3 Thissuggests that tyrosine kinases evolved at about thesame time as metazoa, and implies that the existence ofa tyrosine kinase receptor may have been a prerequisite
for the emergence of true metazoans which require coordinated intercellular communication. The emergenceof tyrosine phosphorylation as a signaling mechanism
also depended upon the evolution of other componentsof the current systems used to propagate signals generated by tyrosine phosphorylation, including SH2
domains, which bind to phosphorylated tyrosines, andprotein-tyrosine phosphatases (PTPs).
The nematode C. elegans, a complex multicellularorganism, has about three times as many total genes asbudding yeast and about three times as many protein
kinase genes, which again comprise about 2% of all
genes. In this organism, tyrosine kinases now form amajor distinct family, as shown by dendrogram analysis,
and represent about a ﬁfth of all protein kinases. Interestingly, D. melanogaster, even though it is a more
complex organism, has fewer genes and fewer protein
kinases than C. elegans, with relatively fewer tyrosine
kinases, but in toto the protein kinases again compriseabout @2% of all genes.4
So what about H sapiens? The total number of genes
is somewhat in dispute. The numbers have ranged from

Let me begin by expressing my gratitude to the Keio
Medical Science Fund for the award of the 2001 Keio
Medical Science Prize. This is indeed a great honor.
I will start by discussing how the recent genomics
revolution affects the way one thinks about cell signaling, particularly signaling through tyrosine phosphorylation. Then I will tell you three short stories aboutsome recent work from members of my group on howtyrosine phosphorylation is used in signal transduction
and how it can be used to regulate the actin cytoskeleton.
Protein Kinase Genomics
Almost exactly 15 years ago, I was rash enough topredict that the human genome might encode as manyas 1,001 protein kinases.1 That prediction was based ona graph I made in 1986, which showed that the total
number of distinct protein kinases had been increasingalmost exponentially, beginning in 1976 or 1977 whenmolecular cloning ﬁrst became feasible. Extrapolatingfrom this curve, I came up with a predicted total number of protein kinases that sounded outrageously highat the time, but one that actually has proved to be notso far from the truth.
As you know, a number of genomes have now been
completely sequenced and one can scan the predicted
open reading frames to identify protein kinases using
the signature motifs that are present in all membersof this superfamily of serine and tyrosine kinases. S.
cerevisiae was the ﬁrst eukaryotic genomic sequence tobe completed and it has around 6,000 genes. Our analysis, done in collaboration with the bioinfomatics groupat Sugen, has identiﬁed 116 protein kinase genes inbudding yeast, which represents about 2% of all genes.2

Presented at the commemorative lecture of the award ceremony of the 2001 Keio Medical Science Prize, November 29, 2001.
Reprint requests to: Dr. Tony Hunter, Molecular and Cell Biology Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla,
CA 92037, telephone 858-453-4100 ext 1385, facsimile 858-457-476, e-mail: hunter@salk.edu
6162

Hunter T: Tyrosine phosphorylation in signaling and diseasekinases and 90 tyrosine kinases. And among the 90
tyrosine kinases, 58 are receptor tyrosine kinases and
32 are non-receptor tyrosine kinases. For comparison,
there are 42 predicted PTPs, which is about half thenumber of tyrosine kinases, 32 serine/threonine phosphatases and 46 phosphatases in the dual-speciﬁcity
phosphatase family, which is distantly related to thetyrosine phosphatase family. In total, then, there are
upwards of 600 human genes recognizably involved in
the phosphorylation and dephosphorylation of proteins:the large number underscores the importance of thisreversible protein modiﬁcation for regulating proteinfunction in eukaryotic cells.
Transmembrane Signaling by
Tyrosine Phosphorylation
Fig. 1

Human protein kinase dendrogram.

30,000, based on gene predictions from genomic sequences, to as many as 150,000, based on cDNA sequencing, but I think it is probably going to be nearer to
30,000 genes. The analysis is still not yet complete, but
our current estimate is that there are @500 protein
kinase genes, including 90 tyrosine kinase genes, andabout 100 pseudogenes, which are recognizable copies
of protein kinase genes that for one reason or anotherappear unlikely to be able to produce a functional product.5 Because the sequence is not yet complete, and theassembly is not perfect, there could be a few missing
protein kinase genes. Of course, the total number ofdistinct protein products derived from these genes ismany more than 500, because a signiﬁcant fraction express alternatively spliced products. If there are 30,000
genes, this means that about 2% of all human genes
encode protein kinases, and about 20% of these protein
kinases are tyrosine kinases. A dendrogram of the human protein kinases shows the prominence of the tyrosine kinase (TK) branch (Fig. 1).
As a sidelight, it is interesting to compare the humangenome with that of A. thaliana, the favorite genetic
organism for plant biologists. Arabidopsis has around
25,000 genes, but it has over a 1,000 protein kinase
genes, which is relatively more than in humans, although, it lacks bona ﬁde tyrosine kinase genes. The
greater frequency of protein kinase genes may seem
somewhat paradoxical, but it makes sense when one
realizes that many of these protein kinases are used torespond to external stimuli and pathogens, which isparticularly important for an organism that is sessile.
If we examine in more detail the complement ofprotein kinases encoded by the human genome – what
we have called the kinome – out of the @500 conventional protein kinases, there are 410 serine/threonine

One of the major functions of tyrosine phosphorylation is in transmembrane signaling. There are two
types of receptor system that use tyrosine phosphorylation. There are receptors with intrinsic tyrosine
kinases activities, like the EGF/PDGF receptors, andthere are binary or bimolecular receptors, where thesurface receptor subunit lacks catalytic activity but
associates with a non-receptor tyrosine kinase, whichacts as a signaling subunit. Examples of this type are theinterferon receptors and the T and B cell antigen
receptors. Activation of both types of receptor tyrosine
kinase occurs through ligand-induced dimerization,
which juxtaposes the catalytic domains either in thereceptors themselves or the associated non-receptor
tyrosine kinase, resulting in transphosphorylation, initially in the activation loop and then subsequently inregions lying outside the catalytic domain, and in somereceptors in an insert in the catalytic domain.
The phosphotyrosines generated by transphosphorylation then create binding sites for target proteins thathave SH2 (Src homology 2), or PTB domains. Both ofthese are sequence speciﬁc phosphotyrosine (P.Tyr)binding domains; SH2 domains recognize P.Tyr andfrom 1–6 residues on its C terminal side, and PTB
domains recognize P.Tyr and up 5 residues on its N
terminal side. In addition to SH2 or PTB domains tyrosine kinase target proteins commonly have other protein or lipid interaction domains, such as SH3 and PH(pleckstrin homology) domains. Some of these targets
act as adaptor proteins, such as the Grb2 SH2/SH3 proteins, and others have enzymatic activities, like phospholipase Cg (PLCg), Ras-GAP, and a subset of the
PTPs. SH3 domains play a critical role in tyrosine phosphorylation based signaling, recognizing short prolinerich sequences, and increasingly important are thephospholipid binding domains. In many cases, theserecognize 3 0 phosphoinositides that are generated by
PI-3 0 kinases; such domains include the PH domain, theKeio J Med 2002; 51 (2): 61–71

recently identiﬁed PX domain, and the FYVE domain.
A recent analysis of the numbers of these domains
encoded by the human genome predicts there to be 126
SH2 domains, 264 SH3 domains and 223 PH domains.6
Activated receptor tyrosine kinases stimulate or trigger a plethora of signaling pathways through recruitment of SH2 or PTB-domain proteins to speciﬁc autophosphorylated residues. For example, in the case of
the PDGFb receptor, which has at least 9 identiﬁed
autophosphorylation sites, PI-3 0 kinase binds to two
phosphorylated tyrosines in the kinase insert via theSH2 domains in its p85 regulatory subunit. Recruitmentof the PI-3 0 kinase complex to the plasma membraneputs it in apposition with its substrate, PIP2 , which it
phosphorylates to yield PIP3 . PIP3 can in turn be hydrolyzed to generate PI34 P2 ; both PIP3 and PI34 P2 interact
with a subset of PH domains, including those of the
PDK1 and Akt/PKB protein kinases, which act in series
to constitute a signaling pathway. Likewise, phosphorylated PLCg can be recruited to the PDGFb receptor at
the membrane, and there it hydrolyzes PIP2 , generating
DAG and IP3 , which releases Ca 2þ ; together DAG and
Ca 2þ stimulate protein kinase C (PKC). The Grb2 SH2/SH3 adaptor protein can also be recruited to the receptor, bringing along the Ras exchange factor Sos; Sos
then activates Ras, which in turn activates the ERK
MAP kinase pathway. The cellular response to PDGF
reﬂects an integration of all of these pathways, many ofwhich lead to the nucleus, and induce gene transcription.
Dysregulation of Tyrosine Phosphorylation
and Disease
We are increasingly aware that mutations or changes
in the expression of protein kinases and phosphatases
play an important role in human disease. Many hereditary diseases are due to mutations in protein kinases
and phosphatases. Many of the genetic changes in cancers that play a causal role in the cancer phenotype involve mutations of protein kinases and phosphatases.
There has been a lot of progress in the development ofspeciﬁc inhibitors for protein kinases and phosphatases
involved in disease.7 And some of these inhibitors havebeen recently approved for the use in humans for treatments of cancer. I am sure that many more protein kinases and phosphatases inhibitors will become approved
drugs in the next few years.
I would like to spend a little more time to illustratesome examples where mutations or ampliﬁcation andoverexpression of protein kinase genes, and tyrosine
kinase genes in particular, are causal in human disease.
Many of the tyrosine kinases, when overexpressed or
activated by mutation play a role in cancer (Table 1A
and B). For instance, ErbB2, is commonly ampliﬁed

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and overexpressed in breast cancer, as is the related
EGF receptor, which is overexpressed in several typesof cancer. A number of the receptor and non-receptor
types of kinases are activated by chromosomal translocations, which generate chimeric fusion proteins. For
instance, fusion of the PDGF receptor with TEL, an Ets
family member, which provides a dimerization domain
and elicits constitutive ligand independent activation,
gives rise to chronic myelomonocytic leukemia.
The stem cell factor (SCF) receptor tyrosine kinase(KIT), which I will talk brieﬂy about later, is activatedby point mutations or small deletions in leukemia andgastrointestinal stromal tumors (GIST). Conversely, inactivation of KIT by germline mutations results in hereditary piebaldism. The FGF receptor tyrosine kinase
family is interesting, because they are mutationally activated in hereditary syndromes that affect skull andbone development (craniosynostosis). FGF receptors
are also activated sporadically in somatic cells by chromosomal translocations in some types of leukemia.
Other receptor tyrosine kinases that are activated bychromosomal translocations in leukemia include ALK
and TRK family members; in general these translocations result in gene fusions that encode chimeric proteins containing a dimerization domain from the fusion
partner and the cytoplasmic domain, including the catalytic domain, from the receptor tyrosine kinase. Lossof function mutations also play a role in human diseasesuch as in diabetes, where inactivating mutations in theinsulin receptor are responsible; and in the case of RET,
the GDNF receptor, loss of function mutations resultin the hereditary intestinal disease, Hirschsprung’s syndrome.
Non-receptor tyrosine kinases can also be either activated or inactivated by mutation in a variety of diseases(Table 1B). For instance, the SRC family kinases arecommonly overexpressed in a wide range of carcinomas, and the ABL tyrosine kinase is activated by achromosomal translocation which fuses it to BCR,
creating the activated BCR-ABL protein that causes
chronic myelogenous leukemia (CML). As we will hearlater today, loss of function mutations in a non-receptor
tyrosine kinase, such as ZAP70, are causal in humanimmunodeﬁciency syndromes.
There is also evidence that PTPs play a role inhuman disease, again largely based on mutational data
(Table 1C). For example, loss of function mutations in
CD45, a receptor-like PTP, causes severe combined
immune deﬁciency disease. Interestingly, it has recently
been reported that the PRL-3 dual-speciﬁcity phosphatase is speciﬁcally overexpressed in metastatic colon
carcinoma, which suggests an important role for phosphatases in metastasis. The PTEN/MMAC-1 dualspeciﬁcity phosphatase, which dephosphorylates 3 0
phosphoinositides, such as PIP3 , rather than phospho-

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Table 1

Hunter T: Tyrosine phosphorylation in signaling and disease
Involvement of Tyrosine Kinases and Phosphatases in Human Disease

A. Receptor tyrosine kinases
EGF receptor
ERBB2/ERBB3/ERBB4PDGF receptor b
CSF-1 receptor
KIT (SCF receptor)
FGF receptor 1
FGF receptor 2
FGF receptor 3
VEGF receptor 3
MET (HGF receptor)
AXL
RSE/SKY
MER
RET (GDNF receptor)
ALK
TRK,TRKC (NGFR)ROS
TIE2 (angiopoietin receptor)
Insulin receptor

mutated, ampliﬁed and overexpressed in cancer
ampliﬁed and overexpressed in breast cancer
activated by chromosomal translocation in leukemia
ectopic expression in cancer
inactivated by mutation in hereditary piebaldism
activated by mutation in leukemia
activated by mutation in craniosynostosis syndromes affecting skull/bonedevelopment (e.g. Crouzon/Pfeiffer syndromes, thanatophoric dysplasia)
FGFR1/3 activated by chromosomal translocation in leukemia/myeloma
inactivated by mutation in hereditary lymphedema
activated by mutation, and ampliﬁed/overexpressed in sporadic/hereditary cancer
overexpressed in myeloid leukemias
overexpressed in breast cancer
inactivated in retinal dystrophy (defect in retinal pigment epithelial cell phagocytosis)
inactivated by mutation in hereditary intestinal diseaseactivated by mutation in hereditary cancer syndromes
activated by chromosomal translocation in leukemia
activated by chromosomal translocation in cancer
activated by chromosomal translocation in glioblastoma
activated by mutation in hereditary vascular dysmorphogenesis disease
inactivated by mutation in non-insulin dependent diabetes

B. Nonreceptor tyrosine kinases
SRC
YES
LCK
ABL
FAK
JAK2
JAK3
BTK
ZAP70

mutated, activated and overexpressed in colon and other cancers
activated and overexpressed in cancer
activated by mutation and chromosomal translocation in leukemia
activated by chromosomal translocation to BCR (BCR-ABL) in chronic myelogenous leukemia (CML)
overexpressed in metastatic cancer
activated by chromosomal translocation in leukemia
inactivated by mutation in severe combined immune deﬁciency disease (SCID)
inactivated by mutation in hereditary agammaglobulinemia
inactivated by mutation in severe combined immune deﬁciency disease (SCID)

C. Protein-tyrosine phosphatases
Shp-1 (mouse)
SHP-2 (human)
DEP-1/RPTPh
CD45
KAP/Cdi1
PRL-3
PTEN/MMAC-1

EPM2
myotubularin/MTM
MTMR2
YopH1 (bacterium)

SH2 domain PTP inactivated by mutation in immune hyperproliferative diseaseSH2 domain PTP mutationally activated in Noonan syndrome
receptor PTP deleted in human thyroid cancer; suppresses thyroid cell growth
receptor PTP inactivated by mutation in severe combined immune deﬁciency disease (SCID)
dual-speciﬁcity protein phosphatase mutated in cancer
dual-speciﬁcity PTP overexpressed in metastatic colon carcinoma
3 0 phosphoinositide (PIP3 ) phosphatase inactivated by mutation in sporadic breast and prostate cancer
and in Cowden’s hamartoma hereditary cancer syndrome; may also dephosphorylate P.Tyr in protein
substrates e.g. FAK
dual-speciﬁcity phosphatase inactivated by mutation in myoclonus epilepsy (Lafora’s disease)
3 0 PI (PI-3P) phosphatase inactivated in X-linked myotubular myopathy
3 0 PI (PI-3P) phosphatase inactivated in Charcot-Marie-Tooth disease 4B peripheral neuropathy
PTP encoded by the virulence plasmid of Yersinia (plague bacterium) that is required for pathogenesis– acts on FAK and p130Cas

proteins, is inactivated in sporadic cancers as well as
in hereditary cancer, resulting in increased levels of
PIP3 , which lead to persistent activation of downstream
signaling.
The clear cut involvement of dysregulated tyrosine
phosphorylation in disease has led to a major effort

on the part of the pharmaceutical industry to develop
inhibitors of tyrosine kinases and phosphatases. A
number of TKIs – tyrosine kinases inhibitors – are inclinical trials for cancer therapy (Table 2).8 Gleevec TM ,previously called STI571, an ABL/PDGFR/SCFR TKI
has been approved by the US FDA, and is a very potent

Keio J Med 2002; 51 (2): 61–71
Table 265

Tyrosine Kinase Inhibitor Drugs

EGF receptor inhibitors (ZD1839/Iressa TM ;OSI774/Tarceva TM )EGFR/ErbB2 inhibitor (CI1033; EKB569;
GW2016; PKI166)
VEGF receptor inhibitors (ZK222584;
ZD6474)VEGFR/FGFR/PDGFR inhibitors(SU6668; SU11248; PTK787)
SCFR/Kit inhibitor (SU11248)
NGF receptor (CEP2583)
Abl/PDGFR/SCFR inhibitor (STI571/
Gleevec TM )anti-EGF receptor MAbs (MAb225/
Erbitux TM )anti-ErbB2 MAbs (MAb4D5/Herceptin TM )Stem Cell Factor Receptor Signaling
NSC lung/pancreaticcancer
NSC lung cancer
angiogenesis
angiogenesis
AML
prostate cancer
CML, GIST
colorectal cancer
breast cancer

Gleevec TM and Herceptin TM are approved for clinical use. The otherdrugs are in clinical trials.
Avastin TM , an anti-VEGF MAb, is also in late stage clinical trials forbreast cancer.

and effective treatment for CML and GIST.9 A neutralizing anti-VEGF monoclonal antibody (MAb) isproving very effective as an anti-angiogenic agent incancer clinical trials, and this MAb is likely to be thesecond MAb approved for clinical use that targets
tyrosine kinase signaling (the ﬁrst was Herceptin TM , theanti-ErbB2 MAb).
There are several ongoing efforts to develop additional types of compound that inhibit or activate speciﬁc tyrosine kinases and phosphatases for treatmentof other types of disease. Many companies are trying
to ﬁnd inhibitors of SRC family kinases, for instanceto treat osteoporosis, or LCK, which is exclusively expressed in T cells; an LCK TKI could be used as an
immunosuppressant. CD45 receptor PTP inhibitors arealso being developed to act as immunosuppressants.
JAK family kinases are very important in cytokine
responses, and JAK TKIs could be used to repress undesirable cytokine responses. And then, for the treatment of type II diabetes, there are programs to develop
inhibitors of the major insulin receptor PTP, PTP1B,
with the goal of potentiating insulin receptor signaling.
In fact, PTP1B inhibitors are already in clinical trials.
An alternative insulin-independent method of enhancing insulin receptor signaling would be through insulinmimetic dimerizers, and some success in this direction
has been reported, with the development of a small
membrane permeant molecule that activates the insulin
receptor by artiﬁcially dimerizing the cytoplasmic catalytic domain. Already a large number of potentially
important new drugs based on tyrosine kinases andphosphatases have been developed, and in the next few
years we can look forward to many more such drugs
becoming available for treatment of human disease.

Now I will tell you brieﬂy about one project in myown group, where the goal has been to analyze the signaling functions of the SCF receptor/Kit, by making
germ line mutations in the mouse. SCFR/Kit is a member of the PDGF-receptor family, having 5 immunoglobulin folds in its extracellular domain, and with acatalytic domain that is characterized by a large kinase
insert.
The SCF receptor is encoded by the dominant whitespotting (W) locus in the mouse, which was identiﬁed
nearly 75 years ago now, through screens looking forovert genetic phenotypes. These mice have a white spot
on their belly due to a pigmentation defect. The W
alleles are all loss of function mutations in SCFR/Kit
that decrease kinase activity. Analyses of these mice
have shown that the SCFR/Kit is critical for the growth,survival and differentiation of stem cells in a series oflineages, including the hematopoietic, melanogenic, andgametogenic lineages. There are also loss of function
mutations in the KIT gene in humans, which cause acondition known as piebaldism; piebald individuals are
anemic and infertile, and have an obvious pigmentation
defect. Interestingly, as I indicated, there are also gainof-function mutations in the KIT gene, found in certain
human leukemias. These include point mutations,which lie in the activation loop of the catalytic domain,
particularly D816V/H that causes mastocytoma andmyeloid leukemia through constitutive activation of thekinase (Fig. 2). Additionally, there are a series of small
in-frame deletions in the cytoplasmic juxtamembrane
domain that also constitutively activate the receptor
and cause GIST in affected individuals.
Let me start by reviewing what is known about signaling downstream of the activated SCF receptor (Fig.
3). One of the major autophosphorylation sites is tyrosine 719, which recruits PI-3 0 kinase and activates anumber of downstream signal pathways, including one
that indirectly leads to the activation of PKC, throughphospholipase D-mediated generation of diacylglycerol.
Activated PKC then phosphorylates serines 739 and
744 in the kinase insert, and this reduces SCFR/Kit signaling. On this basis we had proposed that PKC phosphorylation acts as a negative feedback loop to limit
SCFR/Kit signaling in response to ligand. To study thephysiological importance of these serine phosphorylations, Peter Blume-Jensen, a postdoctoral fellow in mygroup, developed a knock-in mutant mouse in which heconverted the codons serines 739 and 744 to alanine in
the c-kit gene in the mouse germline, using the same
strategy we had used to make a Tyr719Phe mutant
mouse.10 If our model were correct, we might expectthat this double mutation would lead to gain of function
phenotypes, such as tumorigenesis.

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Hunter T: Tyrosine phosphorylation in signaling and disease

Homozygous S739/744A double mutant mice are viable, fertile, and show no obvious phenotypes at birthor when young. However, these mice begin to develop
what appear to be gastrointestinal stromal tumors,with reasonable frequency between 12 and 18 months.
Strikingly, this is the disease that humans with activating mutations in SCFR/Kit develop, and we conclude
based on this that protein kinase C phosphorylation does exert a negative feedback which plays a rolein inhibiting Kit function in vivo. These S739/744A
mice could serve as a model for the human GIST syndrome. And we would predict, although we still need to
do the experiment, that these mice might be cured bytreatment with Gleevec. I think this underscores theimportance of being able to develop mouse models forhuman disease, which one can then use for testing candidate drugs prior to embarking on full scale clinical
trials.
Eph Receptor Tyrosine Kinase Signaling

Fig. 2

SCF receptor/Kit mutations in human cancers.

Fig. 3

SCF receptor/Kit signaling.

The second story I have to tell you concerns the Ephreceptor tyrosine kinase family. This is the largest of
the receptor tyrosine kinase families, with 14 members.
Eph receptor tyrosine kinases are conserved in evolution, with a single Eph gene being found in C. elegans.
The ligands for Eph receptors are called ephrins; there
are two main classes – A-type ephrins, which are GPIlinked, and B-type ephrins, which are transmembraneproteins. In this case, the ligands are presented on thesurface of a neighboring cell, rather than being soluble,
secreted molecules. A-type ephrins interact with EphA
subfamily receptors, and B-type ephrins interact with
the EphB subfamily receptors. Genetic analysis in manydifferent organisms has shown that Eph/ephrin signaling is required for compartment boundary formation, as
well as for cardiovasculogenesis, and is particularly important for axonal pathﬁnding in the CNS, where Eph/ephrin signaling provides a repulsive signal for axonal
growth cones. Eph signaling is still rather poorly understood, but requires clustering of several Eph molecules –a dimer is not sufﬁcient and a higher order oligomer
is needed for a productive Eph signal. In addition, interaction with Eph allows the ephrins themselves, particularly the B-type ephrins, to generate signals in theephrin-expressing cell, establishing a bidirectional signaling system. Autophosphorylation of Eph triggers therecruitment of a number of SH2-containing proteins,
including Nck, RasGAP, and Shp2. In addition, Ephreceptors interact with and can somehow regulate the
FAK tyrosine kinase, p62Dok and ephexin, which is a
Rho family GTP exchange factor (GEF). It was discovered early on that Eph signaling does not stimulate
cell proliferation nor does it activate PI-3 0 kinase, andmore recently it has been shown to inhibit ERK MAP

Keio J Med 2002; 51 (2): 61–71

kinase, possibly via p62Dok, which is the exact opposite
of how a growth factor receptor signals.
The one agreed Eph signaling response is regulationof the actin cytoskeleton. Evidence from a number ofgroups shows that Eph activation causes a transientcollapse of the neuronal growth cone, whose forward
movement depends upon actin polymerization, and thispotentially provides a repulsive mechanism.11 Receptor
tyrosine kinase induced assembly of the actin cytoskeleton generally requires activation of Rho family proteins, which promote F-actin assembly. Of relevance isthe existence of an EphA-associated Rho family GEF
called ephexin, which responds to ephrin binding. In
PC3 carcinoma cells, Eph activation causes dephosphorylation of FAK, loss of F-actin and cell rounding.
In endothelial cells, however, Eph stimulation activates
integrin and promotes cell attachment and F-actin assembly.
Nigel Carter, a postdoctoral fellow in my group, became interested in studying ephrin-Eph signaling, andhe chose to look at ﬁbroblasts. Although ﬁbroblasts are
not normally thought of as a cell type that uses Ephsignaling, many years ago when we ﬁrst cloned EphA2
we found that mouse ﬁbroblasts express high levels of
this receptor tyrosine kinase. To study EphA2 signaling, we developed an assay in which recombinant ephrin A1 is coated onto a tissue culture dish, and then
NIH3T3 mouse ﬁbroblasts are plated onto the coateddish in the absence of serum.12 NIH3T3 cells attach
to the ephrin A1-coated dish, and begin to spread out,
and by 60 minutes many of the cells are well spread.
NIH3T3 cells plated onto polylysine-coated dishes attach but do not spread at all, whereas cells plated onto
ﬁbronectin, an extracellular matrix protein, attach andspread rapidly as expected. If one examines the actin
cytoskeleton in these cells by staining with ﬂuorescentphalloidin, in the cells plated on ephrin A1 the actin
cytoskeleton shows an unusual morphology, with verystrong circumferential staining of short actin bundles.
That is in contrast to the cells plated on ﬁbronectin,
where there is some peripheral F-actin staining, butalso bundles crossing the cell, which is typical. We also
looked at other proteins that are implicated in the actin
cytoskeleton and cell adhesion in this system. For instance, the focal adhesion protein paxillin is localized todiscrete dots lying just beyond the F-actin ring – these
could be proto-focal adhesion structures. One can also
see p130Cas, activated FAK and phosphotyrosine staining in dots outside of the actin ring. A microtubule
network forms between the F-actin ring and the nucleus. We conclude that plating mouse ﬁbroblasts onephrin A1 stimulates phosphotyrosine signaling at theleading edge of the spreading cell with concomitantformation of focal adhesions, and inside that zone theassembly of a very striking annulus of F-actin.

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To test whether actin reorganization is directly due
to EphA signaling, we generated an activated mutant
form of EphA2, which was expressed stably in NIH3T3
cells following infection with a retrovirus vector. To
make the activated mutant, we fused the 14 amino acids
c-Src myristoylation sequence, which will dictate Nterminal myristoylation and plasma membrane association, followed by a constitutive dimerization domain,comprised of the GCN4 transcription factor leucine zipper, to the entire cytoplasmic domain of human EphA2.
We expected that Myr-GLZ-EphA2 would localize tothe plasma membrane, and dimerize through the GCN4
leucine zipper, which would promote transphosphorylation and lead to constitutive activation. As a control wemade and expressed a kinase-dead Myr-GLZ-EphA2
mutant. The wild type but not the kinase-dead MyrGLZ-EphA2 contains high levels of P.Tyr, consistent
with it being constitutively active. When NIH3T3 cells
expressing activated Myr-GLZ-EphA2 are plated onpolylysine these cells attach, spread, and exhibit aperipheral actin ring similar to that seen in cells plated
on ephrin A1, whereas cells expressing the kinase-dead
Myr-GLZ-EphA2 do not. We conclude that EphA2
signaling per se can induce cell spreading and F-actin
reorganization.
What sort of signaling events occur in ephrin A1stimulated ﬁbroblasts? When one analyzes tyrosine
phosphorylation by blotting with anti-P.Tyr antibodies,
phosphorylation of several proteins, including FAK and
p130Cas, is stimulated when cells are plated on ephrin
A1. In addition, p130Cas association with Crk is induced. To determine whether FAK and p130Cas are
required for ephrin A1-stimulated cell spreading, we
tested a panel of knockout mouse embryo ﬁbroblasts(MEF) for their ability to spread on ﬁbronectin and on
ephrin A1. Wild type and FAK/ MEFs spread on
ﬁbronectin. However, although wild type MEFs are wellspread by 60 minutes on ephrin A1, FAK/ MEFs
attach but fail to spread. But, when FAK is reexpressedin the FAK/ cells, they spread normally. Likewise,
MEFs deﬁcient for p130Cas spread on ﬁbronectin but
fail to spread on ephrin A1, unless p130Cas is reexpressed. This type of analysis has told us that FAK, Src
family kinases and p130Cas are critical for spreading on
ephrin A1, whereas they are not essential for spreading
on ﬁbronectin. Similar analysis showed that c-Abl/Arg
are not required for spreading on ephrin A1, which will
be important for what I will tell you next. Inhibitory
antibodies to a number of integrins that are expressedon ﬁbroblasts block spreading on ephrin A1, indicatinga requirement for integrins. For instance, spreadingof AG1523 human ﬁbroblasts on ephrin A1 is inhibitedby the RGDS peptide, which antagonizes most of theintegrins, and also by anti-a4 and anti-a5 integrin subunit antibodies.

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Hunter T: Tyrosine phosphorylation in signaling and disease

Fig. 4Ephrin A/EphA signaling to the actin cytoskeleton.

We conclude that in ﬁbroblasts ephrin A/EphA signaling promotes F-actin polymerization at the boundaryof the spreading cell. Based on the results we obtainedwith the constitutively active form of EphA2, we deduce that F-actin organization is a direct consequenceof EphA2 signaling. However, we do not yet have aprecise model for how this happens (Fig. 4). With regard to the players downstream of EphA, we havelearned that FAK, Src family kinases and p130Cas are
essential. Integrins are somehow activated in this process and are required for spreading, but we do notknow what ligand the integrins bind to, if anything. We
know that ephrin A, which in vivo would be presented
on an adjacent cell, causes clustering and activation of
EphA, leading to FAK activation and processes downstream of FAK. We ﬁnd that p130Cas becomes associated with Crk, and therefore integrin activation could
be occurring through Crk-associated C3G, a GTP exchange factor for R-Ras, which is known to activate
integrins. p62Dok bound to RasGAP may act downstream of EphA to inhibit ERK MAP kinase. In addition, EphA may provide more direct signals to actin
through association with an ephexin-like Rho family
GEF, which could activate Rac and Rho leading toactivation of the Pak and ROCK kinases and increased
F-actin formation. F-actin assembly may also be triggered through Nck, whose SH2 domain binds to

p130Cas when it is tyrosine phosphorylated. Nck associates via its SH3 domains with N-WASP, which in turnbinds to the Arp2/3 complex that initiates F-actin assembly and branching. Clearly, we still have a lot more
to do to understand exactly what happens when EphA
has been activated in ﬁbroblasts.
On the face of it our results are in contrast to thecurrent model for what occurs when a growth cone ofan extending axon encounters an ephrin-expressing cell,
where disassembly of F-actin is promoted and thegrowth cone stops or turns away. To reconcile this discrepancy we propose that ephrin/Eph signaling regulates local F-actin dynamics, and that depending onother factors, such as ephrin/Eph signal strength, andwhich Rho family GEFs are expressed, it can promotelocal assembly or disassembly of F-actin, and that the
net consequence is reorganization of the actin cytoskeleton.
Abl Tyrosine Kinase and the Actin Ctoskeleton
Let me tell you one ﬁnal story concerning the Abltyrosine kinase. The domain structure of the N-terminal
half of c-Abl and the closely related Arg tyrosine kinase
is rather similar to that of the Src-family kinases, with
SH3 and SH2 domains followed by the catalytic domain. But in contrast to Src family kinases, c-Abl/Arg

Keio J Med 2002; 51 (2): 61–71

lack a regulatory tyrosine residue at the C terminus andhave a very long C terminal extension, which contains
several SH3 binding sites, a DNA-binding domain, anda G-actin binding domain, and at the very C-terminus
an F-actin binding domain. c-Abl is present both in the
nucleus and the cytoplasm. c-Abl has three NLS and anuclear export signal, which allow shuttling of c-Abl
between the cytoplasm and the nucleus. F-actin andG-actin can both bind to the C terminal region, and anumber of SH3-containing proteins, including Abi, Crk,
Grb2, and Nck interact with PXXP motifs in the
proline-rich domain. The SH2-catalytic domain linker
region is important in negative regulation of kinase
activity, and very recent evidence suggests that the N
terminus also plays a key role in negative regulation bybinding back to the SH3 and SH2 domains.
A number of factors that regulate c-Abl kinase
activity have been identiﬁed. Cell adhesion, which I am
going to discuss, and growth factors like PDGF bothactivate cytoplasmic c-Abl. Indeed, there is evidence
that c-Abl is required downstream for the PDGF mitogenic response. DNA damage activates nuclear c-Abl,and nuclear c-Abl is activated in the cell cycle in the Sphase, when Rb is phosphorylated. Oncogenic constitutively active forms of Abl can be created by deleting the SH3 domain or by fusing the Bcr oligomerization domain in place of the N-terminal regulatory‘‘cap’’. Knockout phenotypes have been quite informative: c-Abl knockout mice are runted, and most die
postpartum; the survivors develop lymphoid defects.
c-Abl/Arg double knockout mice die during embryogenesis at around E9-11 due to a neurulation defect. cAbl is expressed ubiquitously and colocalizes with Arg
and F-actin at the apical surface of the developing neuroepithelium.
I am going to focus on the interactions between cAbl and F-actin. There is a lot of evidence connecting Abl and F-actin. For instance, c-Abl/Arg double
knockout embryos have disruptions in the F-actin latticework of the neuroepithelium. And c-Abl knockoutﬁbroblasts show delayed rufﬂing and F-actin ring formation in response to PDGF. PDGF also induces colocalization of WAVE1, one of the WASP family members,
with c-Abl at rufﬂing cell membranes. Genetic evidence
from Drosophila also provides a connection between Factin and Abl. Enabled, a relative of vertebrate Menaand VASP, is a suppressor of loss of function Dabl
mutations, and negatively regulates cell motility andpotentiates outgrowth of actin-rich structures.
Pam Woodring, a postdoctoral fellow in my group,
has been trying to understand how c-Abl is regulated byadhesion and whether c-Abl in turn regulates adhesion.
In particular, she has examined whether F-actin might
regulate c-Abl, and conversely whether c-Abl modulates F-actin assembly. We started from the observa-

69tion made over ﬁve years ago that when ﬁbroblasts
are detached from the culture dish c-Abl kinase activity
is signiﬁcantly reduced, when immunoprecipitates areassayed using RNA polymerase II CTD as a substrate.13 Conversely, when detached cells are replated
on ﬁbronectin, c-Abl activity is increased.
Because there is an F-actin binding domain in c-Abl
and because the actin cytoskeleton undergoes rearrangement when adherent cells are detached, we set out
to test whether F-actin itself might regulate Abl kinase
activity. We found that puriﬁed F-actin inhibits the kinase activity of puriﬁed Abl in vitro in a dose-dependent
fashion.14 A c-Abl mutant that lacks the C terminal Factin binding domain is not inhibited by F-actin. Further analysis showed that the binding and inactivationof c-Abl by F-actin requires both the F-actin binding
domain and the SH2 domain. Moreover, when various
c-Abl mutants are stably expressed in Abl/ MEFs,
deletion of either the F-actin-binding domain or theSH2 domain is sufﬁcient to prevent the inactivation ofc-Abl upon detachment. We are not exactly sure how
F-actin inhibits catalytic activity. No structure of fulllength c-Abl is available, and there are only individual
structures of the catalytic domain and the SH2 and SH3
domains. In consequence, we cannot say how this verylarge protein might fold up to adopt an inhibited conformation in the presence of F-actin. There is evidence
that the SH3 domain binds to the SH2 catalytic domain
linker in a proline-dependent fashion and that thiscontributes to the inhibitory mechanism, and bindingof the N terminal cap to the SH3 and SH2 domains
is also important. Perhaps, F-actin binding to the Cterminal domain stabilizes the inhibited conformation
through interaction with the SH2 domain. A possiblemodel for F-actin regulation of c-Abl is shown in Figure
5A.
After obtaining evidence that c-Abl activity was
inhibited by F-actin in vitro, we next wanted to try toshow that this also happens in the cell. For this purpose,
we did three types of experiment.15 First, we showedthat when ﬁbroblasts are detached the amount of actin
present in c-Abl immunoprecipitates increases withtime, and that this parallels the loss in c-Abl kinase
activity. In addition, using immunoﬂuorescence staining
we could show that there is signiﬁcant colocalizationof F-actin and Abl around the periphery of detachedcells, whereas there is relatively little colocalization in
attached cells. Finally, we obtained compelling evidence that F-actin actually does inhibit c-Abl activity
in vivo by using latrunculin-A, an inhibitor of F-actin
polymerization that acts by binding to actin monomers
and preventing them from incorporating into growing chains. When detached cells are treated with thelatrunculin-A, c-Abl kinase activity is restored, and at
the same time there is a decrease in actin association.

70

Hunter T: Tyrosine phosphorylation in signaling and disease

Fig. 5A

c-Abl regulation by F-actin.

Moreover, latrunculin A treatment abolishes the colocalization of F-actin and c-Abl in detached cells. Thus,
we conclude that F-actin is a negative regulator of cAbl in the cell.
What about the converse possibility that c-Abl
activity regulates the actin cytoskeleton? One piece ofevidence in favor of this idea is that c-Abl/ ﬁbroblasts are delayed in spreading on ﬁbronectin comparedto c-Ablþ/þ cells, and normal spreading is restored
when an endogenous level of c-Abl is reexpressed.15
When ﬁbroblasts spread on ﬁbronectin they send out
ﬁlopodial surface microspikes that contain F-actin.
Abl/ MEFs send out fewer microspikes than cells
expressing wild type c-Abl, whereas cells expressing theDF-actin c-Abl mutant, which is resistant to F-actin inhibition, show a more extensive array of F-actin-positive
surface protrusions. Even more strikingly, expression of
DF-actin c-Abl mutant induces the formation of F-actin
positive surface ﬁlopodia in detached cells, whereas expression of wild type c-Abl does not. The STI571 Abl
inhibitor blocks peripheral microspike formation in detached cells expressing DF-actin c-Abl proving that thisphenotype is due to Abl kinase activity. Interestingly,
when these cells are stained not only for F-actin, but also
for P.Tyr, the very tips of the microspikes are strongly
positive for P.Tyr, suggesting that Abl-dependent tyrosine phosphorylation occurs at the tips of these microspikes, and may be responsible for their formation. We
are now trying to identify the protein(s) that are phosphorylated by c-Abl in order to promote the protrusionof ﬁlopodia.
Based on these experiments, we conclude that c-Abl
can be negatively regulated in vitro and in vivo by Factin. We also conclude that Abl kinase activity canpromote F-actin assembly in ﬁbroblasts, and that c-Abl/
Arg activity is required for efﬁcient cell spreading andF-actin assembly. To extend our studies we turned tothe interaction of c-Abl and F-actin in neurons, where
there is strong genetic evidence from mouse and Drosophila mutant phenotypes of the importance of thesetyrosine kinases in regulating F-actin and neuronal
function. In collaboration with David Litwack and
Dennis O’Leary we examined E18 rat embryonic cortical neurons where after two days in culture longextended neurites send out F-actin containing side
branches or ﬁlopodia.15 In contrast when these cells aregrown in the presence of STI571 to inhibit c-Abl andArg kinase activity, the formation of these branches isalmost completely blocked. Conversely, when DF-actinc-Abl is transiently expressed in E18 cortical neurons,
the number and length of the branches is greatly increased compared to those in neurons exogenously
expressing wild type c-Abl. Quantiﬁcation shows that
there are on average 2.7 one mm branches per 100 mm
neurite length in c-Abl expressing cells. Whereas cells
expressing DF-actin c-Abl have 8.0 branches. As analternative approach we made use of c-Abl knockout
mice to see whether we could establish genetically arequirement for c-Abl in neurite branching. Ablþ/þ
E17 cortical neurons had on average 7.0 branches per

Keio J Med 2002; 51 (2): 61–71

71
Abl and F-actin interactions was carried out by Pam Woodring. Hisamaru Hirai provided the p130Cas knockout ﬁbroblasts, and the Ablexperiments were carried out in collaboration with David Litwack and
Dennis O’Leary at the Salk Institute, and Jean Wang at UCSD. The
bioinformatics analysis of the kinome was conducted by Gerard Manning, Ricardo Martinez, David Whyte and Sucha Sudarsanam at
SUGEN. Research in my group is supported by USPHS grants fromthe National Cancer Institute. T.H. is a Frank and Else Schilling
American Cancer Society Research Professor.

References
Fig. 5B

Reciprocal regulation of Abl and F-actin.

100 mm, Ablþ/ neurons had 5.3 branches, and Abl/
neurons had 3.0 branches Clearly, there is a c-Abl genedosage dependent increase in branching frequency,
consistent with the data obtained with STI571.
Let me summarize the relationships we have discerned between F-actin and c-Abl. In ﬁbroblasts, expression of activated forms of Abl results in an increasein peripheral active microspike formation; and conversely loss of Abl function decreases this. The same
appears to hold true for neurite branching in corticalneurons, where inhibition of c-Abl/Arg or reduction ofc-Abl; level results in reduced branching, and expression of activated forms of c-Abl stimulates branching.
Surface microspike or ﬁlopodium formation is believed
to be important for the exploratory behavior of cells as
they migrate, and in the case of neurons these ﬁlopodia
may make contact with neighboring axons, and be theprecursors to synapses formed by dendritic spines.
Overall, we conclude that there is bidirectional regulation of Abl and the F-actin cytoskeleton. Active Ablcan stimulate an F-actin polymerization, whereas F-actin
reduces Abl kinase activity and may act as a feedback
mechanism to restrict the action of c-Abl in promoting
F-actin assembly (Fig. 5B).
Epilogue
Let me conclude by saying that it is a rare privilege
to have witnessed the events and the progress that haveled from discovery of tyrosine phosphorylation over 20
years ago to the development of drugs that block theactivity of oncogenic tyrosine kinases and are being usedsuccessfully to treat cancer and other human disease.
Acknoweldgements: The work on the SCFR/Kit mutant mice was doneby Peter Blume-Jensen, with help from Steven Kwoh. The EphA2
signaling project was carried out by Nigel Carter, and the work on

1. Hunter T: A thousand and one protein kinases. Cell 1987; 50:
823–829
2. Plowman GD, Sudarsanam S, Bingham J, Whyte D, Hunter T:
The protein kinases of Caenorhabditis elegans: a model for signal transduction in multicellular organisms. Proc Natl Acad Sci
USA 1999; 96: 13603–13610
3. King N, Carroll SB: A receptor tyrosine kinase from choanoﬂagellates: molecular insights into early animal evolution. Proc
Natl Acad Sci USA 2001; 98: 15032–15037
4. Manning G, Plowman GD, Hunter T, Sudarsanam S: Evolutionof protein kinase-based signaling. Trends Biochem Sci 2002 (inpress)
5. Manning G, Whyte DH, Martinez R, Hunter T, Sudarsanam S:
Diversity and novelty in the kinase complement of the humangenome. Science 2002 (in press)
6. Copley RR, Doerks T, Letunic I, Bork P: Protein domain analysis in the era of complete genomes. FEBS Lett 2002; 513: 129–
134
7. Cohen P: Protein kinases: the major drug targets of the twentyﬁrst century? Nat Rev Drug Discov 2002; 1: 309–315
8. Shawver LK, Slamon D, Ullrich A: Smart drugs: tyrosine kinase
inhibitors in cancer therapy. Cancer Cell 2002; 1: 117–123
9. Druker B, Sawyers CL, Kantarjian H, Resta DJ, Reese S, Ford
JM, Capdeville R, Talpaz M: Activity of a speciﬁc inhibitor of
the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 2001; 344: 1038–1042
10. Blume-Jensen P, Jiang G, Hyman R, Lee KF, O’Gorman S,
Hunter T: Kit/stem cell factor receptor-induced activation ofphosphatidylinositol 3 0 -kinase is essential for male fertility. Nat
Genet 2000; 24: 157–162
11. Schmucker D, Zipursky SL: Signaling downstream of Ephreceptors and ephrin ligands. Cell 2001; 105: 701–704
12. Carter N, Nakamoto T, Hirai H, Hunter T: Ephrin A1-induced
cytoskeletal reorganisation requires FAK and p130cas. Nat Cell
Biol 2002 (in press)
13. Lewis JM, Baskaran R, Taagepera S, Schwartz MA, Wang JY:
Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic-nuclear transport. Proc Natl Acad Sci USA 1996; 93:
15174–15179
14. Woodring PJ, Hunter T, Wang JYJ: Inhibition of c-Abl tyrosine
kinase activity by ﬁlamentous actin. J Biol Chem 2001; 276:
27104–27110
15. Woodring PJ, Litwack ED, O’Leary DD, Lucero GR, Wang JY,
Hunter T: Modulation of the F-actin cytoskeleton by c-Abltyrosine kinase in cell spreading and neurite extension. J Cell
Biol 2002; 156: 879–892