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Oncogene (2001) 20, 6348 ± 6371
2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00
www.nature.com/onc
Crk family adaptors ± signalling complex formation and biological roles
Stephan M Feller*,1
1
Cell Signalling Laboratory, Imperial Cancer Research Fund, University of Oxford, Institute of Molecular Medicine, John Radcli€e
Hospital, Headington, Oxford OX3 9DS, UK
Crk family adaptors are widely expressed and mediate
the timely formation of signal transduction protein
complexes upon a variety of extracellular stimuli,
including various growth and di€erentiation factors.
Selective formation of multi-protein complexes by the
Crk and Crk-like (CRKL) proteins depends on speci®c
motifs recognized by their SH2 and SH3 domains. In the
case of the ®rst SH3 domains [SH3(1)] a P-x-x-P-x-K
motif is crucial for highly selective binding, while the
SH2 domains prefer motifs which conform to the
consensus pY-x-x-P. Crk family proteins are involved
in the relocalization and activation of several di€erent
e€ector proteins which include guanine nucleotide
releasing proteins like C3G, protein kinases of the Abland GCK-families and small GTPases like Rap1 and
Rac. Crk-type proteins have been found not only in
vertebrates but also in ¯ies and nematodes. Major insight
into the function of Crk within organisms came from the
genetic model organism C. elegans, where the Crkhomologue CED-2 regulates cell engulfment and phagocytosis. Other biological outcomes of the Crk-activated
signal transduction cascades include the modulation of
cell adhesion, cell migration and immune cell responses.
Crk family adaptors also appear to play a role in
mediating the action of human oncogenes like the
leukaemia-inducing Bcr ± Abl protein. This review summarizes some key ®ndings and highlights recent insights
and open questions. Oncogene (2001) 20, 6348 ± 6371.
Keywords: v-Crk; c-Crk; CRKL; SH2; SH3
Historical beginnings
A chicken tumour sample originally collected in the
1930s by Albert Claude at the Rockefeller Institute in
New York was ®nally, in the 1980s, `recycled' by Bruce
Mayer, a graduate student from the laboratory of
Saburo Hanafusa, in the search for novel oncogenes
(Mayer et al., 1988). Surprisingly, the ®brosarcomainducing oncogene identi®ed from this tumour material
did not contain an oncogene with recognizable
catalytic activity, but rather encoded for a fusion gene
of the viral gag gene (which encodes major structural
proteins of the virion) and a cellular gene which
encodes two domains, initially only recognized by their
*Correspondence: SM Feller; E-mail: [email protected]
homology to regions of other signalling proteins like
non-receptor tyrosine kinases and phospholipase C-g.
Despite the absence of an apparent catalytic domain
in the oncogene, Mayer and colleagues noted a
remarkable increase of tyrosine-phosphorylated proteins in chicken embryo ®broblasts transformed by this
virus. This seemed to suggest that the new oncogene
was activating a cellular tyrosine kinase and led to the
name crk (for chicken tumour virus no. 10 [CT10]
regulator of kinase; Mayer et al., 1988).
Shortly after the v-crk gene was isolated from the
CT10 retrovirus, an independent viral isolate named
avian sarcoma virus 1 (ASV-1; Tsuchie et al., 1989)
was found to contain a nearly identical oncogene. Both
oncoproteins di€er only in a few amino acids, which do
not appear to be crucial for the biological potency of
the tumor virus, although a direct comparison of both
oncogenes has not been published and ultrastructural
data do suggest signi®cant di€erences in protein
binding properties (Wu et al., 1995; discussed further
below). Only a single study so far used cells carrying
the ASV-1 or CT10 oncogenes side-by-side (Sakai et
al., 1994b). However, these cells were from di€erent
species and the focus of the study was on the
puri®cation of a v-Crk associated phosphotyrosylprotein, now known as p130Cas.
Following the sequencing of the cellular cDNA of
chicken Crk (Reichman et al., 1992), Crk family
adaptor cDNAs have been sequenced from di€erent
higher eukaryotes (Matsuda et al., 1992; KizakaKondoh et al., 1996; Evans et al., 1997; Galletta et
al., 1999; Reddien and Horvitz, 2000). Human and
mouse Crk-like (CRKL), the products of Crk-homologous but distinct genes, were also cloned shortly after
the ®rst c-Crk sequences had been published (ten
Hoeve et al., 1993; de Jong et al., 1995a). An alignment
of most protein sequences can be found in Galletta et
al. (1999). Unlike CRKL, c-Crk is expressed as two
distinct proteins of 28 and 40 kDa, respectively
(Matsuda et al., 1992). c-Crk I consists of one SH2
and one SH3 domain, while c-Crk II has an additional
SH3 domain separated by a `spacer region' of
approximately 60 amino acids.
Identi®cation of the viral Crk oncoprotein (v-Crk;
p47gag ± crk) and cloning of its cellular counterparts, as
well as the subsequent analyses of their biological roles,
fundamentally changed our view on intracellular signal
transduction. It became clear that the transmission of
signals within cells heavily relies on the formation of
Crk family adaptors
SM Feller
protein complexes which are based on selective protein
domain interactions (Pawson, 1995; Bork et al., 1997;
Pawson and Scott, 1997; Pawson and Nash, 2000).
Very early on, it was shown that the CT10-derived vCrk protein associates with `type I' phosphatidylinositol (PI) kinase activity (Fukui et al., 1989) although
these experiments have not been followed-up much
until quite recently (Akagi et al., 2000), and there is at
present no evidence in the literature indicating that the
activation of tyrosine kinase(s) by v-Crk critically
depends on the action of PI3 kinase.
However, it was rapidly noted that v-Crk forms
stable complexes with two prominent tyrosine-phosphorylated proteins of 70 and 135 kDa in CT10infected chicken ®broblasts (Mayer et al., 1988; details
to follow). Since then, the identi®cation of these and
other Crk/CRKL binding proteins has greatly improved our insight into the components and functional
details of the cellular signalling machinery.
Several reviews have so far discussed the accumulated knowledge or certain aspects of Crk family
adaptors (Feller et al., 1994a, 1998; Birge et al., 1996;
Kiyokawa et al., 1997; Sattler and Salgia, 1998; Buday,
1999; Kolibaba et al., 1999; Platanias and Fish, 1999;
Stupack et al., 2000; Furge et al., 2000; Voss et al.,
2001). However, Crk family proteins continue to be a
focus of considerable research activity, with over one
hundred new studies published per year on their
various isoforms, homologues and binding partners.
In the last few years major advances in understanding
the biological function of Crk adaptors have been
made, for example through the analysis of genetic
model systems like C. elegans and Drosophila and,
most recently, the targeted disruption of the crkl gene
locus in the mouse (Guris et al., 2001). This review's
aim is to summarize key ®ndings and to highlight
recent progress in understanding the biological functions, binding proteins and mechanistic details of Crk
adaptor signal transmission.
Protein ± protein interaction domains
A comparison of the amino acid sequences of Crk
family adaptors (Galletta et al., 1999) shows a high
homology between the family members c-Crk and
CRKL, as well as between di€erent eukaryotic species
within the regions of the protein now widely known as
Src homology 2 and 3 (SH2 and SH3) domains. These
domains are globular protein interaction modules
found in many cellular proteins (Musacchio et al.,
1994; Pawson, 1995; Cohen et al., 1995; Kuriyan and
Cowburn, 1997; Mayer and Gupta, 1998). They were
initially discovered by homology comparision (Sadowski et al., 1986; Mayer et al., 1988). The N- and Ctermini of these domains are in close proximity, which
allowed them throughout evolution to be newly
inserted into proteins without too much disruption of
the overall protein structures. SH2 and SH3 domains
are also the almost exclusive building blocks of a group
of small adaptor proteins, which includes in addition to
the Crk and CRKL proteins the Grb2 and Nck
adaptor families, known to play important roles in
the regulation of cell proliferation, di€erentiation,
immune receptor signalling etc. (Li and She, 2000;
Norian and Koretzky, 2000; Li, 2001, this issue;
McGlade, 2001, this issue; for domain composition of
small SH2/SH3 adaptors see Feller et al., 1998). The
SH2 domains of these adaptors are often the
connection to the upstream partner(s) within a
signalling cascade, while SH3 domains serve in many
cases to couple a protein to its e€ector(s).
6349
SH2
SH2 domains bind to protein motifs which usually
contain a phosphorylated tyrosine residue followed by
speci®c residues (often in positions +2 to +5). The
SH2 domain interacting with tyrosine-phosphorylated
proteins was ®rst recognized through the analysis of
the v-Crk protein, which stably associates with heavily
tyrosine-phosphorylated proteins in v-Crk-transformed
®broblasts (Mayer et al., 1988; Mayer and Hanafusa,
1990; Matsuda et al., 1990, 1991). Those ®ndings were
rapidly con®rmed for other SH2 domains by many
subsequent studies. One hundred and nineteen genes
with SH2 domains exist in the human genome
according to one study (Lander et al., 2001) while
another predicts a minimum of 141 (SMART program,
EMBL website; Schultz et al., 2000). For the Crk SH2
domain the preferred binding motif is pY-x-x-P. This
preference was initially identi®ed by an in vitro
approach (Songyang et al., 1993) and subsequently
con®rmed by mutational analyses of several Crk SH2
domain binding partners. Multiple pY-x-x-P motifs are
present in prominent Crk SH2 binding proteins like
paxillin (p70; Birge et al., 1993; Schaller, 2001, this
issue), p130Cas (Sakai et al., 1994a; Bouton, 2001, this
issue) and its homologues, the ubiquitin-ligase c-Cbl
(p120; Smit and Borst, 1997; Tsygankov, 2001, this
issue) and the large docking protein Gab1 (GarciaGuzman et al., 1999; Sakkab et al., 2000). The binding
speci®city of the highly homologous SH2 domains of
Crk and CRKL is apparently quite similar (Feller et
al., 1995) and many of the proteins shown to bind to
the c-Crk and v-Crk SH2 domains also co-immunoprecipitate with CRKL (de Jong et al., 1995b; Salgia et
al., 1995, 1996; and others). Surprisingly, a somewhat
di€erent binding motif was reported for the Xenopus
Crk SH2 domain which recognizes two Y-x-x-L motifs
in the apoptosis-inducing cell cycle regulator XWee-1
(Smith et al., 2000).
SH3
The ®rst publication implicating SH3 domains as
protein ± protein interaction domains, was a report
that a protein named 3BP-1 (SH3 binding protein 1)
bound to the Abl SH3 domain (Cicchetti et al., 1992)
in an expression library screen using the SH3 domain
Oncogene
Crk family adaptors
SM Feller
6350
Oncogene
as a probe and in other in vitro binding assays. 3BP-1
is a GTPase-activating protein (GAP) for Rac
(Cicchetti et al., 1995). At present it is believed that
the interaction of 3BP-1 with the Abl SH3 domain
does not occur in physiological situations. Shortly after
the 3BP-1 identi®cation as Abl SH3 binder, it was
shown that a short, proline-rich protein fragment of
3BP-1 is necessary and sucient to bind the Abl SH3
domain (Ren et al., 1993). It is now known that SH3
domains often bind to proline-rich protein motifs and
peptides conforming to a core consensus P-x-x-P plus
some speci®city-determining additional residue(s) in the
vicinity. Nevertheless, some prominent examples are
known which deviate from this common theme: the
tumour suppressor gene product p53 forms a complex
with an SH3-containing protein named p53BP2
(Gorina and Pavletich, 1996), with the SH3 domain
of p53BP2 recognizing a bipartite motif in the `L3loop' of p53. The SH3 domain of the Eps8 protein,
which regulates EGF receptor signalling and tracking
(Lanzetti et al., 2000) is apparently only functional as
an intertwined SH3 domain dimer (Kishan et al., 1997)
and recognizes a P-x-x-D-Y motif (Mongiovi et al.,
1999). Another example is the SH3 domain of the
immune cell adaptor FYB (also known as SLAP),
which binds a R-K-x-x-Y-x-x-Y motif of SKAP55, yet
another immune cell adaptor protein (Kang et al.,
2000). The SH3 domains of the tyrosine kinases Fyn
and Lck also bind to this motif, albeit 10-fold weaker.
Most recently the Grb2 SH3(C) domain was shown to
bind to the core motif P-x-x-x-R-x-x-K-P, which is
present in adaptors and large docking proteins like
SLP-76, Gab1 and Gab2 and AMSH (Lock et al.,
2000; Lewitzky et al., 2001).
At present more than 800 SH3s and SH3-like
domains are contained in the various databases, over
200 in the human genome alone (Lander et al., 2001).
SH3-like folds are not only found in intracellular
proteins of eukaryotes but also in extracellular proteins
like MIA (Stoll et al., 2001), in virus genes and even in
prokaryotes (Whisstock and Lesk, 1999). The DNAbinding domain of the HIV-1 integrase has an SH3-like
structure (Eijkelenboom et al., 1995; Lodi et al., 1995).
The same is true for the metal binding domain of the
cobalt-activated diphtheria toxin repressor-DNA complex (Pohl et al., 1999).
The binding speci®city of the ®rst SH3 domain of cCrk was unravelled through analysing its complex
formation with the highly selective CrkSH3(1) binding
protein C3G, a guanine nucleotide releasing protein for
the small GTPase Rap1. C3G contains in its central
region several motifs which conform to the consensus
P-x-x-P-x-K (Knudsen et al., 1994; Tanaka et al.,
1994). Subsequent studies showed that the lysine
following in position +2 of the P-x-x-P motif is crucial
for the high anity interaction (Knudsen et al., 1995;
Wu et al., 1995). The remarkable anity and selectivity
created by the lysine residue in the P-x-x-P-x-K motif is
explained by the unique structure of the lysine-binding
pocket of the Crk SH3(1) domain. The positive charge
at the end of the lysine side chain is balanced by three
negative residues arranged in a highly symmetric
fashion, while the hydrophobic part of the lysine side
chain stacks against the hydrophobic ring structure of
a tryptophan. Switching the lysine to an arginine not
only strongly reduces the anity of the C3G-derived
peptide for the SH3 domains of c-Crk and CRKL, but
also switches its binding speci®city, thereby generating
a core binding motif for the N-terminal SH3(N)
domain of the adaptor protein Grb2 and for the
second SH3 domain of the adaptor Nck.
In this context it is interesting that the v-Crk protein
encoded by the ASV-1 retrovirus has a Crk sequence
identical to the chicken c-Crk sequence, while CT10
has several point mutations. A molecularly signi®cant
change occurs for example in the `RT-loop' of the
CT10 v-Crk SH3(1) domain: glutamic acid 384, one of
the three negatively charged residues which make
contact to the positive charge of the lysine in the Px-x-P-x-K motif, is changed to glycine. This mutation
is predicted to signi®cantly (8 ± 10-fold) weaken the
binding of CT10 v-Crk SH3(1) to the P-x-x-P-x-K
motif. Moreover, if only two of the three negatively
charged residues can make contact to the lysin, the
preferential binding of lysine vs arginine is lost,
according to computer modelling results (Wu et al.,
1995). Whether this CT10 SH3(1) domain mutation
also leads to a switch in the biological pathways used
by CT10 vs ASV-1 is not yet studied. Due to the great
abundance of viral Crk protein in virus-infected
®broblasts, even such a signi®cant mutation may be
more than compensated by sheer mass production of
the viral oncoprotein. It is also possible that the crk
gene-carrying retroviruses never use c-Crk/CRKLactivated pathways and simply `spill over' into other
signalling pathways.
There is at least one other SH3 domain in which a Px-x-P-x-K motif is part of the binding consensus,
namely the SH3 domain of the 85 kDa cytoskeletal
protein cortactin which localizes to the sites of actin
assembly (Sparks et al., 1996; Weaver et al., 2001;
reviewed in Weed and Parsons, 2001, this issue).
However, cortactin seems to preferentially recognize
sequences which have a proline in position +3 of the
core P-x-x-P motif and/or tryptophan and/or leucine in
positions +5 and +6 (P-x-x-P-x-K-P-x-W-L; Sparks et
al., 1996). The cortactin SH3 domain does not bind to
high anity CrkSH3(1) binding peptides (Oehrl, 1997)
but some overlap of CrkSH3(1) and cortactin SH3
binding using in vitro binding assays has been reported
(Yamabhai Kay, 1997). No examples of a biologically
relevant competition of the Crk and cortactin SH3
domains for the same binding partner are currently
known.
Parrini and Mayer have recently generated a
temperature-sensitive version of the CrkSH3(1) domain, which may open new ways of looking at Crk
signalling mechanisms (Parrini and Mayer, 1999).
Furthermore, the `tool box' for the analysis of SH3
domains from adaptor proteins like Crk and Grb2 has
been extended by the generation of SH3 domain
binding peptides in which natural prolines are replaced
Crk family adaptors
SM Feller
by non-natural amino acid derivatives (Nguyen et al.,
1998, 2000; Tuchscherer et al., 2001). These peptides
bind with higher anities than the corresponding
peptides completely made up of naturally occurring
amino acids. In vivo studies with these compounds are
still missing, but it is already clear from the current
literature that Grb2 and Crk/CRKL SH3 domain
blocker peptides can be carried into cells with peptidevector systems, thereby inhibiting adaptor protein
signalling (Cussac et al., 1999; Kardinal et al., 2000,
2001; Sakkab et al., 2001).
Adaptors and enzymes which interact with Crk/CRKL
SH2 and SH3 domains
p130Cas family
Nearly all proteins which interact with the SH2 domain
of Crk family adaptors have several to many of the
already described Y-x-x-P motifs, which are sometimes
clustered within a distinct region of the binding protein
(for a structural overview see Sakkab et al., 2000). This
is especially true for the long sought p130Cas protein
(130 kDa Crk associated kinase substrate), which is
also a prominent substrate of retroviral tyrosine kinase
oncoproteins like v-Src, and was ®nally cloned by
Sakai et al., in 1994 (Sakai et al., 1994a; reviewed in
Bouton, 2001, this issue). In p130Cas no less than 15
Y-x-x-P motifs are clustered within the 260 amino acid
`substrate region' of the protein. The p130Cas protein
family now includes the homologous proteins Hef1/
Cas-L and Efs/Sin (O'Neill et al., 2000). The overall
structural organization of p130Cas-family proteins is
quite similar to other large docking proteins like
insulin receptor substrates (IRS1 to 4), Gab (Grb2
associated binder) family proteins, their Drosophilarelative Daughter of sevenless (Dos) etc. (see also
Schlessinger (2000) for structural compositions). At the
N-terminus a single SH3 domain (in the case of Cas
family proteins) or a pleckstrin homology (PH) domain
(in Gab, IRS and DOS proteins) can be found. They
function as protein or phosphatidylinositol interaction
domains. In addition there are numerous tyrosines
clustered or scattered throughout the proteins which
serve as docking sites for various SH2 domains.
Besides Crk family adaptors, other adaptors and a
variety of enzymes are recruited to these multi-docking-site proteins, including tyrosine kinases of the FAK
and Src families, tyrosine phosphatases and, in the case
of p130Cas, the Crk SH3(1) domain-binding guanine
nucleotide exchange factor C3G which activates the
Rap1 GTPase (Kirsch et al., 1998, and references
therein).
The exact nature of the multiprotein complexes
assembled on the p130Cas family protein sca€olds
presumably varies signi®cantly, depending on cell type
and stimulus. While the SH2 domain of Crk has a
particularly great preference for the proline in position
+3 of the phosphotyrosine residue, a considerable
number of SH2 domains also display a slight
preference for proline in the same position (Abl, Nck
etc.; Songyang et al., 1993). This could lead to
competitive situations between several SH2 domaincontaining proteins in vivo unless additional conformational constraints or other not yet well de®ned
parameters further specify a certain Y-x-x-P motif for
exclusive binding of a single SH2 domain.
For a better understanding of how such large
signalling complexes are assembled, coordinated and
used to generate speci®c signals, it will be necessary to
perform in depth biochemical analyses and reconstitution experiments, similar to studies previously done
with equally complex systems, for example the basal
transcription machinery. To complicate things, it may
be necessary to include membrane and cytoskeletal
components or even focal adhesion-like structures into
such assays in order to allow the proper assembly of
the signalling complexes. These biochemical reconstitution experiments have to be accompanied by the
simultaneous observation of multiple tagged proteins
in living cells in order to validate in vitro ®ndings in
vivo.
Many of the Crk/CRKL ± p130Cas protein family
related signalling studies have been summarized elsewhere (Feller et al., 1998; Sattler and Salgia, 1998;
Cary and Guan, 1999; Schlaepfer et al., 1999; O'Neill
et al., 2000; Stupack et al., 2000; Bouton, 2001, this
issue). p130Cas-de®cient mice die `in utero' and have
severe defects in cardiovascular development and actin
reorganization (Honda et al., 1998). According to a
plethora of reports using various cell lines, p130Cas
and its siblings play in concert with Crk family
adaptors and di€erent enzymes a major role in the
reorganization of the cytoskeleton during cell shape
changes and cell migration (Klemke et al., 1998;
Cheresh et al., 1999; and others) and in mediating
signal transmission from integrins (Nojima et al.,
1995). Signals from soluble growth factors like
bombesin also require Crk ± Cas complexes (Rozengurt, 1998; Posern et al., 2000, and references therein;
see also Table 1 for other factors). Within the year
2000, reports from one group have indicated that the
human p130Cas homologue, which they renamed
BCAR1, plays a role in the antiestrogen resistance of
breast cancer cells (Brinkman et al., 2000; van der Flier
et al., 2000a,b). Also implicated in tamoxifen-resistance
of breast cancer cells is BCAR3 (van Agthoven et al.,
1998), which was also cloned under the name of AND34, binds to p130Cas and is an activator of Ras-like
GTPases including Rap1 (Cai et al., 1999; Gotoh et al.,
2000). p130Cas not only binds to AND-34 but was
also proposed to regulate its GEF-activity.
6351
Paxillin
Similar to the p130Cas protein family, the 68 ± 70 kDa
protein paxillin is a substrate for several normal and
oncogenic tyrosine kinases. It is thought to function as
a sca€old or adaptor to organize signal transduction
proteins into a large complex at `focal adhesions',
major contact points between plasma membrane and
Oncogene
Crk family adaptors
SM Feller
6352
the cytoskeleton and a site of integration for growth
and adhesion signals (reviewed in Turner, 2000;
Schaller, 2001, this issue). Paxillin also regulates cell
morphology and motility (Nakamura et al., 2000; Petit
et al., 2000 and references therein).
Several p130Cas binding proteins have been reported
to also bind to paxillin, including Src and FAK family
kinases. p130Cas and paxillin are thought to be
physically linked ± at least indirectly ± since they are
both parts of a large multiprotein complex (Turner,
2000). Recent data led to the hypothesis that paxillin
and p130Cas may oppose each other functionally in
regulating the movement of certain cells (Yano et al.,
2000b). Paxillin is attached to the cell membrane via its
C-terminal LIM domains (Dawid et al., 1998) and
binds to a4-integrins (Liu et al., 1999a). It also binds to
the Arf-GAP protein p95PKL (paxillin ± kinase linker)
via a leucine-rich repeat (LD4 motif) which leads to its
indirect interaction with PAK (p21 GTPase-activating
kinase), Nck and PIX (PAK-interacting exchange
factor) (Turner et al., 1999).
Moreover, paxillin also contains two motifs (Y31
and Y118) which conform to the Y-x-x-P consensus
and are functional Crk/CRKL SH2 domain binding
sites (Schaller and Parsons, 1995; Petit et al., 2000).
Binding of c-Crk and CRKL to these sites after their
phosphorylation leads to an accumulation of Crk
e€ectors bound via the Crk/CRKL SH3 domains at
sites of paxillin abundance. Paxillin is also strongly
hyperphosphorylated in v-Crk-transformed cells and
was identi®ed as a binding partner of the v-Crk SH2
domain (Birge et al., 1993). It appears in several splice
variants which di€er at their N-termini. Whether the
di€erential splicing a€ects Crk/CRKL binding has yet
to be studied. Furthermore, several paxillin-homologous proteins exist, but they do not seem to interact
with Crk family adaptors (Thomas et al., 1999).
Paxillin-de®cient mice su€er from early embryonic
lethality indicating that the other paxillin family genes
(Hic-5, leupaxin) have distinct functional roles from
paxillin (Turner, 2000).
Cbl and Cbl-b
p120 c-Cbl is another Crk/CRKL SH2 binding protein
which forms tyrosine phosphorylation-dependent, inducible or constitutive complexes, depending on the
cell type and situation studied (reviewed in Feller et al.,
1998; Lupher et al., 1999; and others). In Abltransformed cells, two phosphorylated Y-x-x-P motifs
(Y700, Y774) were identi®ed as the major CRKL SH2
domain docking sites (Andoniou et al., 1996). T- and
B-lymphocytes were found in many studies to
phosphorylate c-Cbl and to build complexes of Cbl
with di€erent adaptor proteins, including Crk and
CRKL, upon activation of the T- or B-cell receptors
although the function of these complexes is not well
understood. Cbl association with CRKL has been
suggested to play a role in the maintenance of T-cell
non-responsiveness (anergy) which occurs when the Tcell receptor is stimulated in the absence of a co-
Oncogene
stimulus. It is thought that this e€ect is accomplished
by blocking IL-2 action through a TCR ± Cbl ±
CRKL ± C3G ± Rap1 pathway (Boussiotis et al.,
1997). However, this ®nding has not yet been further
con®rmed by other studies.
Certain cytokines and di€erentiation factors, for
example erythropoietin (Epo) also lead to Cbl
phosphorylation and the formation of Cbl ± CRKL
complexes (Barber et al., 1997; Chin et al., 1997; and
others). More recently, Cbl phosphorylation and
complex formation was also reported for non-haematopoietic cells in response to hepatocyte growth factor
(HGF)-activation of the c-Met tyrosine kinase receptor
and in cells transfected with the transforming fusion
protein Tpr-Met (Garcia-Guzman et al., 2000).
Until two years ago, c-Cbl was believed to be
basically a large adaptor protein not unlike p130Cas or
paxillin. Then it was discovered that the Ring ®nger
(RF) domain of c-Cbl has in fact E3-type ubiquitin
ligase activity which plays an important role in the
proteolytic down-modulation of cell surface receptor
tyrosine kinases by attaching the 76 amino acid
polypeptide ubiquitin onto the receptor protein, thereby marking the receptors for proteasomal destruction
(Joazeiro et al., 1999; Levkowitz et al., 1999; reviewed
in Sawasdikosol et al., 2000). In other cases,
ubiquitination may also have di€erent functions, for
example to serve as a sorting signal in endocytosis or in
DNA repair (Dubiel and Gordon, 1999; Rotin et al.,
2000; Jentsch and Pyrowolakis, 2000). Could c-Crk
and/or CRKL serve to present proteins to Cbl for
ubiquitination and would this lead to protein degradation or the regulation of endocytosis? No answers to
these questions can be given at this point, although
p130Cas has recently been reported to function in
integrin-mediated endocytic adenovirus entry (Li et al.,
2000b).
Latest studies indicate that Cbl associates with
specialized membrane structures called `lipid rafts'
upon certain stimuli (Baumann et al., 2000; Lafont
and Simons, 2001; Saltiel, 2001). If, and how this
a€ects or involves Crk adaptor family signalling also
remains to be studied. A Cbl-relative, Cbl-b, which was
also reported to interact with Crk (Elly et al., 1999) can
function as a negative regulator of lipid raft formation
in T cells (Krawczyk et al., 2000).
IRS proteins
The insulin receptor and the IGF-1 receptor phosphorylate upon stimulation a family of multi-site docking
proteins which are known as insulin receptor substrate
(IRS) proteins and include four members (IRS-1 to
IRS-4; recently reviewed in White, 1998; Giovannone
et al., 2000; Whitehead et al., 2000; Bevan, 2001; Burks
and White, 2001). As brie¯y mentioned above, IRS
proteins are characterized by the presence of Nterminal PH domains which bind to phosphoinositides
(Razzini et al., 2000). These are followed by phosphotyrosine binding (PTB) domains which recognize
speci®c phosphotyrosine-containing epitopes, for ex-
Crk family adaptors
SM Feller
Table 1 Selected examples of oncogenes, receptor ligands and other stimuli proposed to induce/alter Crk/CRKL complex formation
Ligand/stimulus
Receptor/oncogene
via SH2a
via SH3a
Reference(s)
v-Crk
paxillin,
p130Cas
c-Abl
C3G
DOCK180
PI kinase
Bcr-Abl
paxillin,
c-Cbl,
SHIP1
Bcr-Abl
SoS
Fukui et al., 1989
(Birge et al., 1992)b
Birge et al., 1993
Knudsen et al., 1994
Feller et al., 1994b
Sakai et al., 1994a
Kiyokawa et al., 1998
ten Hoeve et al., 1994b
Oda et al., 1994
de Jong et al., 1995b
Salgia et al., 1995
Sattler et al., 1996
Sattler et al., 2001
Voss et al., 2000
Bocciardi et al., 1997
Tel-Abl
Retm
paxillin
Angiopoietin-1
Angiotensin II
anti-BCR
Tek/Tie2
Bombesin
(*)
anti-CD2
EGF
bombesinR
p130Cas, Pax
C3G
CD2
EGF R.
Cbl
p130Cas,
Cbl
PI3 kin. p85
Epo
Epo R.
Cbl,
STAT5,
Gab2
C3G
FGF
Fibronectin,
vitronectin,
anti-integrin,
anti-LFA1
FGF R.
Integrins
p130Cas,
paxillin,
Cbl
DOCK180
B cell R. (BCR)
GM-CSF
p62dok
p130Cas
Cbl,
Hef-1
Tel-Abl
C3G
STAT5
GDNF
c-Ret
p130Cas,
paxillin
p130Cas
Gab1,
Cbl
Growth horm. (GH)
HGF
GH R.
c-Met
IGF-1
IGF-1 R.
IRS-4
Immunoglobulins
Fc R.s.
Cbl
Interferons
IFN R.s.
.
Cbl,
STAT5
C3G
C3G
6353
Jones and Dumont, 1998
Takahashi et al., 1998
Smit et al., 1996a
Smit et al., 1996b
Ingham et al., 1996
Astier et al., 1997
Casamassima and Rozengurt, 1997
Posern et al., 2000
Lin et al., 2001
(Birge et al., 1992)
Ribon and Saltiel, 1996
(Kizaka-Kondoh et al., 1996)
Khwaja et al., 1996
Rani et al., 1997
Ojaniemi and Vuori, 1997
Katayama et al., 1999
Barber et al., 1997
Chin et al., 1997
Oda et al., 1998
Ota et al., 1998
Ozaki et al., 1998
Nosaka et al., 1999
Larsson et al., 1999
(Nojima et al., 1995)
(Hamasaki et al., 1996)
(Vuori et al., 1996)
Petruzelli et al., 1996
Manie et al., 1997
Sattler et al., 1997
Hunter and Shimizu, 1997
Uemura and Griffin, 1999
Albert et al., 2000
Ota et al., 1998
Ozaki et al., 1998
Murakami et al., 1999
Zhu et al., 1998
Garcia-Guzman et al., 1999
Sakkab et al., 2000
Garcia-Guzman et al., 2000
Riordan et al., 2000
Sakkab et al., 2001
Beitner-Johnson and LeRoith, 1995
Butler et al., 1997
Qu et al., 1999
Jin et al., 2000
Park et al., 1999
Izadi et al., 1998
Ahmad et al., 1997
Platanias et al., 1999
Fish et al., 1999
Alsayed et al., 2000
Grumbach et al., 2001
Continued
Oncogene
Crk family adaptors
SM Feller
6354
Table 1 (Continued )
Ligand/stimulus
Receptor/oncogene
via SH2a
Insulin
IRS-1
Interleukin-2
STAT5,
PI3K. p85,
Cbl, Gab2
STAT5
Gab2
Interleukin-3
Interleukin-5
MGSA
NGF
IL-8 rec. cl. II
TrkA
p130Cas
FRS2,
paxillin
PDGF
PDGF R.
IRS-1,
p72
SCF
c-Kit
p62dok
SDF-1
anti-TCR
CXCR4
T cell R. (TCR)
Cbl
Cbl
Thrombopoietin
(TPO)
VEGF
Retinoic acid (ATRA)
Reactive oxygen spec.?
(Src kinases)
Sphingosylcholine/
sphingosine-1-P
Vanadate/PAO*
STAT5
VEGFR-1, Flt-1
Cbl
p130Cas
paxillin
via SH3a
Reference(s)
Beitner-Johnson and LeRoith, 1995
(Beitner-Johnson et al., 1996)
Okada and Pessin, 1997
Gesbert et al., 1998a,b
Oda et al., 2000
C3G
c-Abl
C3G
Barber et al., 1997
Chin et al., 1997
Ozaki et al., 1998
Nosaka et al., 1999
Du et al., 2000
(Schraw and Richmond, 1995)
(Tanaka et al., 1993)
(Hempstead et al., 1994)
(Matsuda et al., 1994)
(Torres and Bogenmann, 1996)
Ribon and Saltiel, 1996
York et al., 1998
(Meakin et al., 1999)
Escalante et al., 2000
Rani et al., 1997
Hansen et al., 1997
Yokote et al., 1998
Sorokin et al., 1998
Sattler et al., 1997
van Dijk et al., 2000
Chernock et al., 2001
Sawasdikol et al., 1995
Buday et al., 1996
Reedquist et al., 1996
Boussiotis et al., 1997
Ozaki et al., 1998
Oda et al., 2000
Schulze et al., 2000
Ito et al., 1998
Alsayed et al., 2000
Yoshizumi et al., 2000
Casamassima and Rozengurt, 1997
Blakesley et al., 1997
Schaller and Parsons, 1995
Erdreich-Epstein et al., 1997
a
Domains of v-Crk, c-Crk and/or CRKL. bReferences in brackets refer to in vitro data, overexpression studies, preliminary ®ndings etc.
*Inhibitors of tyrosine phosphatases. (*)Other stimuli also signalling through 7-TM receptors include: bradykinin, endothelin, LPA,
vasopression
ample N-x-P-pY motifs present on the insulin and
other receptors (Gustafson et al., 1995; He et al., 1995;
Wolf et al., 1995; Sawka-Verhelle et al., 1996), thereby
anchoring the IRS protein at the receptor. Throughout
the rest of the protein, a fairly large number of tyrosyl
residues is present which can become phosphorylated
upon certain stimuli.
IRS proteins bind distinct, if partially overlapping
sets of signal transducers including PI3 kinase, SHP2,
adaptor proteins like Grb2, Nck, Crk and CRKL and
others. They have also been shown to exist in distinct
subcellular localizations (Anai et al., 1998; Giovannone
et al., 2000). IRS-1-de®cient mice are quite small,
indicating that IRS-1 is important for cell growth. IRS1(7/7) mice do not succumb to a severe diabetes-like
disease (Araki et al., 1994). By contrast, IRS-2-de®cient
mice develop severe hyperglycemia and diabetes early
in life, hence IRS-2 appears to be critical for normal
Oncogene
carbohydrate metabolism (Withers et al., 1998). The
IRS-3 protein is mainly expressed in adipocytes of
white fat tissue, and mice lacking a functional gene for
IRS-3 synthesis have no apparent defects in growth or
glucose metabolism (Liu et al., 1999b). The phenotype
of IRS-4 knockout mice (Fantin et al., 2000) is
relatively mild. A slight growth retardation and minor
defects in glucose homeostasis were observed. Taken
together, these animal studies document that the IRS
family proteins have quite distinct biological roles and
functional redundancy does not appear to be a major
issue in this protein family. A recent study even
suggests that the IRS-3 and IRS-4 proteins can impair
rather than complement the signalling of their relatives
IRS-1 and -2 (Tsuruzoe et al., 2001).
Of the four family members, IRS-1 and IRS-4 have
so far been shown to interact w. Crk/CRKL (BeitnerJohnson et al., 1996; Sorokin et al., 1998; Koval et al.,
Crk family adaptors
SM Feller
1998b; Butler et al., 1998; Qu et al., 1999). If indeed all
Crk/CRKL ± IRS complexes are mediated by the direct
binding of SH2 domains from Crk family adaptors
remains to be established. Although the human IRS-4
does not contain a single Y-x-x-P motif, IRS-4 from
IGF-1-stimulated human embryonic kidney (HEK293)
cells was reported to bind to the SH2 domains of both
c-Crk and CRKL in vitro (Koval et al., 1998b). On the
other hand, rat IRS-3 has three Y-x-x-P motifs (mouse
IRS-3 has two) but no evidence for Crk/CRKL SH2
domain binding to IRS-3 is yet published.
The functional consequences of Crk/CRKL complex formation with the IRS-1 and IRS-4 proteins are
largely unclear. Since both of these IRS proteins
apparantly have some role in regulating cell proliferation, such complexes may act in concert with other
well known binders of IRS proteins like PI3 kinase,
SHP2 and Grb2 to generate appropriate signals
downstream from insulin, IGF-1, PDGF and other
growth factors in a speci®c cell context (Sorokin et
al., 1998; Nakashima et al., 1999; Qu et al., 1999).
Other roles, for example as a signal mediator in
adipocyte di€erentiation, have also been recently
suggested (Jin et al., 2000). Sorokin and Reed have
reported a switch of Crk from p130Cas to which it is
initially bound onto the IRS-1 protein in response to
insulin stimulation (Sorokin and Reed, 1998). If and
how this a€ects the transmission of insulin signals to
Crk e€ector molecules remains to be clari®ed. A
similar switch from p130Cas to another large docking
protein, Gab1, has been reported for cells transformed by the Tpr-Met oncoprotein (Lamorte et al.,
2000).
Gab proteins
In addition to the IRS family, a group of similarly
structured proteins which may be considered `distant
cousins' of IRS proteins have been identi®ed. These
Gab proteins also possess one N-terminal PH domain
and multiple SH2 domain docking sites but lack a
PTB domain (Hibi and Hirano, 2000; Schlessinger,
2000). So far, only the sequences of Gab1 and Gab2
have been published (Holgado-Madruga et al., 1996;
Gu et al., 1998; Nishida et al., 1999; Zhao et al.,
1999) but Gab3 was already cloned (LR Rohrschneider, personal communication; Miyakawa et al., 2001)
and at least one additional Gab protein seems to
exist. Human and mouse Gab3 proteins do not
contain Y-x-x-P motifs conserved in both species
(there is one Y-x-x-P motif in mouse which is not
present in human Gab3), so it seems unlikely that
Gab3 is a direct binder of the Crk and CRKL SH2
domains. Gab proteins have been shown to form
complexes with Crk family adaptors in various cell
types.
Gab1 contains six potential Crk/CRKL SH2 domain
binding sites (see Sakkab et al., 2000). Upon HGFstimulation of epithelial cells (HEK293; HeLa etc.),
Crk and CRKL form complexes with Gab1 (GarciaGuzman et al., 1999; Sakkab et al., 2000, 2001;
reviewed in Furge et al., 2000). Deletion of the 6(Yx-x-P)-region in Gab1 abolishes binding of CRKL.
Stimulation of epithelial cells with HGF leads to
activation of the GTPase Rap1 and c-Jun N-terminal
kinases (JNKs). Both of these HGF-activated pathways depend on complexes built by Crk/CRKL. The
functional outcome of these signals include the
upregulation of certain matrix metalloproteinases and
reduced cell adhesion (Garcia-Guzman et al., 1999;
Sakkab et al., 2001).
The phenotype of mice with a disrupted gab1 gene
indicates that it has developmental functions in organs
like placenta, heart and skin (Itoh et al., 2000). This
pathology is similar to that of mice with de®ciencies in
growth factors like HGF, PDGF or EGF. Gab1de®cient mice also have a reduced liver size and various
cell migration defects, resulting in disturbed muscle
development and reminiscent of the defects observed in
c-met (7/7) mice (Sachs et al., 2000). Since Crk and
CRKL are also implicated in the regulation of cell
migration events and c-Met signalling, it should be
interesting to cross and analyse heterozygous and
homozygous animals of the recently generated crklknockout (Guris et al., 2001) with c-Met and Gab1
(+/7)- or (7/7)-animals to look for phenotype
enhancement.
Gab2, initially designated p97, is prominently
expressed in haematopoietic cells. It forms complexes
with CRKL in interleukin-2 (IL-2)-stimulated T-cells
(Gesbert et al., 1998b). In erythroid cells stimulated
with Epo, Gab1 and Gab2 are also heavily tyrosinephosphorylated (Wickrema et al., 1999) and recruited
to the Epo receptor-connected signalling complex.
CRKL is also recruited into this complex (reviewed
in Wojchowski et al., 1999). The situation is therefore
reminiscent of c-Cbl, which also binds to Crk when it
is tyrosine phosphorylated after Epo-stimulation of
cells (Barber et al., 1997; Chin et al., 1997). Again the
functional consequences of Gab ± Crk/CRKL complexes in haematopietic cells are not well understood
yet. One protein which is recruited to the activated Epo
and T-cell receptors is HPK1 (Nagata et al., 1999;
Hehner et al., 2000; Liou et al., 2000; Ling et al., 2001),
a serine/threonine kinase of the germinal centre kinase
(GCK) subfamily, which in turn belong to the PAK
kinase superfamily (Sells and Cherno€, 1997). HPK1
binds to the SH3(1) domains of Crk and CRKL via a
proline-rich motif in its noncatalytic region and can for
example activate JNK (Oehrl et al., 1998; and others).
The downstream signals generated by Crk/CRKL ±
HPK1 complexes remain, however, at present largely
elusive.
So far many experiments studying the function of
Epo have been done with erythroid cell lines. This
sometimes leads to con¯icting results, depending on the
cell line used. To gain further insight into the possible
biological roles of complexes between Crk and Gab
family proteins, it should be useful to analyse primary
haematopoietic cells, even if it is more demanding to
study crucial enzymatic activities like GTPases, kinases
and phosphatases in primary isolates.
6355
Oncogene
Crk family adaptors
SM Feller
6356
STAT5
Epo stimulation of erythroid cells also leads to the
activation of the STAT5 transcription factors (for
reviews see Wojchowski et al., 1999; Co€er et al.,
2000; Smithgall et al., 2000). Besides the well-known
STAT dimers, heterodimers of STAT5 and CRKL
were reported upon Epo-treatment of cells in several
studies, and also with other cell stimuli (Oda et al.,
1998; Ota et al., 1998; Ozaki et al., 1998; Fish et al.,
1999; and others). Whether STAT5 ± CRKL complexes are crucial for signal transmission of Epo in
vivo is still an open question. STAT5 exists in two
forms, STAT5a and STAT5b, which are products of
highly related but distinct genes. STAT5a,b doublede®cient mice have a normal adult haematopoiesis but
appear to have defects during their embryonic
haematopoiesis, which can apparently be compensated
later in development under laboratory conditions
(Socolovsky et al., 1999), although this could lead
to a signi®cantly lower viability in free nature. If
STAT5 ± CRKL complexes occur during embryonic
development and whether they are then part of a
functionally important signalling cascade remains to
be studied. It is also not known, though tempting to
speculate, whether a CRKL ± STAT5 heterodimer
formed via their SH2 domains would allow the
CRKL SH3 domains to bring additional enzymes,
for example kinases like c-Abl or HPK1 into the
nucleus. c-Abl is well known to have nuclear
functions (Shaul, 2000; Wang, 2000) and both
HPK1 and c-Abl can phosphorylate c-Jun in vitro
(Barila et al., 2000; EK Schmidt and SM Feller,
unpublished data). Furthermore, c-Abl and HPK1
were found to bind to each other upon experimentally-induced DNA-damage (Ito et al., 2001). Complexes of STAT5, CRKL and kinase(s) binding to
transcription-regulating elements could certainly open
up interesting new ways of controlling STAT5dependent transcription events.
SHIP and DOKs
DOK (downstream of tyrosine kinases) proteins are
generally characterized by an N-terminal PH domain
followed by a PTB domain, which both show about
50 ± 60% homology between DOK family members,
and then a more diverse C-terminal region with
multiple SH2 domain docking sites. Dok proteins are
known to bind to p120RasGap, the tyrosine kinases
Abl, Lyn and Tec and the inositol 5-phosphatase SHIP
(Carpino et al., 1997; Yamanashi and Baltimore, 1997;
Brauweiler et al., 2000; van Dijk et al., 2000). p62dok
(DOK1), the ®rst family member to be cloned (Carpino
et al., 1997; Yamanashi and Baltimore, 1997) is heavily
tyrosine-phosphorylated in Bcr ± Abl- and v-Abl-expressing haematopoietic cells. Mouse and human
p62dok both have 6 Y-x-x-P motifs. In p56dok-2
(DOK-R, DOK2) four Y-x-x-P motifs are conserved
between the human and mouse proteins (Di Cristofano
et al., 1998; Jones and Dumont, 1998). DOK-3,
Oncogene
however, although otherwise similar in structure, lacks
Y-x-x-P motifs (Lemay et al., 2000).
Crk has been reported to bind constitutively to DokR, presumably via its proline-rich tail (Jones and
Dumont, 1998). On the other hand, tyrosine-phosphorylated p62dok in Bcr ± Abl kinase-expressing cells
binds to CRKL via its SH2 domain (Bhat et al., 1998).
SHIP1 (for review on SHIPs see Rohrschneider et al.,
2000) can apparently also bind to both, CRKL and
DOK1 (Sattler et al., 2001). The interaction of SHIP1
and DOK1 occurs via the DOK PTB domain with
Y917 and Y1020 of SHIP1. The binding site of the
CRKL SH2 domain to SHIP has yet to be mapped but
candidate Y-x-x-P motifs are present in SHIP1. In the
same publication, a constitutive association of PI3
kinase with CRKL, presumed to occur via the CRKL
SH3 domains, was also documented. This brings up the
possibility that CRKL could serve in certain situations
to stabilize a SHIP ± PI3 kinase complex and may allow
cross-talk and concerted action of these two phosphoinositide-modifying enzymes. Additional stability for
such a ternary complex could come for example from a
PI3 kinase p85 binding motif present in SHIP1 (Gupta
et al., 1999; Lucas and Rohrschneider, 1999).
ZAP-70
ZAP-70 (T cell receptor zeta-chain associated protein
of 70 kDa) is a cytoplasmic tyrosine kinase implicated
in T-cell receptor function (Chan et al., 1992, reviewed
in Chu et al., 1998). It was originally identi®ed in
Jurkat T-cells and contains two SH2 domain which
precede the catalytic domain. One study has reported
that in T-cell receptor-stimulated Jurkat cells, Crk coimmunoprecipitates with active ZAP-70 kinase (Gelkop
and Isakov, 1999). A direct binding of the CrkSH2
domain is suspected, presumably to one of the three
ZAP-70 Y-x-x-P motifs (Y221, Y315, Y319). ZAP-70
phosphorylation on these sites probably depends on
the Src family kinase Lck or a similar kinase.
Functional outcomes of the Zap70-Crk interaction
could include the recruitment of substrates like Cbl or
Cbl-b to ZAP-70 via the Crk SH3 domain(s) (Elly et
al., 1999) or a protection of ZAP-70 from dephosphorylation by PTPases.
C3G
C3G was the ®rst Crk SH3 domain binding protein
identi®ed (Knudsen et al., 1994; Tanaka et al., 1994).
The binding sites of C3G for Crk and CRKL have
been mapped to the already mentioned P-x-x-P-x-K
motifs in the centre of the protein (Knudsen et al.,
1995). Both Crk and CRKL exist in constitutive
complexes with C3G in many cell types, but a
regulation of these complexes has also been reported
in response to insulin receptor activation, integrin
stimulation and other stimuli (Okada et al., 1997, 1998;
Buensuceso and O'Toole, 2000). The situation is
somewhat similar to the dissociation of the Rasactivating Grb2 ± SoS complex. The di€erence is that
Crk family adaptors
SM Feller
the exchange factor SoS is phosphorylated for example
by MAP kinases or RSK-2 leading to dissociation from
Grb2, while the crucial phosphorylation event required
for c-Crk II ± C3G dissociation after insulin-stimulation
of Chinese hamster ovary cells seems to be c-Crk II
(Y221)-phosphorylation (Okada et al., 1998) which
allows the intramolecular binding of the c-Crk II SH2
domain to pY221 (Feller et al., 1994b; Rosen et al.,
1995). C3G can also become tyrosine-phosphorylated,
but this phosphorylation is thought to upregulate its
activity rather than to lead to dissociation from Crk
(Ichiba et al., 1999). It is clear that c-Crk I ± C3G
complexes cannot be regulated by this mechanism,
since the truncated c-Crk I protein ends before the
Y221 residue. Possible speci®c functions of c-Crk I ±
C3G complexes or other c-Crk I complexes have not
yet been analysed in any detail.
C3G appears usually as multiple bands in Western
blots and several splice forms exist (Knudsen et al.,
1994; Shivakrupa and Swarup, 1999). Their functional
di€erences are not yet studied. It is, however, now well
established that C3G is a guanine nucleotide releasing
protein which removes GDP from the small GTPase
Rap1 allowing its spontaneous reloading with GTP
(Gotoh et al., 1995; van den Berghe et al., 1997) which
leads to the recruitment of e€ector molecules like BRaf and active signalling. Binding of the Crk/CRKL
SH2 domains to newly generated target sites on
activated receptors and their substrate proteins leads
to a redistribution of C3G within the cell, presumably
to sites where Rap1 is located. When C3G comes into
the vicinity of activated tyrosine kinases, phosphorylation of the regulatory tyrosine residue (Y504) could
occur, although C3G ± Y504 phosphorylation was so
far only observed in certain oncogene-transformed cells
with hyperactive tyrosine kinases (Ichiba et al., 1999).
Integrin stimulation-dependent tyrosine phosphorylation of C3G was also reported (de Jong et al., 1998)
but the exact site has not been mapped so far.
For several years, C3G was the only identi®ed
activator of Rap1 but now a considerable and still
growing number of Rap1-activating proteins which do
not interact with Crk family adaptors is known
(reviewed in Zwartkruis and Bos, 1999; see also
Dodelet et al., 1999; Clyde-Smith et al., 2000; Gotoh
et al., 2000; Rebhun et al., 2000; Yamashita et al., 2000
and others). However, inducible Crk/CRKL complex
formation and activation of Rap1 has been observed in
di€erent cell types and with several di€erent stimuli
(Ichiba et al., 1997; Garcia-Guzman et al., 1999;
Alsayed et al., 2000; Du et al., 2000; Posern et al.,
2000; Sakkab et al., 2000, 2001; Xing et al., 2000). In
some of these cases, for example in HGF-stimulated
human embryonic kidney epithelial cells and Swiss 3T3
mouse ®broblasts stimulated with bombesin, it was
clearly shown that Rap1 activation is dependent on
Crk family adaptors (Posern et al., 2000; Sakkab et al.,
2000, 2001). Other Ras-superfamily GTPases like RRas and TC21(R-Ras2) also appear to be targets of
C3G (Gotoh et al., 1997; Ohba et al., 2000) although
the evidence is still less striking.
The downstream targets of GTP-loaded Rap1 are in
most cases not well characterized, with one exception:
the cytoplasmic kinase B-Raf has been implicated as a
Rap1-target in many studies (Ohtsuka et al., 1996;
Vossler et al., 1997; York et al., 1998; Dugan et al.,
1999; Czyzyk et al., 2000; Grewal et al., 2000; Schmitt
and Stork, 2000; and others). This leads to activation
of the MAPK/Erk kinases and subsequent activation
of the transcription factor Elk-1. However, many of
these studies report Crk/CRKL-independent activation
of Rap1 and the B-Raf protein is not expressed in
many cell types where Crk ± C3G ± Rap1 signal signalling activity has been detected. Other possible
candidates need to be analysed in much more detail,
for example the Ral ± GDS-like proteins RGL and
RGL-2 (Ikeda et al., 1995; Peterson et al., 1996) and
the Ral ± GDS-like factor (Rlf) (Wolthuis et al., 1996),
but also more recently found candidates like the cell
junctional multidomain protein AF-6 (Boettner et al.,
2000) and RGS14, a member of the `regulator of Gprotein signalling' family (Traver et al., 2000).
Activation of the `stress kinase' JNK by Crk and
C3G (Tanaka et al., 1997; Dol® et al., 1998; Tanaka
and Hanafusa, 1998; Zhu et al., 1998) has also been
reported, but how this exactly occurs remains to be
de®ned although a recent report suggests that R-Ras
rather than Rap1 may be responsible for this.
Besides its catalytic GNRP (syn. GEF) domain at
the C-terminus and a Ras-exchanger motif (REM;
Guerrero et al., 1998), C3G probably has presumably
other functionally important domains (Guerrero et al.,
1998) which are not well characterized yet. For
example, C3G binds to the large docking protein
p130Cas which also interacts with AND-34, another
already mentioned Rap1 activator (Kirsch et al., 1998)
but it is not clear whether ternary C3G ± p130Cas ±
AND-34 complexes can occur.
6357
DOCK180
DOCK180, a prominent 180 kDa Crk SH3(1) domain
binding protein initially detected in `Far Western'
overlay blots (Feller et al., 1995) was cloned by Miki
Matsuda's laboratory (Hasegawa et al., 1996). Homologous proteins were also found in C. elegans (CED-5;
Wu and Horvitz, 1998) and Drosophila (MBC;
Erickson et al., 1997), two organisms that have
provided important clues for Crk signalling functions
in living organisms.
Importantly, it was quickly found that DOCK180
binds and activates the GTPase Rac but not the related
GTPase Cdc42 (Kiyokawa et al., 1998; Nolan et al.,
1998) making it very clear that Crk e€ector proteins
are important regulators of multiple Ras-family
GTPases. Rac is known to play a key role in
controlling actin dynamics, integrin-mediated cell
adhesion and cell shape changes, but also in the
engulfment of dead cell bodies (Giancotti, 1997;
Cheresh et al., 1999; Schmitz et al., 2000; Bishop and
Hall, 2000; Albert et al., 2000; Reddien and Horvitz,
2000; Savill and Fadok, 2000; Tosello-Trampont et al.,
Oncogene
Crk family adaptors
SM Feller
6358
2001). Very recent ®ndings are the recruitment of
DOCK180 to membranes by binding of its C-terminus
to phosphatidylinositol (3,4,5) trisphosphate (PIP3) and
the observation that increased expression of PI3 kinase
translocates DOCK180 to the membrane (Kobayashi
et al., 2001). This opens up interesting new possibilities
for a crosstalk of PI3 kinase with DOCK180dependent signals to Rac. According to other very
recent results, DOCK180 also binds with its C-terminal
region to the second and third SH3 domain of the
Nck-like adaptor Grb4 (Nck-2, Nck-b, Nckb; Tu et al.,
2001) but functional consequences of this interaction
are not known. DOCK2, a DOCK180-relative prominently expressed in haematopoietic cells and also
known to activate Rac (Nishihara et al., 1999), has
no obvious Crk binding motifs and no Crk binding
was detected.
The Abl family
c-Abl and Arg (Abl related gene) are large nonreceptor-type tyrosine kinases which exist in several
spliceforms and appear in multiple locations within
cells. According to the results obtained with knockout
mice and other studies, c-Abl and Arg transmit key
signals during the embryogenic development of nervous
system (Koleske et al., 1998; Liebl et al., 2000) and
bone formation (Li et al., 2000a). Many of the key
functions of Abl family kinases may thus be already
completed in adult animals or individuals. Indirect
support for this hypothesis comes from the results of
clinical trials with STI 571 (CGP57148B, Glivec), an
Abl and Arg kinase inhibitor compound which shows
promising early results as a therapeutic agent for Bcr ±
Abl-driven chronic myelogenous leukaemia (CML)
(Schindler et al., 2000; Druker and Lydon, 2000;
Okuda et al., 2001). STl571 has, maybe suprisingly,
relatively little severe side e€ects in CML patients,
arguing for a lack of key pathways which are primarily
controlled through Abl family kinases in adults. On the
other hand, nuclear c-Abl pools have been implicated
in the control of DNA-damage sensing by ATM
(Baskaran et al., 1997; Shafman et al., 1997). Adverse
STl571-e€ects on nuclear c-Abl functions may only
become apparent upon long-term treatment of patients.
An in vitro interaction of c-Abl and Arg with c-Crk
is known for quite a few years (Feller et al., 1994b;
Ren et al., 1994), but only recently has c-Abl been
linked to Crk signalling in cell culture model systems
(Escalante et al., 2000; Yano et al., 2000a; Kain and
Klemke, 2001). Possible functions of Crk ± Abl interactions include the negative regulation of ®broblast
migration and di€erentiation induction by nerve
growth factor (NGF). While this manuscript was being
written, Kain and Klemke reported that c-Abl/Arg
double-de®cient mouse ®broblasts display an increased
Crk/Cas signalling and an enhanced cell motiliy (Kain
and Klemke, 2001).
Although c-Abl plays an important role in controlling genomic integrity in the cell nucleus, there is at
present little evidence to strongly support a nuclear
Oncogene
role for Crk family adaptors in mammals, despite the
already mentioned STAT5 ± CRKL complexes and
results from biochemical experiments with Xenopus
oocyte extracts which found co-immunoprecipitation of
Crk with the cell cycle regulator XWee1 (Smith et al.,
2000). Given the defects found in the Abl/Arg-`knockouts' but also in CRKL-de®cient mice (Guris et al.,
2001), embryonic development seems to be the best
place to ®nd additional clues for major roles of Crk/
CRKL ± c-Abl/Arg complexes in intracellular signal
transmission.
The oncogenic v-Crk protein co-precipitates with cAbl from chicken embryo ®broblast lysates which has
led to the suggestion that c-Abl (and Arg) could be a
major kinase relevant for v-Crk transformation (Feller
et al., 1994b) but this hypothesis is questioned by other
®ndings which suggest the Src family kinase Fyn to be
more important for tyrosine phosphorylation of
p130Cas in v-Crk-expressing mouse ®broblasts (Sakai
et al., 1997). Nevertheless, v-Crk and c-Crk were just
shown to trans-activate the c-Abl kinase (Shishido et
al., 2001) in 293T cells. With the availability of the Abl
and Arg kinase-inhibiting molecule STI571 (which also
blocks the tryosine kinases PDGF receptor and c-Kit
but not Src family kinases) or similar compounds, it
may now be possible to get some new answers in
search for the kinase primarily responsible for the
massive hyperphosphorylation of tyrosyl residues in
p130Cas, paxillin and other proteins of v-Crk-transformed chicken embryo ®broblasts and chicken
tumours. Abl and Arg double-de®cient mouse ®broblasts may also be a useful tool for such studies.
Human leukaemic Abl kinases
Bcr ± Abl and Tel ± Abl, the leukaemia-inducing counterparts of the c-Abl kinase, arise from chromosomal
translocations and are well known to phosphorylate
CRKL and c-Crk II (Voss et al., 2000, and references
therein). The Bcr ± Abl fusion protein is found in more
than 95% of human adult CMLs. This constitutively
active kinase has been the focus of a great number of
studies which showed that numerous cellular signalling
pathways are a€ected by its expression (reviewed in
Sattler and Salgia, 1998; Faderl et al., 1999; Sawyers,
1999; Voss et al., 2001). Functional consequences of
the aberrantly high tyrosine kinase activity permanently maintained by Bcr ± Abl and Tel ± Abl include
reduced cell adhesion, increased proliferation and
protection against cell death.
The human gene encoding the CRKL protein was
found due to its localization near the bcr locus
(breakpoint cluster region) on chromosome 22 (band
q11) (ten Hoeve et al., 1993). Furthermore, CRKL
forms stable complexes with Bcr ± Abl (ten Hoeve et
al., 1994b) and is a prominent tyrosine phosphorylated
protein in neutrophils from CML patients (Oda et al.,
1994; ten Hoeve et al., 1994a; Nichols et al., 1994). The
direct interaction of CRKL and Bcr ± Abl is, at least in
part, mediated by a proline-rich region in the Cterminal tail of the Abl kinase which is localized near
Crk family adaptors
SM Feller
the nuclear localization signal of the Abl sequence and
binds to the ®rst SH3 domain of CRKL. The
inhibition of complexes mediated by the CRKLSH3(1)
domain in CML cell lines and primary isolates from
patient cells can inhibit the proliferation of these cells
(Kardinal et al., 2000). By contrast, enhanced expression of CRKL has just the opposite e€ect: mice
transgenic for the p190 Bcr ± Abl protein developed
leukaemia more rapidly when CRKL was overexpressed four- to sixfold (Hemmeryckx et al., 2001).
HPK1 and KHS
Based on homology comparisons, the already mentioned haematopoietic progenitor kinase 1 (Hu et al.,
1996; Kiefer et al., 1996) and its relative KHS (kinase
homologous to SPS1/STE20; also cloned as germinal
centre kinase related kinase [GCKR]; Shi and Kehrl,
1997; Tung and Blenis, 1997) are members of the still
growing superfamily of PAK-related serine/threonine
kinases. They lack the CRIB domain found in some
other family members which is known to mediate
binding to the GTPase Rac. Instead, the N-terminal
kinase domain of HPK1 (amino acids 17 ± 274) and
KHS is followed by a large C-terminal part which has
multiple putative SH3 domain binding sites (reviewed
in Kyriakis, 1999). HPK1 and KHS both bind to the
®rst SH3 domain of Crk and CRKL (Oehrl et al.,
1998; Ling et al., 1999, 2001; Shi et al., 2000) and may
thus be yet another (C3G-independent) link from Crk/
CRKL to JNK activation.
Binding of HPK1 to the Abl SH3 domain in a yeasttwo-hybrid screen was already described a few years
ago (Kiefer et al., 1996). A brand new study now
reports that HPK1 and c-Abl do indeed bind to each
other in response to genotoxic stress induced by
chemical or radiation treatment of Jurkat cells (Ito et
al., 2001). If and how Crk family adaptors are involved
here remains to be investigated.
HPK1 and KHS also have in their noncatalytic parts
(amino acids 500 ± 805 of HPK1) a citron homology
region (CNH). It is named after the Rac and Rho
binding protein citron (Madaule et al., 1995) which
appears due to di€erential splicing also as citron kinase
and regulates neuronal cytokinesis (Madaule et al.,
1998, 2000; Di Cunto et al., 2000). The function of this
CNH region, which is sometimes also called CMG
(Citron ± MRCK ± Gek) domain and occurs in over 60
proteins of diverse origin, remains to be clari®ed.
HPK1 is expressed exclusively in haematopoietic
cells while KHS was also found in some nonhaematopoetic cells, for example epithelial cells and
®broblasts. Certain cell lines, which require Epo for
proliferation (FD ± Epo) or di€erentiation (SKT6) were
shown to depend on HPK1 for their Epo-response
(Nagata et al., 1999) but other Epo-dependent cells
(like UT-7 and TF-1) apparently do not show this
e€ect. Therefore, these results should be followed up
by performing experiments with primary cell isolates.
HPK1 activity is also induced by T-cell and B-cell
receptor engagement (Liou et al., 2000) and down-
regulates Erk2 and AP-1 activity in T cells. It may thus
function as a negative regulator of the TCR response.
The protein complexes assembled in order to activate
HPK1 appears to involve multiple adaptor proteins
like LAT, SLP-76, Blnk, Grb2 and GRAP, but also
several tyrosine kinases (Src, Syk, ZAP-70). Interestingly, one study which looked at TCR-stimulation in
Jurkat T cells just found tyrosine phosphorylation of
HPK1 and inducible binding of HPK1 to c-Crk, but
also a constitutive complex of HPK1 and CRKL (Ling
et al., 2001). Further details of these interactions and
possible functional consequences are currently unclear.
The HPK1 gene locus has been disrupted by gene
targeting several years ago but apparently no phenotype compared to wildtype mice was observable and
the results were not published. Whether detailed
analyses of HPK1(7/7) mice can ®nally unravel a
rather subtle phenotype or whether HPK1 function can
be fully compensated by other kinases of this relatively
large kinase family remains an open question.
6359
PI3 kinase
After the early ®nding of Fukui et al. (1989) that v-Crk
associates with phosphatidylinositol kinase activity,
only few studies investigated a functional connection
between Crk and phosphatidylinositol kinases. The p85
alpha and beta subunits of PI3 kinase contain P-x-x-Px-K motifs which could serve as Crk/CRKL SH3(1)
domain binding sites (reviewed in Feller et al., 1998)
but there are only few reports suggesting a stable
interaction (Sattler et al., 1997, 2001; Gesbert et al.,
1998a). Ternary complexes which do not require a
single high anity interaction but rely on multiple
intermediate to low anity binding sites are of course
also a possibility; the SH2 domains of the p85 subunit
bind with high anity to pY-(M)-x-M motifs of Cbl-,
Gab- and IRS-family proteins which allow binding of
Crk/CRKL via their pY-x-x-P motifs. This could allow
Crk/CRKL to bind to p85 with their SH3(1) domains,
thereby possibly modulating complex stability or
enzyme function. Along these lines, a recent study
shows that the viral oncoprotein v-Crk activates PI3
kinase (Akagi et al., 2000). This leads to activation of
the kinase c-AKT (PKB) but does apparently not e€ect
the JNK or MAP kinase pathways. Inhibition of PI3
kinase activity with the inhibitor compound LY294002
suppressed v-Crk-induced cell transformation.
Regulation of the c-Crk II protein conformation by
tyrosine phosphorylation
In the search for Crk interacting proteins, it was found
early on that c-Crk II binds to the c-Abl kinase. c-Crk
II is also phosphorylated by c-Abl on tyrosine 221, a
residue absent in the shorter c-Crk I protein (Feller et
al., 1994b). The c-Crk II Y221 sequence motif is
conserved in the CRKL protein and the corresponding
tyrosine (Y207) is the major tyrosine phosphorylation
site of CRKL in Bcr ± Abl-expressing cells (de Jong et
Oncogene
Crk family adaptors
SM Feller
6360
Oncogene
al., 1997). Crk Y221 and CRKL Y207 conform to the
Y-x-x-P motif found as preferred Crk/CRKL SH2
domain binding motif by Songyang et al. (1993).
Moreover, c-Crk II was found in non-complexed,
tyrosine-phosphorylated form in HeLa cell protein
extracts. The same extracts contained proteins which
bound eciently to bacterially expressed, not tyrosylphosphorylated c-Crk II protein. These results led to
the hypothesis that the phosphorylated Y221 of c-Crk
II can bind to its own SH2 domain, presumably in an
intramolecular fashion, and that this somehow blocks
the SH3(1) domain of c-Crk II from binding to other
proteins (Feller et al., 1994b). The model is similar to
the regulatory folding model long proposed for Src
family kinases and ®nally con®rmed for c-Src and Hck
by crystallographic and functional analyses (Moare® et
al., 1997; Sicheri et al., 1997; Xu et al., 1997).
Based on the `Crk folding hypothesis', an ultrastructural study using NMR was carried out which
further supported this idea but fell short of providing
further mechanistic details (Rosen et al., 1995).
Subsequently, several reports accumulated evidence
using cell culture models which support this hypothesis,
initially based only on biochemical in vitro data. In
response to EGF, insulin or NGF-treatment of certain
cells, a phosphorylation of c-Crk II on Y221 which is
followed by dissociation of complexes mediated by the
SH3(1) domain of c-Crk II has been repeatedly found
(Beitner-Johnson and LeRoith, 1995; Okada et al.,
1997, 1998; Koval et al., 1998a; Hashimoto et al., 1998;
Nakashima et al., 1999; Escalante et al., 2000; Kain
and Klemke, 2001). These events are probably cell
type-speci®c and can be only analysed on a case by
case basis. Moreover, di€erences between c-Crk II and
CRKL have been found. For example Butler et al.
(1997) report upon IGF-1 stimulation of cells an
increase in c-Crk II ± paxillin complexes but a decrease
in CRKL ± paxillin complexes. Taken together, the
current data show that two very di€erent outcomes
with respect to Crk signalling can occur after the
activation of intracellular tyrosine kinases. On one
hand, tyrosine phosphorylation of Y-x-x-P-motif-containing proteins generates CrkSH2 domain binding
sites, relocating CrkSH3(1) domain-interacting enzymes
and leading to the active transmission of signals. On
the other hand, tyrosine phosphorylation of c-Crk II
on Y221 somehow leads to the dissociation of
CrkSH3(1) bound proteins and downregulates Crk
signals. In principle, both of these events can take place
in temporal succession, ®rst an active signal and
subsequently downregulation of the signal by c-Crk
II folding, resulting in a transient activation peak
followed by desensitization of the Crk signalling
pathway. Since Rap1 and Ras can compete for e€ector
proteins like B-Raf, switching-o€ Crk signals may
allow the Grb2 ± Sos ± Ras ± B-Raf pathway to signal
more eciently. The desensitization period could,
however, also be avoided if the `folded-up' c-Crk II
is rapidly reopened and new complexes are established,
allowing for example a rapid switch from one binding
partner to the next.
There are still many open questions regarding the
regulation of Crk signalling by folding: Can Y221phosphorylation lead to intramolecular CrkSH2 domain interactions even if the SH2 domain is initially
bound to other proteins like p130Cas or Gab1? How
does Y221-phosphorylation lead to dissociation of
CrkSH3(1) binding proteins? Are there other tyrosines
also phosphorylated and functionally important for
Crk and CRKL folding? What are the di€erences
between c-Crk II and CRKL with respect to folding
regulation by Y221 and Y207, respectively? Is the
`folded-up' Crk protein further utilised in vivo by
reactivation through a tyrosine phosphatase? How is it
decided which of the several SH3(1) domain binding
proteins expressed in cells is actually recruited to form
an active `pre-complex'? Some new insight should come
from a detailed ultrastructural analysis of Y221phosphorylated c-Crk II by X-ray crystallography
and/or NMR, followed by mutational analyses of
other crucial Crk residues thus identi®ed.
Many of the studies which have looked at Crk
folding in living cells used cell lines transfected to
express certain receptors in high numbers. The results
may therefore not re¯ect the true behaviour of the Crk
protein in vivo, making it desirable to also look at
primary cells which should re¯ect more closely the in
vivo situation. When feasible, such experiments with
cultured cells should be followed by `knock in' studies,
for example expression of a c-Crk II Y221F- or
CRKLY207F-mutant protein into mice with a disruption of the corresponding gene.
Crk functions in Drosophila melanogaster
While most of the Crk/CRKL-related studies have
been done with mammalian and avian cells, it is now
known that Crk does not only have functions in
amphibians (Evans et al., 1997; Smith et al., 2000) but
proteins homologous to Crk and its e€ector proteins
(C3G, DOCK180, c-Abl) are indeed conserved and
functionally important throughout very di€erent higher
eukaryotes. In the fruit ¯y, genes for Crk (Galletta et
al., 1999), DC3G (Ishimaru et al., 1999) and the
DOCK180-homologue MBC (myoblast city; Erickson
et al., 1997; Nolan et al., 1998) were cloned and
functionally characterized. MBC is important for
myoblast fusion, dorsal closure and neural development, phenotypes which may be linked at least in part
to a role of MBC in the migration of speci®c cells.
MBC is presumed to signal through the Drosophila
GTPase Rac1, but does not seem to be crucial for the
activation of JNK, although JNK is one target of Rac
in mammalian cells. DC3G appears to play a role in
the regulation of cell fates and proliferation since a
membrane-tagged full-length DC3G, which is believed
to function as a constitutively active form of DC3G,
leads to phenotypes mimicking an overactivation of the
Ras1 ± MAPK pathway. A truncated (dominant negative) version, lacking the catalytic domain, leads to
morphogenetic defects (Ishimaru et al., 1999). Reduc-
Crk family adaptors
SM Feller
tion of the gene doses of Rap1 or Ras diminished the
e€ects induced by expression of `overactivated' DC3G.
Another study, which also analysed Rap1, found that
it is critical for morphogenesis of the ¯y eye, the ovary
and the embryo, and that Rap1 mutations disrupt cell
migration and normal cell shapes. They found no
results to implicate Rap1 in cell proliferation or cellfate speci®cation (Asha et al., 1999).
Crk functions in Caenorhabditis elegans
Several proteins homologous to Crk and its downstream signalling partners have been identi®ed as the
genes behind a number of cell engulfment defect (`ced')
mutants (reviewed in Blelloch et al., 1999; Liu and
Hengartner, 1999; May, 2001). It is now clear that
corresponding signalling cascades comprised of CED-2
(c-Crk II) - CED-5 (DOCK180) and CED-10 (Rac)
exist in C. elegans and mammals. Moreover, CED-12,
which is not yet de®ned in its molecular nature, has
been implicated in the C. elegans pathway by genetic
analyses (Chung et al., 2000).
A key ®nding that illuminated the molecular nature
of a cell engulfment signalling cascade and also
migration regulation of two gonadal tip cells in C.
elegans is described in the work of Wu and Horvitz
(1998). In this paper, CED-5 was cloned and found
to be highly homologous to the Rac-activating Crk
SH3 domain binder DOCK180. Later on it was
shown in the same laboratory that CED-2 (Crk) and
CED-10 (Rac) do in fact control phagocytosis and
cell migration (Reddien and Horvitz, 2000). In
mammals, phagocytosis is prominently controlled by
speci®c integrins expressed on phagocytosing cells. In
C. elegans, CED-1 was just recently cloned and
shown to be a receptor related to the mammalian
SREC protein (scavenger receptor from endothelial
cells; Zhou et al., 2001). It is functionally linked to
CED-7 an ABC transporter protein. Genetic evidence
suggests that there are two functionally distinct
groups of ced genes which regulate cell engulfment,
the ®rst comprised of ced-1, ced-6 and ced-7 and the
second encompassing the genes ced-2, ced-5, ced-10
and ced-12.
The upstream molecules which activate the CED-2,
CED-5 and CED-10 pathway remain unknown.
However, in recent phagocytosis studies with mammalian cells ± which were inspired by the ®ndings in C.
elegans ± it was documented that at least the intracellular portion of the cell engulfment signalling cascade
is very similar to the nematodal signalling pathway
(Albert et al., 2000; Savill and Fadok, 2000; ToselloTrampont et al., 2001).
Crk in mammals
A very large number of stimuli a€ect Crk adaptors in
mammalian cells. In many of the cases it is not well
understood if c-Crk and/or CRKL are merely `in-
nocent bystanders' or if they are indeed crucial players
regulating the elicitation of biological responses. An
(incomplete) list of stimuli reported to a€ect Crk
family adaptors is given in Table 1. Of these just two
examples will be discussed.
6361
Cell migration
Directional movement of speci®c cells is crucial during
embryo development in higher eukaryotes but also
postnatally throughout the life of a complex organism.
Constant remodelling of tissues, replenishment of
peripheral blood cells from the bone marrow, maturation of B-cells and T-cells, function of dendritic cells
and other immune defence cells, wound repair and
in¯ammatory reactions are just a few of the migrationdependent events in mammals and other complex
metazoa which are needed to maintain functional
integrity and to fend o€ pathogens. Diseases caused
by certain bacterial infections, tumour growth and the
spreading of tumour metastases can also only occur
because abnormal migratory events of cells are
induced. The many di€erent migration events are for
example regulated by gradients of motility inducing
factors and cell ± cell contacts but also by cell
interactions with extracellular matrix (ECM) proteins.
This creates a versatile regulation system for the
delicate signalling networks which ®ne tune the
responses of the huge numbers of cells permanently
on the move.
Since v-Crk is a potent oncoprotein in chicken, it
could be expected from the beginning that v-Crk also
a€ects cell signalling events which regulate cell
migration. Nevertheless, only relatively few studies
have been conducted which begin to address the
details of v-Crk induced migration processes (Liu et
al., 2000 and references therein). Possible functions of
cellular Crk and CRKL proteins in cell migration
have, on the other hand, received more attention and
there is now good evidence that c-Crk and CRKL are
important regulators of physiological cell migration in
mammalian cells (reviewed in Hanks and Polte, 1997;
Stupack et al., 2000). Migration-regulating Crk/CRKL
signals are generated downstream of migration-inducing factors like HGF and bombesin and upon
activation of integrins. Integrins are a large family
of cell surface receptors which make contacts with
other cells but also with ECM proteins like ®bronectin
and collagens (Giancotti and Ruoslahti, 1999; O'Neill
et al., 2000). Stimulation of cells with bombesin or
integrin activation leads to tyrosine phosphorylation
of the p130Cas protein (Vuori et al., 1996; Casamassima and Rozengurt, 1997) allowing the SH2 domains
of Crk and other signal transducers to dock and
leading to the recruitment of multiple enzymes. Cas ±
Crk coupling appears to be a `molecular switch' which
induces cell migration and may enhance invasivity of
some tumour cells although other signalling pathways
which lead to the activation of MAP kinase and
myosin light chain kinase (MLCK) are equally
important in controlling cell motion (Klemke et al.,
Oncogene
Crk family adaptors
SM Feller
6362
1998; Cheresh et al., 1999; Cho and Klemke, 2000).
The current data support a model for ECM ± integrin
downstream signalling through a FAK/Src ± p130Cas ±
Crk/CRKL ± DOCK180 ± Rac cascade which a€ects
actin cytoskeleton changes and also cell survival. In
addition, other CrkSH3(1) domain binding proteins
could play a role in the control of cell motion, for
example c-Abl which seems to function as a negative
regulatory protein (Kain and Klemke, 2001).
HGF (syn. `scatter factor') triggers in epithelial
cells the formation of a di€erentially composed
signalling complex consisting of c-Met, Gab1 and
Crk/CRKL. This leads to the activation of the
GTPases Rap1 and Rac (Garcia-Guzman et al.,
1999; Sakkab et al., 2000, 2001; reviewed in Furge
et al., 2000) and results in the dispersion of
epithelial cell clusters (scattering). Since most human
tumours are of epithelial origin and disruption of
intact epithelial layers is clearly a prerequisite for
tumour invasion/metastasis, it seems worthwhile to
investigate in the future possible roles for c-Crk and
CRKL in mediating the invasiveness of certain
tumour cells.
Nerve growth factor
Whether NGF relies on Crk family adaptors for signal
transmission in animals is currently not certain.
Forced expression or microinjection of v-Crk or cCrk proteins has signi®cant e€ects on factor responsiveness and development of the neuronal phenotype
in NGF-sensitive cells (Tanaka et al., 1993; Hempstead et al., 1994; Matsuda et al., 1994), but whether
this re¯ects a natural role for cellular Crk proteins in
NGF-signalling remains to be proven. Most studies in
this context have so far been conducted with the PC12
rat pheochromocytoma cell line model system. These
cells respond by an extensive outgrowth of neurite-like
structures in the continuous presence of NGF. York
et al. (1998) proposed a model of Rap1 activation by
C3G that depends on c-Crk/CRKL and leads to
sustained activation of MAP kinase via complex
formation of GTP-loaded Rap1 and B-Raf. However,
this publication relied much on transfected PC12 cells.
Several other research groups have failed to detect
Rap1-activation after NGF-treatment of PC12 cells
before and after this report, possibly due to clonal
drift and resulting variations between the PC12 cells
used in the di€erent laboratories. Overexpression of
the tyrosine kinase GTK (also called BSK, IYK, FRK
or RAK) or other proteins like Shb in PC12 cells
apparently favours Crk-dependent Rap1 activation
(Anneren et al., 2000; Lu et al., 2000,). An
involvement of PI3 kinase in NGF-induced Rap1
activation has also been described (York et al., 2000).
PC12 cells also have NGF- and Crk-independent
means of activating Rap1 and B-Raf (Vossler et al.,
1997; Grewal et al., 2000). On the other hand, support
for
the
NGF ± TrkA ± Crk ± C3G ± Rap1 ± B-Raf ±
MEK ± MAPK cascade model now comes from
another study just published (Kao et al., 2001).
Oncogene
The FRS2/SNT protein, originally cloned as a Grb2
binding protein involved in FGF-receptor signalling
(Kouhara et al., 1997) could be an additional
component of this pathway. It was reported to bind
to Crk in vitro when extracted from NGF-stimulated
cells transfected with TrkA (Meakin et al., 1999) but
detailed functional analyses are yet to be done.
Another suspect that has surfaced more recently in
NGF signalling is c-Abl kinase. Some years ago it was
shown that Bcr ± Abl expression induces neurite outhgrowth in PC12 cells (Maru et al., 1996) and last year
an interaction of TrkA and c-Abl was reported (Brown
et al., 2000; Yano et al., 2000a). Moreover, results
from the Birge laboratory point to a key role of c-Abl
in phosphorylating the regulatory tyrosine 221 of c-Crk
II (Y222 in the chicken protein; Escalante et al., 2000).
The availability of DNA chips allows large numbers
of genes to be analysed simultaneously for expression.
These chips could be used to compare the di€erent
PC12 clones investigated in those studies and may help
to explain some con¯icting results obtained by the
di€erent research groups. Nevertheless, a better idea
may be to move beyond the PC12 cell system and to
also look in detail at NGF-responsive primary cells
derived from developing animal embryos. Since mice
void of the CRKL protein exhibit severe neural
pathology, crucial in vivo signalling events mediated
by Crk family adaptors may await discovery in these
cells.
`Knockout' mice
The targeted disruption of the crkl gene locus (Guris
et al., 2001) was reported just a few month ago.
CRKL-de®cient mice show striking defects in neural
crest development which mimic to some extent a
human neurocristopathy disease known as DiGeorge/
velocardiofacial syndrome (DGS/VCFS). Multiple
knockout mouse tissues derived from the cranial and
cardiac neural crest cells are a€ected at di€erent
stages of embryonic development, which led to the
conclusion that CRKL is an essential developmental
gene with de®ned stage- and tissue-speci®c roles.
Moreover, tissues and organs which depend on neural
crest cells for proper development like thymus,
thyroid and parathyroid glands and head skeletal
structures are also a€ected. Only a small number of
homozygously crkl gene-de®cient mice survive until
birth.
Considering the numerous defects resulting from the
elimination of the CRKL protein it seems very unlikely
that c-Crk and CRKL have redundant functions in
mice and that all the observed defects can be simply
explained as a `gene dosage change' of Crk family
adaptors. The much more likely explanation is that the
c-crk and crkl genes are not functionally as similar as
one may have guessed from results of biochemical
studies looking at Crk and CRKL SH2 and SH3
binding proteins. crkl-knockout mice have also been
generated in the laboratory of Nora Heisterkamp and
John Gro€en where the human CRKL was initially
Crk family adaptors
SM Feller
found. These mice appear to have a similar but not
identical phenotype when compared to the mice
generated by Guris et al. (2001) (N Heisterkamp and
J Gro€en, personal communication).
So far, no mice with a targeted disruption of the crk
gene locus have been described. One study generated
by gene trap insertional mutagenesis a mouse strain
which expresses only a truncated, `c-Crk I-like' Crkprotein, but these mice did not exhibit obvious
abnormalities (Imaizumi et al., 1999).
Crk family adaptors in human disease
There exist only a few reports which indicate that Crk
family adaptors could be functionally linked to human
diseases. The most obvious example at present is the
already discussed connection between CRKL and its
binding partners Bcr ± Abl and Tel-Abl in leukaemias.
In addition, a mutant c-Ret receptor which causes
multiple endocrine neoplasia type 2B (Men 2B) can
phosphorylate Crk binding proteins like paxillin which
are not prominent substrates of the wildtype c-Ret
receptor (Bocciardi et al., 1997). The functional
consequences of these additional substrate phosphorylations are not known. p130Cas ± Crk complexes may
also play a role in the infection of epithelial cells by the
bacterium Yersinia pseudotuberculosis which causes
gastroenteritis (Persson et al., 1997; Weidow et al.,
2000). In the current literature there are many data to
indicate that Crk family adaptors are involved in the
regulation of biological phenomena like cell morphology changes, cell movement, cell proliferation and
di€erentiation but also functional responses of the Band T-cell receptors. It is therefore likely that
additional hints regarding human diseases which a€ect
or utilise Crk adaptor-mediated signals will be found.
Many open questions
After more than a decade of Crk adaptor protein
analyses there are still many more questions than
answers. Some important questions not yet pointed out
relate to the role of the SH3(2) domain in c-Crk II and
CRKL and the possibly quite di€erent roles of the cCrk I and c-Crk II proteins. Several laboratories have
so far failed to detect proteins or even protein motifs
which can stably interact with the SH3(2) domains. As
mentioned above, mice which lack full length c-Crk II
and only express a truncated c-Crk I-like protein
clearly do not have a grossly abnormal phenotype
(Imaizumi et al., 1999). On the other hand, c-Crk I and
c-Crk II show clearly di€erences in their transforming
potency of ®broblast cells (Matsuda et al., 1992) and
the p130Cas tyrosine phosphorylation is much greater
in cells overexpressing c-Crk I compared to cells
overexpressing c-Crk II (Ogawa et al., 1994). This led
to the suggestion that the SH3(2) domain of c-Crk II
could serve as a negative regulatory element, but how
this might be accomplished is not understood. A new
study suggests now that the second SH3 domain of Crk
could function as a trans-acting regulatory element for
the tyrosine kinases FAK and c-Abl (Zvara et al.,
2001).
Other important questions relate to the targets of
Crk-mediated signals downstream of Rap1 and Rac as
well as on the level of gene expression. Changes
presumably induced in the transcriptional programme
of some cells which transmit signals via Crk and
CRKL into the nucleus, like signals generated by
STAT5 ± CRKL heterodimers are completely unknown.
6363
To ¯oat or not to ¯oat
In earlier paragraphs it was summarized how Crk
family adaptors interact with multiple proteins via their
SH2 and SH3 domains. While some complexes appear
to be largely constitutive (for example the Crk ± C3G
interaction), many Crk/CRKL SH2 domain interactions are clearly inducible and transient. This raises the
question whether signalling cascades come into
existence somewhat `randomly', which means that their
formation is mainly driven by statistical probabilities
depending on protein abundancies, binding anities
etc. In other words, the question is whether the Crk
and CRKL adaptor molecules ¯oat freely within the
cytosol and are simply ®shed out whenever a receptor
or receptor cluster starts to signal. Alternatively, there
might be some yet unknown form of higher order
which determines whether a preformed Crk ± C3G or a
Crk ± DOCK180 complex is recruited in a certain
situation. This could occur by restricting the di€usion
of the Crk ± C3G versus the Crk ± DOCK180 complexes to distinct cellular subregions, through low
anity interactions with sca€old proteins or by other
means. The rapidly accumulating knowledge about
cellular ultrastructures, molecular wiring diagrams and
their dynamics provides more and more examples of
highly ordered biological response systems. Seem-ingly
small di€erences in proteins through di€erential
splicing, posttranslational modi®cation etc. may play
key roles in these processes. From the recently emerged
knowledge about the human genome it is clear that
signi®cantly fewer genes than expected exist and that
more protein variants are derived from one gene than
previously anticipated. It is certainly true for the
analysis of Crk family adaptor signalling that di€erences between existing splice variants of the involved
proteins have so far been hardly looked at.
Do they need to meet?
Another important question is, how crosstalk between
Crk-mediated pathways and other pathways occurs
and if it is functionally important that multiple Crk/
CRKL molecules and their binding partners are
recruited to `intracellular meeting points' like the
15(Y-x-x-P) cluster in p130Cas. In other words: are
Oncogene
Crk family adaptors
SM Feller
6364
Crk/CRKL and its binding partners components of
large `signalosomes'?
It has become apparent in recent years that many
intracellular signalling components are con®ned to
speci®c subcellular locations and form large complexes
either by interacting with sca€old proteins, lipid rafts,
microdomains and caveolae or by assembling into
multi-protein structures that have been called `signalosomes', `transducisomes' etc. (Kurzchalia and Parton,
1999; Burack and Shaw, 2000; Schillace and Scott,
1999; Simons and Toomre, 2000). Considering the Crk/
CRKL SH2 binding proteins with numerous Y-x-x-P
motifs already known, it is not unlikely that Crk
adaptors are important components of such large
signal transduction complexes. Studies using overexpression systems of wildtype or mutant Crk/CRKL
binding proteins could therefore lead to experimental
e€ects which are dicult to understand in terms of
their true biological signi®cance. One possibility to
overcome some of the limitations of forced protein
expression is the use of small inhibitory molecules
which only block the interaction(s) of a single protein
domain. Inhibitory molecules for SH2 and SH3
domain binding pockets are currently being developed
in the form of cell-penetrating peptides and small
compounds (Cody et al., 2000; Kardinal et al., 2000,
2001). Their usefulness will critically depend on binding
anities but also on their ability to block with great
selectivity only one or very few of the more than 300
SH2 and SH3 domains which exist in human cells.
Mining the upcoming information from genome
sequencing projects
With the ongoing e€orts to sequence many vertebrate
genomes, it becomes increasingly feasible to obtain a
list of new candidate proteins which could recruit Crk
family adaptors by simple sequence analysis of
publically accessible data bases. This belief is based
upon the observation that many Crk/CRKL SH2
binding proteins have multiple Y-x-x-P motifs clustered
within a speci®c region of the protein. Examples are
the previously discussed proteins of the Cas-, Gab- and
IRS-families. Looking through the database, the
occurrence of four or more Y-x-x-P motifs per
100 kDa of protein chain length is relatively rare. If
one further excludes extracellular proteins from the list,
the number of `hits' is not too large. Proteins with a
known or suspected function in signal transduction
deserve, of course, special attention. The best way to
screen the initial candidates for their actual binding
properties may be to select or generate cell lines which
express the candidate protein plus a constitutively
active tyrosine kinase which eciently phosphorylates
Y-x-x-P motifs, for example v-Src, v-Abl or Bcr-Abl.
Lysates from these cells can then be used to perform
co-immunoprecipitation experiments.
By contrast, recognizing good candidates for novel
Crk/CRKL SH3(1) domain interacting proteins may
not be quite so simple. The currently well known
binding proteins have at most four, but usually only
one or two binding sites and there are simply too many
proteins with a P-x-x-P-x-K motif in the databases to
use a simple `one-dimensional' approach. Nevertheless,
by taking advantage of the available high resolution
structure of the SH3(1) domain (Wu et al., 1995) and
information in some cases available on the folding of
candidate Crk SH3(1) binding molecules, it may be
possible to develop suitable molecular modelling
programmes which narrow down the number of
preliminary suspects considerably. Last not least, cell
types newly implicated by gene knockout studies may
be a good ®shing ground to catch novel signalling
partners and de®ne yet unknown signalling pathways
of Crk family adaptors.
Note added in proof
An important new study just documented that insulinstimulated Glut4 glucose transporter translocation requires
a Crk ± Cbl complex-mediated signal independently of PI3
kinase ± IRS complexes (Chiang et al., 2000).
Acknowledgements
I thank Marc Lewitzky for critical reading of the manuscript.
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