From the
The Crk protein belongs to the family of proteins consisting of
mainly Src homology 2 and 3 (SH2 and SH3) domains. These proteins are
thought to transduce signals from tyrosine kinases to downstream
effectors. In order to understand the specificity and effector function
of the SH3 domain of Crk, we screened an expression library for binding
proteins. We isolated Eps15, a substrate of the epidermal growth factor
receptor (EGFR) tyrosine kinase, and Eps15R, a novel protein with high
sequence homology to the carboxyl-terminal domain of Eps15. Antibodies
raised against a fragment of the Eps15R gene product immunoprecipitated
a protein of 145 kDa. Eps15 and Eps15R bound specifically to the
amino-terminal SH3 domain of Crk and coprecipitated equivalently with
both c-Crk and v-Crk from cell lysates. The amino acid sequences of
Eps15 and Eps15R featured several proline-rich regions as putative
binding motifs for SH3 domains. In both Eps15 and Eps15R, we identified
one proline-rich motif which accounts for their interaction with the
Crk SH3 domain. Each binding motif contains the sequence
P-X-L-P-X-K, an amino acid stretch that is highly
conserved in all proteins known to interact specifically with the first
SH3 domain of Crk. Furthermore, we found that immunoprecipitates of
activated EGFR-kinase stably bound in vitro-translated Eps15
only in the presence of in vitro-translated v-Crk. Crk might
therefore be involved in Eps15-mediated signal transduction through the
EGFR.
The protooncogene products of v-crk, c-Crk-II and
c-Crk-I, belong to a new family of proteins consisting primarily of Src
homology 2 and 3 (SH2 and SH3)
Src homology domains have been found
in a wide range of proteins involved in cell signaling and are
suggested to act as molecular adaptors linking and regulating the
subcellular localization and enzymatic activity of functionally diverse
molecules(5, 6) . Binding of SH2 domains to
tyrosine-phosphorylated regions of growth factor receptors is thought
to provide a common mechanism by which regulatory proteins interact
specifically with growth factor receptors and thereby couple growth
factor stimulation to intracellular signaling pathways (7). SH3 domain
interactions have been implicated in the targeting of proteins and in
the regulation of small GTP-binding proteins(8, 9) .
Thus, SH2 and SH3 domains within one adaptor protein may collaborate to
assemble a signaling cascade by recruiting upstream and downstream
enzymatic activities into a ternary complex. Grb2 was shown to be
complexed via its SH3 domain with Sos, a guanine-nucleotide releasing
protein for Ras. Upon growth factor stimulation, the Grb2-Sos complex
is recruited from the cytosol to the plasma membrane to activate Ras
(10, 11). v-Crk which has been shown to interact via its SH2 domain
with tyrosine-phosphorylated epidermal growth factor receptors (EGFR)
might have a similar function as Grb2 (12).
The molecular nature of the
interactions between the SH2 and SH3 modules of Crk and their
respective protein ligands is likely to determine signal specificity
and therefore effector function. SH2 domain-phosphopeptide interactions
have been well characterized, predicting that a phosphotyrosine residue
is required for binding and that neighboring residues confer
specificity. The Crk SH2 domain was shown to preferentially bind
peptides that contain a phosphorylated Y-X-X-P
motif(21, 22) . Progress toward the definition of
binding motifs for SH3 domains and their functions has been made by
screening expression libraries for SH3 domain binding proteins. The
first such protein described was 3BP1(23) , which bound to the
SH3 domain of Abl. The binding motif for the SH3 domain has been
localized to a sequence rich in proline residues(24) . Similar
results have been obtained from screens of combinatorial peptide
libraries (25). Solution and crystal structures of SH3 domains revealed
that the core of the domain consists of two perpendicular,
anti-parallel, three-stranded
Identification of additional Crk SH3
domain binding proteins and determination of their respective binding
motifs may help to elucidate the cellular effector function of Crk and
further substantiate the P-X-L-P-X-K(R) motif as a
specific Crk SH3 domain binding sequence. In the present work, we
cloned two proteins which interact specifically with the SH3 domain of
Crk by screening an expression library. One protein was identified as
Eps15, an EGFR tyrosine kinase substrate which is involved in the
control of cell proliferation(34) . The second protein exhibits
a novel sequence related to Eps15 which we called Eps15R (R for
related). Both proteins interact with the SH3 domain of Crk via a
consensus P-A-L-P-P-K binding motif. Furthermore, we present evidence
for a prolonged stable association of Eps15 with the stimulated EGFR in
the presence of v-Crk.
Plasmids containing full-length eps15 and v-crk were transcribed in vitro,
and the purified RNAs were translated in rabbit reticulocyte lysates in
the presence of [
Eps15 phosphorylation is not
sufficient for binding as demonstrated by the fact that when
kinase-inactive EGFR is incubated with phosphorylated Eps15 and v-Crk,
no binding was detected.
We identified by expression cloning two new targets of the
SH3 domain of Crk. Our first clone, eps15, or EGFR pathway
substrate 15(34) , was isolated as a protein that was
phosphorylated on tyrosine upon stimulation of the EGFR. Overexpression
of eps15 led to transformation of NIH3T3 cells suggesting a
possible role of Eps15 in a mitogenic EGFR signaling
pathway(34) . The human homologue of eps15 bears 89%
similarity to murine eps15 and has been mapped to chromosome
1p31-p32.(46) . This region is a hot spot for nonrandom
chromosomal abnormalities, exhibiting deletions in neuroblastoma as
well as translocations in acute lymphoblastic
leukemia(47, 48) . Indeed, two translocations t(1;11)
(p32;q11) detected in myeloid leukemias fuse the HRX gene to
AF-1p(49) , the latter being identical with human Eps15 by
sequence comparison(46) . HRX (also called MLL, ALL-1, HTRX) is
a putative transcription factor containing DNA binding domains,
AT-hooks, zinc fingers, and methyltransferase
regions(50, 51) . The structural features of the Eps15
protein suggested its subdivision into three domains(34) .
Domain I contains a candidate tyrosine phosphorylation site and EF-hand
helix-loop-helix calcium binding motifs. The amino acid sequence of
domain I in Eps15 is 88% similar to the amino-terminal region of End3p,
a protein required for the internalization step of endocytosis and for
actin cytoskeletal organization in yeast(52) . Domain II
includes a coiled-coil region, while domain III has both proline rich
regions and repeats of aspartic acid-proline-phenylalanine, the latter
suggestive of methyltransferase activity. It is intriguing that both
HRX and Eps15 contain putative methyltransferase regions. The
oncogenicity of HRX-Eps15 may be related to the fusion protein's
ability to methylate DNA and thereby affect transcription. Nonetheless,
neither the physiological function of Eps15 nor its role in neoplastic
transformation has been elucidated in any detail.
Our second clone
contained the partial sequence of a protein that is highly homologous
to the carboxyl-terminal end of Eps15. We named this protein Eps15R for
Eps15-related. Antibodies generated against this new protein
immunoprecipitated a 145-kDa protein which was also detected by serum
generated against the Eps15 protein. Anti-Eps15 antibodies recognized a
major species migrating at 145 kDa and a minor species at 155 kDa. The
molecular nature of those two proteins is not clear. One possibility is
that p145 corresponds to Eps15R and p155 to Eps15. It has also been
suggested that the 155-kDa species represents a post-translationally
modified form of the 145-kDa Eps15 species(34) . This would be
consistent with our data if both Eps15 and Eps15R are 145 kDa in the
unmodified state and if Eps15R does not undergo significant alterations
after translation. A final possibility is that Eps15R does not exist in
HeLa cells as a protein and that the antisera generated against the
expressed clone in fact recognizes the 145-kDa species of Eps15.
Full-length sequencing of Eps15R will be necessary to ascertain whether
or not there are functional or structural differences between Eps15 and
Eps15R. We will also need to generate antibodies against unique
epitopes in Eps15R and Eps15.
We identified in the carboxyl termini
of Eps15 and Eps15R proline-rich motifs, prm2 and prm2R, which mediated
the association of these proteins to the SH3 domain of Crk. prm2 and
prm2R contain a sequence of identical amino acids,
P
However,
a characteristic binding motif is not synonymous with an in vivo interaction. Although Eps15/15R and C3G share the same
proline-rich binding motif, we failed to coimmunoprecipitate Eps15/15R
with c-Crk or v-Crk from cell lysates. In addition, stimulation of the
EGFR had no impact on the ability of native Eps15/15R to bind Crk. Only
amino-terminal GST- or T7 gene10 protein-tagged Eps15/15R bound to Crk;
we thus assume that the native conformations of Eps15 or Eps15R do not
expose their Crk-SH3 binding motifs, whereas amino-terminal extensions
or truncations might permit a surface presentation of these motifs.
In vivo, the conformation of Eps15 might change during
transient interaction with the EGFR kinase, fostering an association
with v-Crk within a heterotrimeric complex. Our data, using in
vitro-translated,
Useful suggestions and technical assistance provided
by Eduardo Fajardo, Akiko Hata, Stephan Feller, and Mutsuko Ohuchi are
gratefully acknowledged. We thank Ray Birge for comments and help on
the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)domains while
lacking any catalytic domain(1, 2) . Nck and Grb2 are
two other members of this emerging family of so-called ``adaptor
proteins''(3, 4) . The widely expressed c-Crk-II
protein contains an amino-terminal SH2 domain followed by two SH3
domains. The c-Crk-I protein, which is found in embryonic lung cells,
and v-Crk do not contain the second SH3 domain. In addition, v-Crk has
an amino-terminal Gag region.
(
)Sos
and C3G, a cytosolic protein with homology to Ras guanine-nucleotide
releasing protein, were recently shown to bind to the first SH3 domain
of c-Crk(14, 15) . The demonstration that binding of the
Grb2 SH2 domain to phosphotyrosine motifs did not change the binding
affinity of the SH3 domain to Sos (16, 17) leads to the suggestion that
SH2 and SH3 domains are not allosterically coupled within one adaptor
molecule. The SH2 and SH3 domains of Crk, however, may be
interdependent, the binding of one domain to its target eventuating in
a new interaction involving the adjacent domain. The amino-terminal SH3
domain of c-Crk was shown to target the Abl family tyrosine kinases
c-Abl and Arg. The subsequent phosphorylation of the c-Crk protein
generates a binding motif for the SH2 domain of
Crk(18, 19, 20) .
-sheets. The most highly conserved
residues form a hydrophobic surface which has been identified as ligand
binding site (26, 27). SH3 domains recognize proline-rich motifs
possessing the left-handed type II polyproline helix conformation. The
pseudosymmetry of the polyproline type II helix explains the
observation that proline-rich motifs interact in both axial
orientations with SH3 domains(28, 29) . The polyproline
helix has three residues per turn. Every third residue, that is
residues at positions i and i + 3 lie on the
same face of the helix(30, 31) . Two proline residues
spaced by two amino acids, the P-X-X-P motif (i, i + 3), directly intercalate between the
aromatic residues on the hydrophobic surface of the SH3 domain. Other
prolines in the ligand appear to promote the helix formation, whereas
neighboring non-proline residues are thought to determine
specificity(25, 32) . The binding motif
P-X-L-P-X-K(R) is present in all proteins
demonstrated to interact with the amino-terminal Crk SH3 domain: Sos,
Abl, Arg, and C3G(33) .
SH3 Domains Containing GST-Fusion Proteins
The
expression vectors for glutathione S-transferase (GST),
GST-Crk[SH3], GST-Crk[SH3][SH3], and
GST-Nck have been described(18) . The expression vector for
GST-Grb2 was a gift from Tadaomi Takenawa(35) , and the
GST-Src[SH3] construct was kindly provided by David Baltimore
(23). Expression and purification of the GST-fusion proteins was done
as described(36) . S-Labeling of
GST-Crk[SH3][SH3] was performed as
detailed(37) .
Library Screening
A 16-day stage mouse embryo cDNA
expression library constructed in phage EXlox vector (Novagen) was
screened for Crk SH3 domain binding proteins. Binding to expressed
proteins was detected with
S-labeled
GST-Crk[SH3][SH3] for the first screen and
unlabeled GST-Crk[SH3][SH3] followed by anti-GST
antibody and
I-labeled Protein A (Amersham) for
subsequent screens. 13 clones were purified from approximately 8
10
plaques. Automatic conversion of phage
recombinants to plox plasmids was generated by infection of a bacterial
host expressing the P1 cre recombinase (BM25.8 bacterial strain from
Novagen), which in turn recognizes the loxP sites and forms the plasmid
by site-specific recombination. Terminal sequencing of the plasmid
preparations by the dideoxy-chain termination method using a commercial
kit (UBI) revealed 6 independent clones. 4 clones were identified by a
GenBank
search using the BLAST program(38) , while
1 clone was determined by Southern hybridization. The final clone
revealed a novel nucleotide sequence.
Eps15 and Eps15R GST-Fusion Constructs
The 1.6-kb
partial cDNA clone of eps15 was excised from plox with EcoRI and HindIII and subcloned into pBluescript II
SK (Stratagene). A fragment of 0.8 kb was cut
out from the insert with PstI, and the purified vector was
religated. The shortened insert of 0.8 kb was excised from pBluescript
with BamHI and MscI and directionally subcloned into
pGEX-3X to generate pGEX-Eps15aa713-884. The 0.8-kb partial clone
of eps15R was excised from plox with EcoRI and HindIII and directly subcloned into pGEX-1N. The constructs
were sequenced through the junctions to verify sequence fidelity and
orientation. All regions of the eps15 and eps15R nucleotide sequences which encode proline-rich motifs (prm) were
oligo-synthesized and cloned into pGEX-1N digested with EcoRI
to generate pGEX-Eps15prm1, pGEX-Eps15prm2, pGEX-Eps15prm3,
pGEX-Eps15Rprm2R, and pGEX-Eps15Rprm3R. The proline-rich peptide
constructs were sequenced entirely. The full-length construct of eps15, pGEX-Eps15aa1-897, has been
described(34) . Expression and purification of all GST-fusion
proteins was done as detailed(36) . Purity and integrity of the
fusion proteins was assessed by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) (39) and Coomassie Blue staining.
Antisera
The Eps15 antiserum (RF99) was generated
by immunizing rabbits with the GST-Eps15aa713-884 protein. The
Eps15R antiserum (RF148) was produced in rabbits with the cloned
GST-Eps15R fusion protein as immunogen. Polyclonal antibodies against
GST were produced by immunizing rabbits with GST and purifying the sera
through an affinity column. Anti-c-Crk (265) and anti-Gag (3C2)
have been described(40) . A monoclonal anti-EGFR antibody
directed against the extracellular receptor domain was purchased from
Amersham.
Antibody Binding to Eps15 and Eps15R
Antibody
binding to Eps15 and Eps15R was tested by immunoprecipitation and
immunoblotting. HeLa cells were harvested in RIPA buffer containing 20
mM Tris-HCl, pH 7.5, 100 mM NaCl, 2.5 mM EDTA, 1 mM dithiothreitol, 1% (v/v) Triton X-100, 0.5%
(w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, 100
kallikrein inactivating units/ml aprotinin, 1.0 µg/ml leupeptin, 1
mM phenylmethylsulfonyl fluoride, 1 mM sodium
orthovanadate, 10 µM sodium molybdate, and 10 mM sodium fluoride. Lysates were cleared from particulate material by
centrifugation for 10 min at 10,000 g. Protein
concentration of cell lysates was determined by the Bradford method
(Bio-Rad). Precipitations were performed with 0.5 mg of total lysate
proteins and 5 µl of either anti-Eps15 or anti-Eps15R antiserum in
0.5 ml of RIPA buffer. The antibody complexes were immunopurified with
Protein G-Sepharose beads (Pharmacia Biotech Inc.) and washed three
times with RIPA buffer. Proteins were then separated by SDS-PAGE,
transferred onto nitrocellulose, and probed with either anti-Eps15 or
anti-Eps15R serum, each diluted 1:300 in binding buffer. Binding buffer
contained Tris-buffered saline, 1 mM EDTA, 0.1% (v/v) Tween
20, 0.02% (w/v) sodium azide, 2% (w/v) bovine serum albumin, 1 mM dithiothreitol, and the aforementioned protease and phosphatase
inhibitors. Bound antibodies were detected with
I-labeled
Protein A and autoradiography.
Far Western Blot
Expression of EXlox phage
cDNA inserts was induced with
isopropyl-1-thio-
-D-galactopyranoside for 3 h after
infection of a bacterial host strain carrying the gene for T7 RNA
polymerase (BL21[DE3]pLysE bacterial strain from Novagen). 5
ml of bacterial culture were pelleted by centrifugation (3000 rpm for
10 min) and lysed in 0.5 ml of RIPA buffer. Insoluble material was
pelleted by microcentrifugation at 11,000
g for 10 min
and analyzed by SDS-PAGE. Separated proteins were transferred onto
nitrocellulose membranes (Immobilon) and blocked in binding buffer.
Membranes were probed with GST-fusion proteins at 1 µg/ml in
binding buffer for 3 h. Bound GST-fusion proteins were detected with a
polyclonal antiserum to GST followed by
I-labeled Protein
A (Amersham).
Precipitation of Crk from Cell Lysates
Parental
human carcinoma A431 cells and lines overexpressing c-Crk or v-Crk were
cultured as described(41) . v-Crk-transformed rat fibroblasts
and v-Crk expressing 3Y1 cells were also utilized(42) . Cell
lysis was performed in 1% Nonidet P-40 buffer containing 20 mM
Tris-HCl, pH 7.4, 150 mM NaCl, and the inhibitors used for
RIPA buffer. 10 µg of GST-fusion protein was incubated for 3 h at 4
°C with either 1 mg of protein from total A431 cell lysate or 300
µg from total v-Crk/3Y1 cell lysate in 0.5 ml of Nonidet P-40 lysis
buffer. 15 µl of glutathione-Sepharose beads (Pharmacia Biotech
Inc.) were subsequently added for 40 min to collect the protein
complexes. All samples were washed four times with ice-cold Nonidet
P-40 lysis buffer, boiled in electrophoresis buffer, and analyzed by
SDS-PAGE. 50 µg of total cell lysate proteins were subjected to
SDS-PAGE as a control. Western blots were blocked and probed with
appropriate antisera to the Crk proteins. For detection of the primary
antibodies, I-labeled Protein A or
I-labeled antisera specific for mouse immunoglobulins
(Amersham) were used.
In Vitro Transcription and Translation
The
pCEV27-Eps15 plasmid (34) was linearized with SfiI and
blunt-ended with the Klenow DNA polymerase, whereas the
pBluescript-v-Crk plasmid (41) was linearized with XbaI. This allowed for selective transcription of the
respective inserts and not of the remainder of the plasmid. After
phenol extraction and ethanol precipitation of the linearized plasmids,
1 µg of plasmid DNA was used in 50 µl of transcription
reactions using riboprobe reagents (Promega). Transcription reactions
were performed with SP6 RNA polymerase for eps15 and T7 RNA
polymerase for v-crk at 38 °C for 1 h. After
transcription, RNase-free DNase (RQ1; Promega) was added to the
reaction samples to digest plasmid DNA. Following phenol extraction,
the RNAs were precipitated with ethanol and resuspended in 30 µl of
RNase-free sterile water. For the in vitro translation, 10
µl of the in vitro-transcribed RNAs were incubated with
rabbit reticulocyte lysate (Promega) and
[S]methionine (Amersham) in a final volume of 60
µl under the conditions suggested by the manufacturer. Reactions
were analyzed by SDS-PAGE and fluorography using an autoradiographic
image enhancer (National Diagnostics).
Analysis of Eps15 Binding to the EGF Receptor in Presence
of v-Crk
For each binding reaction, 1 µl of anti-EGFR
antibody was incubated with 100 µg of total A431 lysate proteins
for 90 min at 4 °C. Following an additional incubation for 40 min
at 4 °C in the presence of 15 µl of Protein G-Sepharose beads
(Pharmacia), the EGFR immunocomplexes were washed four times with
ice-cold HNTG buffer containing 20 mM HEPES, pH 7.4, 150
mM NaCl, 0.1% Triton X-100, and 10% glycerol and all
previously mentioned protease and phosphatase inhibitors. The EGFR
immunocomplexes were thereupon incubated with 5 µl of the in
vitro-translated proteins (v-Crk and/or Eps15) in a final volume
of 100 µl for 90 min at 4 °C. As indicated in the figure
legends, EGFR tyrosine kinase was either activated by addition of 7
mM MnCl and 60 µM ATP followed by an
incubation for 30 min at 30 °C or inactivated by the addition of 10
mM EDTA. Bead-coupled EGFR complexes were finally regenerated
by centrifugation and washed four times with HNTG buffer. All binding
reactions were analyzed by SDS-PAGE and fluorography.
Isolation of cDNA Clones Encoding Binding Proteins for
the SH3 Domain of Crk
A commercial 16-day-old mouse embryo cDNA
expression library was screened for Crk SH3 domain binding proteins.
Six cDNA clones were purified and terminally sequenced, four of which
were identified by computer-assisted sequence homology search with the
GenBank data base. One cDNA clone of 1.6 kb corresponded
to the newly discovered EGF receptor pathway substrate, Eps15, a
145-kDa protein phosphorylated by the activated EGFR tyrosine kinase
(34), whereas three clones were translational artifacts of prothymosin
(43), skeletal muscle actin(44) , and
-1-globin(45) , respectively. Translational frame shifts of
these clones led to artificial synthesis of Crk SH3 domain binding
sequences. One cDNA clone was identified by Southern hybridization as
the recently cloned Crk SH3 domain binding protein,
C3G(15, 33) . The sixth cDNA clone of 0.8 kb revealed a
unique nucleotide sequence which was entirely determined.
Identification of an Eps15-related Gene
Product
The sequence of the 0.8-kb cDNA clone predicted an open
reading frame of approximately 0.4 kb encoding 139 amino acids. The
novel protein sequence revealed a 45% identity (61% similarity) to the
carboxyl-terminal end of Eps15 and was therefore called Eps15-related,
Eps15R (Fig. 1). To analyze the full-length gene product of this
novel sequence and to compare it with Eps15, we generated polyclonal
antisera against both proteins. The cDNA encoding amino acids
713-884 of Eps15 and the entire clone of Eps15R were expressed as
GST-fusion proteins for use as immunogens. When added to HeLa cell
lysate, the anti-Eps15 serum immunoprecipitated two proteins, a
predominant one of 145 kDa and another of 155 kDa; the serum against
Eps15R reacted with a single protein of 145 kDa (Fig. 2). On
immunoblots, both antisera reacted with the immunoprecipitated 145-kDa
protein species. To further show that these antisera recognize
different epitopes, we immunoblotted the HeLa cell lysates and
supernatants after immunoprecipitation with each antiserum. Anti-Eps15
antisera partly depleted both the 145-kDa and the 155-kDa bands, while
anti-Eps15R antisera partly depleted only the 145-kDa band. The 145-kDa
band might represent comigrating Eps15 and Eps15R proteins. The nature
of the 155-kDa protein is unknown.
Figure 1:
Amino acid
sequence comparison between Eps15 and the partial gene product encoded
by the eps15-related clone. The identity (colons)
between the two amino acid sequences is 45%, while the similarity (colons and dots) is 61%. The boxed proline-rich domains are candidate binding motifs for the SH3
domain of Crk.
Figure 2:
Identification of the Eps15R gene product.
Western analysis using specific antisera noted on the bottom of figure were performed on (i) total HeLa cell lysates, (ii) HeLa
cell lysates immunoprecipitated (IP) with anti-Eps15 or
anti-Eps15R, and (iii) supernatants (SN) of the lysates after
immunoprecipitation. Bound antibodies were detected with I-labeled Protein A. Molecular mass markers in
kilodaltons are shown on the left. The 97-kDa band is
nonspecific.
Specificity of the Isolated Crk-binding Proteins for
Various SH3 Domains
Binding specificity of the expressed phage
cDNA inserts of eps15 and eps15R was assessed by far
Western blots probed with a panel of different SH3 domains fused to GST (Fig. 3). Partial eps15 (1.6 kb) and eps15R (0.8 kb) clones expressed as T7 gene10 carboxyl-terminal fusions
bound strongly to the first SH3 domain of Crk, weakly to full-length
Grb2, and negligibly to the SH3 domains of Nck and Src.
Figure 3:
Binding specificity of cloned Eps15 and
Eps15R gene products to various SH3 domains. Cloned phage inserts
of eps15R and eps15 were expressed as fusions with
260-amino-acid T7 gene10 protein and analyzed by SDS-PAGE. The Western
blots were probed with GST-Crk[SH3], GST-Grb2, GST-Nck,
GST-Src[SH3], and GST as shown in the panels from left to right. Binding was detected with a polyclonal antibody
to GST followed by
I-labeled Protein A. Molecular mass
standards are indicated in kilodaltons.
We then
sought to determine whether Eps15 and Eps15R interact equivalently with
both v-Crk and c-Crk. GST-fusion proteins of Eps15 and Eps15R ligand
precipitated both c-Crk and v-Crk from overexpressing A431 cell
lysates. GST-Eps15 corresponded to the carboxyl-terminal amino acids
713-884 of the full-length Eps15 protein while GST-Eps15R
comprised the carboxyl-terminal 139 amino acids; these represent
homologous regions and contain the putative Crk binding motif. Probing
of the Western blots with specific antibodies to the Crk species
revealed that both of these GST-fusion proteins bind equivalently to
c-Crk and v-Crk (Fig. 4). 5 min of stimulation of A431 cells with
50 ng/ml EGF had no impact on Crk binding to the Eps15 and Eps15R
fusion proteins.(
)
Figure 4:
Precipitation of c-Crk and v-Crk from cell
lysates with GST-Eps15 or GST-Eps15R. A431 cell lysates overexpressing
c-Crk or v-Crk were either ligand-precipitated by GST-fusion proteins
and analyzed by SDS-PAGE or directly separated by SDS-PAGE. GST was
fused to either full-length Eps15 or to the partial Eps15R protein. The
Western blot was probed for the presence of c-Crk with polyclonal
antiserum followed by I-labeled Protein A; the presence
of v-Crk was detected with a monoclonal antibody to Gag followed by
I-labeled sheep anti-mouse antibody. Molecular mass
standards (in kDa) are indicated on the left.
Amino Acid Sequences in Eps15 and Eps15R
Responsible for Binding to the SH3 Domain of Crk
We generated
GST-fusion peptides of proline-rich regions identified within Eps15 and
Eps15R (boxed amino acid sequences in Fig. 1) as
putative binding motifs for the first SH3 domain of Crk. There were
three proline-rich motifs identified in the Eps15 protein sequence: (i)
proline-rich motif 1 (prm1) for amino acids 206-216; (ii) prm2
for amino acids 770-780, and (iii) prm3 for amino acids
781-792. In Eps15R, we found amino acid stretches homologous to
prm2 and prm3 and termed them prm2R and prm3R, respectively. Each
GST-proline-rich motif (GST-prm) was analyzed separately for its
respective ability to precipitate v-Crk from v-Crk expressing 3Y1 cell
lysates (Fig. 5A). Western blots probed with anti-Gag
monoclonal antibody showed that the amino-terminal SH3 domain of Crk
binds preferentially to particular proline-rich motifs (prm2 and prm2R)
within Eps15 and Eps15R, respectively. The amino-terminal proline-rich
motif (prm1) in Eps15 lacks a P-X-X-P binding motif
and consequently did not bind to the Crk SH3 domain (Fig. 5B). prm2 and prm2R, the regions responsible for
the association of Eps15/15R with the SH3 domain of Crk, are highly
homologous regions with identical P-A-L-P-P-K motif. A third
proline-rich motif, prm3 or prm3R, bound only weakly the SH3 domain of
Crk despite the presence of P-X-X-P motifs neighbored
by charged lysine and arginine residues.
Figure 5:
Identification of the binding motif for
the SH3 domain of Crk in Eps15 and Eps15R. A, GST-fusion
protein precipitates of v-Crk expressing 3Y1 cell lysates were
subjected to SDS-PAGE and transferred to nitrocellulose. The Western
blot was analyzed for the presence of v-Crk with a monoclonal antibody
to Gag followed by I-labeled sheep anti-mouse
immunoglobulin. GST was fused to full-length Eps15 protein, to the
partial Eps15R protein, and to proline-rich motifs (prm1, -2, -3, -2R,
and -3R) which had been indicated in Fig. 1. The migration of molecular
mass markers (in kDa) are shown on the left. B,
alignment of the proline-rich motifs (prm) of Eps15 and Eps15R.
Indicated are the amino acid boundaries in the Eps15 protein sequence
and their relative Crk binding affinities.
The binding motifs
identified in Eps15 and Eps15R are remarkably similar to binding
sequences in other known Crk SH3 domain interacting proteins (). They share with all listed proteins a
P-X-L-P-X-K sequence. However, an arginine three
amino acid residues carboxyl-terminal to the P-X-X-P
motif is present in several Crk-binding proteins yet absent in both
Eps15 and Eps15R.
Binding of v-Crk and Eps15 to the EGF Receptor
We
found that the SH3 domain of Crk interacts in vitro with a
specific proline-rich motif (prm2) within Eps15. Eps15 is an EGFR
substrate which gets phosphorylated on tyrosine upon EGFR-kinase
activation. However, a stable association of Eps15 with the EGFR has
not been observed(34) . The SH2 domain of v-Crk binds in
vitro and in vivo to the tyrosine-phosphorylated
EGFR(12, 41) . Therefore, it was of interest
to investigate how v-Crk and Eps15 might modulate each other's
interactions with the EGFR.
S]methionine. The radiolabeled
proteins were then tested for their abilities to bind to
Sepharose-coupled EGFR (Fig. 6). In the absence of v-Crk, the
phosphorylation of Eps15 by the immunopurified EGFR tyrosine kinase
occurred without stable binding of the substrate to the kinase (Fig. 6, lane 1); Eps15 phosphorylation was confirmed by
using radiolabeled ATP.
In contrast, stable binding of
Eps15 to the activated EGFR kinase was observed in the presence of
v-Crk (Fig. 6, lane 2). Following binding to the
autophosphorylated EGFR, v-Crk was phosphorylated by the activated
receptor tyrosine kinase resulting in a doublet (Fig. 6, lanes 2, 4, and 5); the doublet reflects
binding of both phosphorylated and unphosphorylated v-Crk to the
EGFR.
The tyrosine phosphorylation occurs within the Gag
domain of v-Crk.
(
)Both v-Crk bands showed
similar intensities on the autoradiogram suggesting that roughly 50% of
v-Crk bound to the EGFR was phosphorylated under the conditions of our
experiment. However, as demonstrated by intensity differences between
lanes 3 and 4, the total amount of v-Crk that associated with the EGFR
was augmented if v-Crk remained unphosphorylated; phosphorylation of
v-Crk appears to accelerate its dissociation from the receptor.
Figure 6:
Interaction of Eps15 and v-Crk with the
EGF receptor in vitro. In vitro-translated
[S]methionine-labeled Eps15 and v-Crk were
tested for their abilities to bind to Sepharose bead-coupled EGFR under
various conditions. EGFR kinase reaction was initiated by the addition
of ATP and MnCl
in the presence of v-Crk, Eps15, or both.
When indicated, the kinase reaction was terminated through addition of
EDTA. The EGFR beads were then centrifuged and analyzed by SDS-PAGE and
fluorography. Lane 1, kinase reaction with the EGFR and Eps15. Lane 2, kinase reaction with the EGFR, Eps15, and v-Crk. Lane 3, kinase reaction with the EGFR and Eps15 followed by
kinase termination and addition of v-Crk. Lane 4, kinase
reaction with the EGFR and v-Crk followed by kinase termination and
addition of Eps15. Lane 5, kinase reaction with the EGFR,
Eps15, v-Crk, and GST-prm2, the latter being a GST fusion protein of
the 11-amino-acid proline-rich binding motif of
Eps15.
A stable association of Eps15 with the EGFR occurred only in
the presence of v-Crk and an active receptor tyrosine kinase (Fig. 6, lane 2). In lane 3, a kinase reaction
was initiated in the presence of Eps15, and v-Crk was added subsequent
to kinase inactivation with EDTA. The inactivation of the kinase was
complete as shown by lack of a v-Crk band shift. Conversely, in lane 4, a kinase reaction was initiated in the presence of
v-Crk, and Eps15 was added after kinase termination. Therefore, EGFR
kinase inactivation did not prohibit binding of v-Crk; however,
blocking kinase action of the EGFR-v-Crk complex did abolish subsequent
binding of Eps15. Furthermore, phosphorylation of v-Crk was not
sufficient for Eps15 binding as EGFR kinase inactivation after
phosphorylation of v-Crk did not permit association of Eps15 (lane
4). Binding of Eps15 to the EGFR-v-Crk complex was competitively
inhibited by excess GST-prm2 (Fig. 6, lane 5), while GST
alone had no effect.
Therefore, Eps15 bound to the EGFR
only if (i) v-Crk was complexed and (ii) the EGFR kinase was active.
-A
-L
-P
-P
-K
,
which also comprises a Crk-SH3 binding motif in C3G. The crystal
structure of the Crk SH3 domain complexed with the 10-amino-acid
binding motif of C3G (53) showed that the peptide bound in the
same axial orientation as that of a 9-amino-acid Sos1 proline-rich
peptide to the carboxyl-terminal SH3 domain of
Grb2(28, 29) . The hydrophobic surface of the Crk SH3
domain bound the two coplanar positioned proline residues, Pro
and Pro
, of the polyproline type II helix in a
conformational mode referred to as external packing (29). The second
set of coplanar positioned residues, Leu
and
Lys
, contact the SH3 domain of Crk in a mode referred to as
internal packing(29) . The Lys
residue, which is
tightly coordinated by acidic residues in the RT loop of the Crk SH3
domain, is the key determinant of binding orientation, affinity, and
specificity. Amino acids carboxyl-terminal to Lys
do not
appear to strongly affect binding(53, 54) .
S-radiolabeled Eps15 and v-Crk
proteins, supports this concept. Presumably, the EGFR interacts with
the amino terminus of Eps15 through its kinase domain and the SH2
domain of v-Crk through its autophosphorylated tail; this allows the
SH3 domain of Crk to bind to a proline-rich motif (prm2) of Eps15 (see
model, Fig. 7). The EGFR contains at residue 992 a possible
Crk-SH2 phosphotyrosine binding motif, Y-L-I-P(13, 21) .
Eps15 association with the active EGFR-v-Crk complex was abolished in
the presence of competing peptides containing the Crk SH3 domain
proline-rich binding motif. Therefore, we assume that the transition
state of Eps15 with the active EGFR kinase might expose the
proline-rich motif in Eps15 permitting a ``capture'' by the
v-Crk SH3 domain. Our inability to confirm an in vivo association between v-Crk and Eps15 may be largely due to a
disruption of the tenuous Eps15-v-Crk-EGFR complex by the
immunoprecipitation reaction.
Figure 7:
Model
of v-Crk mediated stable association of Eps15 with the stimulated EGF
receptor. Stimulation of the EGFR leads to the recruitment of v-Crk and
Eps15 from the cytoplasm to the membrane. The SH2 domain of Crk binds
to a specific phosphotyrosine binding motif (pYXXP) in the
carboxyl-terminal tail of the autophosphorylated receptor whereas the
SH3 domain of Crk interacts with the proline-rich motif (PALPPK) in the
carboxyl-terminal end of Eps15. The amino-terminal end of Eps15
contains a candidate tyrosine phosphorylation motif (Y) which gets
phosphorylated by the EGFR kinase.
Stimulation of the EGFR should lead to
the recruitment of Crk and Eps15 from the cytosol to the membrane. The
biological consequences of a possible convergence of Eps15 and Crk on
EGFR signaling remain to be investigated. Crk might decrease turnover
of the receptor-substrate complex. In this respect, Crk might foster a
prolonged association of Eps15 and the EGFR, altering the signal
propagating from the complex. Similarly, adaptor proteins are likely to
act as a bridge between the receptor, its substrate(s), and downstream
effectors. Crk and Eps15 might be crucial links in the mitogenic signal
emanating from the EGFR.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.