From the
The Ras GTPase-activating protein (GAP) is a target for protein
tyrosine kinases of both the receptor and cytoplasmic classes and may
serve to integrate tyrosine kinase and Ras signaling pathways. In this
report, we provide evidence that GAP is an SH3 domain-binding protein
and substrate for the Src-related tyrosine kinase Hck, which has been
implicated in the regulation of myeloid cell growth, differentiation,
and function. Wild-type (WT) or kinase-inactive (K269E) mutant Hck
proteins were co-expressed with bovine GAP using the baculovirus/Sf-9
cell system. GAP was readily phosphorylated on tyrosine by WT but not
K269E Hck. GAP was present in WT Hck immunoprecipitates from the
co-infected cells, indicative of Hck
Hck is a member of the Src family of cytoplasmic protein
tyrosine kinases and is expressed primarily in hematopoietic cells of
the myeloid and B-lymphoid lineages(1, 2, 3) .
Previous studies have linked Hck activation to granulocyte-macrophage
colony-stimulating factor signal transduction (4) as well as
macrophage activation by bacterial
lipopolysaccharide(5, 6, 7) . In addition, Hck
expression is significantly enhanced during the terminal
differentiation of myeloid leukemia cells in
vitro(4, 8) . These findings suggest a diverse role
for Hck in signal transduction processes required for the growth,
differentiation, and function of hematopoietic cells. A more recent
study has linked Hck activation to leukemia inhibitory factor signaling
in embryonic stem cells, suggesting that Hck may play a role in early
development as well(9) . However, the cellular targets for the
Hck kinase are currently undefined.
Hck shares several structural
features with Src. These include a C-terminal tail with a negative
regulatory tyrosine residue (Tyr-501), a tyrosine kinase domain,
non-catalytic Src homology 2 and 3 (SH2 and SH3) domains, and a unique
N-terminal domain (reviewed in Ref. 10). SH2 domains have been
postulated to regulate the kinase activity of cytoplasmic protein
tyrosine kinases by binding phosphorylated tyrosine residues in the
catalytic domain and tail region(10) . SH3 domains also help to
regulate tyrosine kinase activity and may cooperate with SH2 domains in
this regard(11, 12, 13) .
SH2 and SH3 domains
are also found in other proteins associated with tyrosine kinase signal
transduction, including phospholipase C-
Several lines of evidence strongly
implicate Ras as an essential downstream component of Src and other
protein tyrosine kinase growth-regulatory signaling pathways. Cells
stimulated with mitogenic growth factors or transformed by v-src and other tyrosine kinase oncogenes contain elevated levels of Ras
in its active GTP-bound form(18, 19, 20) .
Conversely, microinjection of antibodies to Ras blocks cellular
transformation by tyrosine kinase oncogenes as well as the mitogenic
response to epidermal growth factor and platelet-derived growth
factor(21, 22) . Similarly, dominant-negative Ras
mutants block tyrosine kinase-mediated signal transduction(23) .
GAP may serve as part of the biochemical link between protein
tyrosine kinases and Ras signal transduction(24, 25) .
Several members of the Src family have been shown to associate with and
phosphorylate GAP, including v-Src, c-Src, Lck, Yes, Fyn, and
Lyn(26, 27, 28, 29, 30, 31, 32, 33, 34) .
Structurally, GAP consists of a C-terminal catalytic domain that
interacts with Ras and an N-terminal regulatory region containing SH2
and SH3 domains (reviewed in Ref. 35; see Fig. 1). GAP enhances
the intrinsic GTPase activity of Ras, promoting its conversion from the
active GTP-bound form to the inactive GDP-bound
form(24, 25) . Although this catalytic function of GAP
suggests that it is primarily a negative regulator of Ras, other
studies suggest an effector function for GAP as well. For example,
expression of deletion mutants of GAP that lack the C-terminal
catalytic domain can induce gene expression from a Ras-dependent
reporter gene construct (36) and cytoskeletal
reorganization(37) . Furthermore, antibodies to the GAP SH3
domain block Ras-dependent germinal-vesicle breakdown in Xenopus oocytes(38) .
The
To test for the presence of
Hck
SH3 domains contain hydrophobic binding
pockets for the proline-rich motif found in target
proteins(17, 44) . One of these pockets contains the
highly conserved sequence Tyr-X-Tyr (YXY), which is essential for SH3-target interaction(42) . To
establish a role for this motif in GAP recognition by the Hck SH3
domain, we deleted the amino acids YDY in two different Hck SH3 GST
fusion proteins. As shown in Fig. 7, deletion of the YXY
motif from these SH3 proteins almost completely abolished binding to
GAP,
To establish that the N-terminal region of
GAP is involved in SH3 binding, we assessed the ability of an
N-terminal deletion mutant to associate with the Hck SH3 domain. This
mutant (
Identification of an SH3
domain-binding sequence in GAP suggests that GAP may be a target for
other SH3 domains as well. To address this possibility, in vitro binding assays were conducted with the SH3 domains from v-Src,
Lck, and Fyn in comparison to Hck. As shown in Fig. 9, the SH3
domains from all of the Src kinase family members were able to
associate with GAP and the GAP deletion mutants.
Using a baculovirus/Sf9 cell co-expression system, we have
established that the Ras GTPase-activating protein (GAP) is a substrate
for the hematopoietic protein-tyrosine kinase, Hck. Hck is a member of
the Src family of cytoplasmic tyrosine kinases, several of which have
been shown to phosphorylate
GAP(26, 27, 28, 29, 30, 31, 32, 33, 34) .
Tyrosine phosphorylation of bovine GAP by v-Src occurs primarily at Tyr
residue 457 (equivalent to Tyr-460 in the human sequence) both in
vitro and in vivo(27) . While Tyr-457 is likely to
be utilized by Hck as well, data presented here show that other GAP
sites are phosphorylated by Hck. The
Although we have not mapped the additional site(s) of GAP
phosphorylation by Hck and c-Src, some evidence suggests that they may
be localized to the C-terminal catalytic region. Deletion mutants of
human GAP containing N-terminal residues 1-181 or C-terminal
residues 705-1047 were not phosphorylated when co-expressed with Hck in
the baculovirus system (data not shown). Thus, tyrosine phosphorylation
of the bovine GAP
Data
presented here suggest that the SH3 domains of Src family kinases may
be previously unrecognized determinants of GAP interaction. Recombinant
fusion proteins containing the v-Src, Hck, Lck, and Fyn SH3 domains
were able to bind to full-length and mutant forms of GAP. Mutation of a
conserved SH3 sequence essential for target recognition (YXY
motif; Ref. 42) abolished binding of the Hck SH3 domain to GAP in
vitro. Inspection of the N-terminal region shared by the
full-length,
Although data presented here suggest a function for the
SH3 domains of Src family kinases in complex formation with GAP,
previous analyses of GAP-Src interaction indicate important roles for
other Src domains as well. Using chick embryo fibroblasts as a model
system, association of c-Src with GAP was shown to involve tyrosine
phosphorylation of the Src negative regulatory tail (Tyr-527) and an
intact Src SH2 domain(28) . Involvement of Src Tyr-527 suggests
that the C-terminal Src kinase (CSK) may affect GAP-Src interaction,
possibly by creating a binding site for the GAP SH2 domains. However,
autophosphorylation of c-Src was not required, since a kinase-inactive
mutant was observed to associate with GAP as readily as the wild-type
kinase. This result is consistent with our finding that a
kinase-inactive mutant of Hck can associate with GAP to the same extent
as the wild-type kinase (Fig. 3). In another study, individual
deletions of the N- or C-terminal halves of the c-Src SH2 domain or the
entire SH3 domain did not affect tyrosine phosphorylation of GAP by
c-Src in transfected chick embryo fibroblasts (48). This result is
consistent with the idea that more than one domain of c-Src is involved
in the recognition of GAP in vivo. Whether or not the Hck
C-terminal tail or SH2 domain contribute to interaction with GAP is
currently under investigation. Differences in the mechanism of
interaction of individual members of the Src kinase family with GAP are
also possible, which may produce unique effects on GAP function.
The
finding that GAP associates with the SH3 domains of Hck, Src, Lck, and
Fyn adds to a growing list of substrates that interact with Src kinase
family members via this mechanism. Several recent studies have shown
that the 85-kDa subunit of phosphatidylinositol 3`-kinase binds to the
SH3 domains of Src, Fyn, and Lck(49, 50, 51) .
Dynamin, a GTP-binding microtubule-associated protein binds to the SH3
domains of Src, Fyn, and Fgr as well as to the SH3 domains of GRB-2 and
p85(52) . In the case of phosphatidylinositol 3`-kinase and
dynamin, association with the SH3 domain may modulate the biochemical
function of these proteins. In addition, recombinant SH3 domain fusion
proteins from Src, Fyn, and Lyn have been shown to interact with
discrete populations of proteins from
fibroblasts(42, 53) . When similar SH3 domain binding
experiments were conducted with whole cell lysates from Src-transformed
cells, many of the SH3-associated proteins were found to be
phosphorylated on tyrosine. Several of these SH3-binding proteins were
later identified and include SHC, a signaling protein that links
tyrosine kinases to Ras via GRB-2, the GAP-associated protein p62,
heterogeneous nuclear ribonucleoprotein K, and paxillin, a
cytoskeleton-associated protein(42, 53) . These findings
suggest that the SH3 domains of Src and Src family members are
important for substrate recognition in vivo.
Although GAP
is generally regarded as a negative regulator of Ras, some evidence
suggests that GAP is a downstream effector of Ras as well (see
Introduction). Thus, interaction of Hck or other Src kinases with GAP
may influence Ras activity and GAP effector function in several ways.
Phosphorylation of GAP on novel sites within the C-terminal catalytic
region may alter GAP activity toward Ras in vivo.
Alternatively, tyrosine phosphorylation of GAP could influence
interaction with putative effector proteins such as
p62(54, 55) . Another possibility is that binding of the
proline-rich N-terminal region of GAP to the SH3 domain of Hck or other
SH3-containing proteins may influence its catalytic or signaling
functions. Both granulocyte macrophage-colony stimulating factor and
leukemia inhibitory factor have recently been linked to cellular Hck
activation(4, 9) , and granulocyte macrophage-colony
stimulating factor is known to activate Ras through a
tyrosine-kinase-dependent mechanism(56) . Phosphorylation of GAP
may occur as a result of Hck activation in response to these and other
physiological stimuli, suggesting a possible role for GAP in these
signaling pathways.
We acknowledge the expert technical assistance of Jan
Williamson with the peptide synthesis and Dr. Fulvio Perini for
conducting amino acid composition analysis, and Victoria Boryca for
generating the kinase-inactive Hck mutant.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
GAP complex formation.
Unexpectedly, GAP also associated with the kinase-inactive mutant of
Hck, suggesting that tyrosine autophosphorylation of Hck is not
required for complex formation. The WT and K269E forms of Hck also
associated with GAP mutants lacking either the C-terminal catalytic
domain (
CAT) or the Src homology region (
SH), indicating that
these GAP domains are dispensable for complex formation. Recombinant
GST fusion proteins containing the Hck, Src, Fyn, or Lck SH3 domains
associated with full-length GAP,
CAT, and
SH, all of which
share an N-terminal proline-rich region resembling an SH3-binding motif
(PPLPPPPPQLP). Deletion of the highly conserved YXY sequence
from the Hck SH3 domain abolished binding. GAP-SH3 interaction was also
inhibited by the proline-rich peptide GFPPLPPPPPQLPTLG, which
corresponds to N-terminal amino acids 129-144 of bovine GAP. An
N-terminal deletion mutant of GAP lacking this proline-rich region did
not bind to the Hck SH3 domain. These data implicate the Hck SH3 domain
in GAP interaction, and suggest a general function for the SH3 domains
of Src family kinases in recognition of GAP via its proline-rich
N-terminal domain.
, the 85-kDa subunit of
phosphatidylinositol 3`-kinase (p85 subunit), and the Ras
GTPase-activating protein (GAP;
(
)reviewed in
Refs. 14-16). Other SH2- and SH3-containing proteins lack an
associated catalytic function and serve as molecular adaptors to join
proteins with the appropriate Src homology recognition
motifs(15, 16) . SH2 domains bind to
tyrosine-phosphorylated sequences with high affinity and specificity
and help to mediate protein-protein interactions between
autophosphorylated tyrosine kinases and downstream effector proteins.
SH3 domains are also involved in protein-protein interaction. Unlike
SH2 domains, SH3 domains bind to proline-rich sequences in target
proteins in a phosphorylation-independent manner (17). Recent evidence
suggests a central role for SH3 domains in mediating subcellular
localization and substrate recognition by Src-related kinases (see
``Discussion'').
Figure 1:
Structures of
Hck and bovine GAP mutants used in this study. Numbering above the
diagrams indicates the amino acid boundaries of each of the various
domains and the locations of the deletions. SH, Src homology; PPPP, putative proline-rich SH3 target region; CAT,
GAP catalytic domain.
In the present study, we provide evidence
that GAP is a substrate for the Src family kinase, Hck. GAP associates
with Hck in a phosphotyrosine-independent manner involving the Hck SH3
domain and a proline-rich sequence found in the N-terminal domain of
GAP. Identification of an SH3-binding motif within GAP suggests a
previously unrecognized mode of protein-protein interaction for this
critical Ras regulator/effector.
Generation of Recombinant Hck and GAP
Baculoviruses
A full-length human Hck cDNA was obtained from the
American Type Culture Collection (ATCC, Rockville, MD). This clone was
originally isolated by Ziegler et al.(1) , and contains
artifactual 5` sequences that lack proper initiation signal sequences
for translation. These sequences were deleted and a functional
initiation codon was restored using the polymerase chain reaction
(PCR). The conserved kinase domain residue Lys-269 of Hck was converted
to Glu using standard PCR-based techniques. The resulting wild-type and
K269E mutant cDNAs were inserted into the baculovirus transfer vector
pVL1393 (Pharmingen, San Diego, CA) and used to generate recombinant
baculoviruses using Baculogold DNA and the manufacturer's
protocol (Pharmingen).
N GAP mutant was prepared by
digesting a pBlueBac transfer vector (InVitrogen) containing the p120
bovine GAP cDNA with MscI and BglII. A NotI
linker was added to the MscI site of the resulting 1-kilobase
fragment, which was then subcloned into NotI/BglII-digested pBlueBac-p120 GAP transfer
vector. The recombinant
N GAP baculovirus was prepared by
co-transfection of Sf9 cells with the transfer vector and wild-type
baculoviral DNA, followed by plaque purification using standard
protocols.
(
)Preparation of baculoviruses for the
expression of full-length,
SH, and
CAT GAP is described in
detail elsewhere(29) .
Antibodies
Monoclonal antibodies to GAP were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Epitope
mapping experiments (not shown) indicate that this antibody recognizes
part of the GAP SH3 domain, and therefore does not recognize the GAP
SH mutant. To allow detection of the GAP
SH mutant on
immunoblots, rabbit antiserum was raised against a recombinant GST
fusion protein containing the first 181 amino acids of human GAP,
immediately N-terminal to the first SH2 domain (Panigen,
Blanchardsville, WI; see Fig. 1). Antiserum to Hck was purchased
from Santa Cruz Biotechnology. This antiserum recognizes the unique
N-terminal region of Hck. Monoclonal antibody PY20 to phosphotyrosine
was purchased from Transduction Laboratories (Lexington, KY).
Co-expression of Hck and GAP Proteins in Sf9 Insect
Cells
For co-expression studies, Sf9 cells were grown to 50%
confluence in T-25 flasks and infected at a multiplicity of infection
of 5-10 with recombinant Hck and GAP baculoviruses. Forty-eight
hours postinfection, cells were lysed by sonication in 1.0 ml of lysis
buffer (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, 1 mM MgCl, and 0.1% Triton X-100)
supplemented with 25 µg/ml aprotinin, 50 µg/ml leupeptin, 1
mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1
mM Na
VO
, and 50 µM
Na
MoO
. To test for GAP tyrosine
phosphorylation, the GAP monoclonal antibody and protein G-Sepharose
(10 µl of a 50% slurry; Pharmacia Biotech Inc.) were added to the
clarified lysates and incubated at 4 °C for 1 h. Immune complexes
were pelleted by centrifugation and washed with three 1.0-ml aliquots
of RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1%
Triton X-100, 0.1% SDS, 1 mM EDTA, and 1% sodium
deoxycholate). Immunoprecipitated proteins were resolved by SDS-PAGE,
transferred to polyvinylidine difluoride membranes, and probed with
antibodies to phosphotyrosine.
GAP complexes, immunoprecipitates were prepared from lysates
of co-infected Sf9 cells with the anti-Hck antiserum.
Immunoprecipitated Hck proteins were analyzed for associated GAP or the
GAP
CAT mutant by immunoblotting with the anti-GAP monoclonal
antibody or for the presence of the GAP
SH mutant by
immunoblotting with the anti-GAP N-terminal antiserum described above.
Expression of Hck-GST Fusions in Escherichia coli and in
Vitro Association Reactions
Using PCR, the coding sequences of
the Hck unique N-terminal domain (amino acids 1-62), SH3 domain
(amino acids 52-122), SH2 domain (amino acids 123-220), and
various combinations thereof (see Fig. 6) were amplified and
cloned into the bacterial expression vector, pGEX-2T (Pharmacia).
Similarly, the coding sequence of the SH3 domain of v-Src (amino acids
76-147) was amplified by PCR and cloned into pGEX-2T. The
resulting plasmids were used to express these domains as GST fusion
proteins in E. coli(39) . The fusion proteins were
purified using glutathione-agarose beads as described
elsewhere(40, 41) . GST fusion proteins containing the
SH3 domains of Lck and Fyn were purchased from Santa Cruz
Biotechnology. The coding region for the highly conserved YXY
motif found in the SH3 domain of many Src kinase family members (42) was deleted from the Hck SH3 domain coding sequence using
PCR. The resulting Hck SH3 YDY mutant was also expressed as a GST
fusion protein for binding studies.
Figure 6:
The Hck SH3 domain binds GAP, SH, and
CAT in vitro. The noncatalytic domains of Hck
(N-terminal, SH3, and SH2 domains) were expressed either alone or in
the combinations shown as GST fusion proteins in E. coli and
immobilized on glutathione-agarose beads. Immobilized fusion proteins
or GST as a negative control were mixed with lysates from Sf9 cells
expressing full-length GAP or the GAP deletion mutants
CAT,
SH, or
N. Following incubation and washing, bound GAP
proteins were visualized by immunoblotting. The SH3 binding activity of
full-length GAP (A) and the GAP deletion mutants
SH (B),
CAT (C), and
N (D) are shown.
To verify that
N was expressed in part D, an aliquot of
the
N cell lysate was immunoprecipitated with the GAP monoclonal
antibody and immunoblotted in parallel with the other samples (lane
marked CON).
For GST fusion protein binding
experiments, aliquots of clarified lysates from Sf9 cells expressing
full-length and mutant forms of GAP were incubated with Hck-GST fusion
proteins or GST (10 µg) immobilized on glutathione-agarose beads in
a final volume of 1.0 ml of lysis buffer. Following incubation at 4
°C for 1 h, protein complexes were pelleted by centrifugation and
washed three times with RIPA buffer. Associated proteins were eluted by
heating in SDS-PAGE sample buffer, and the presence of GAP or GAP
mutants was determined by immunoblotting with either the GAP monoclonal
antibody (to visualize full-length, CAT and
N proteins) or
the GAP N-terminal antiserum (for
SH GAP mutant).
GAP Proline-rich Peptide Synthesis and Inhibition of SH3
Binding
The proline-rich peptide GFPPLPPPPPQLPTLG, corresponding
to bovine GAP N-terminal domain residues 129-144, was synthesized
by standard solid phase methods on an Applied Biosystems Model 430A
peptide synthesizer. An unrelated peptide with the sequence
KPQIAALKEETEEEV was synthesized for use as a negative control.
Syntheses were performed on a 0.25 mmol scale on
[p-(hydroxymethyl)phenoxy]methylpolystyrene resins
(0.88 meq/g substitution). N-Amino groups were
protected with the base-labile N-(9-fluorenyl)methyloxycarbonyl (Fmoc) group. Side chain
functional groups were protected as follows: Gln (Trt or trityl); Glu
(O-tert-butyl ester); Lys (Boc or tert-butyloxycarbonyl); and Thr (tert-butyl).
Synthesis was initiated by the in situ coupling of the
C-terminal residue to the
[p-(hydroxymethyl)phenoxy]methylpolystyrene resin in
the presence of excess N,N`-dicyclohexylcarbodiimide and
1-hydroxybenzotriazole with 4-(dimethylamino)pyridine as coupling
catalyst. Peptide chain elongation was accomplished by repetitive Fmoc
deprotection in 50% piperidine in N-methylpyrrolidinone
followed by residue coupling in the presence of
2-(1H-benzotriazol-1-yl)-1,1,1,3,3,-tetramethyluronium
hexafluorophosphate. Peptides were purified by high performance liquid
chromatography according to previously published methods(43) .
The acetate salt form of the peptides were generated in each case and
purity (>96%) was assessed as the integrated single peak area on
analytical high performance liquid chromatography. Peptides were
characterized by amino acid compositional analysis. For inhibition
studies, the peptides were added to in vitro association
reactions at various concentrations (see legend to Fig. 8) with
the immobilized Hck GST-SH3 fusion protein and Sf9 cell lysates
containing full-length GAP or the deletion mutants as described above.
Figure 8:
Inhibition of GAP-SH3 interaction in
vitro with a proline-rich peptide corresponding to GAP N-terminal
residues 129-144. In vitro SH3 domain binding assays
were conducted with the immobilized Hck SH3 domain and full-length GAP
and the GAP deletion mutants in the presence of 1, 3, 10, 30, 100, and
300 µM (left to right) of the synthetic peptide
GFPPLPPPPPQLPTLG. This sequence corresponds to residues 129-144
of the bovine GAP N-terminal domain and resembles a proline-rich SH3
domain-binding sequence. The amount of GAP bound in each reaction was
assessed by immunoblotting as described under ``Experimental
Procedures'' and in the legend to Fig. 6. Shown are the effects of
the peptide on the binding activity of full-length GAP (A),
and the GAP deletion mutants SH (B) and
CAT (C). Control lanes include GST without the SH3 sequence (GST), and binding activity in the absence of the peptide (SH3).
Phosphorylation of GAP by Hck in a Baculovirus
Co-expression System
Previous studies have established that GAP
is a target for both normal and transforming cytoplasmic protein
tyrosine kinases of the Src
family(26, 27, 28, 29, 30, 31, 32, 33, 34) .
To determine whether or not GAP is a substrate for Hck as well, we used
a baculovirus/Sf9 cell system for the co-expression of these proteins.
Sf9 insect cells were infected with recombinant baculoviruses
containing the full-length bovine GAP cDNA either alone or in
combination with baculoviruses containing either wild-type Hck (WT) or
a kinase-inactive Hck mutant (K269E; Fig. 1). GAP was
immunoprecipitated from clarified cell lysates, resolved by SDS-PAGE,
transferred to polyvinylidine difluoride membranes, and probed with
antibodies to either phosphotyrosine or GAP. As shown in Fig. 2,
GAP was readily phosphorylated on tyrosine residues in cells in which
it was co-expressed with WT Hck but not in cells where it is expressed
either alone or with the kinase-inactive form of Hck. Control blots
show that the WT and K269E Hck proteins were expressed at approximately
equal levels.
Figure 2:
Phosphorylation of GAP by Hck in a
baculovirus/Sf9 cell coexpression system. Sf9 cells were infected with
a bovine GAP baculovirus alone (GAP) or with the GAP and Hck
wild-type (WT) or Hck kinase-inactive (K269E)
baculoviruses in combination. GAP was immunoprecipitated from the
infected cell lysates with anti-GAP monoclonal antibodies and analyzed
by immunoblotting with either the antiphosphotyrosine antibody, PY20 (left), or the GAP monoclonal antibody (center). To
verify the expression of the Hck K269E mutant, the cell lysates were
analyzed directly by immunoblotting with the anti-Hck antiserum (right).
GAP
Previous studies have shown that GAP is not
only phosphorylated by tyrosine kinases of the Src family, but often
forms complexes with Src kinases as
well(26, 27, 28, 29, 30, 31, 32, 33, 34) .
To investigate the possibility of HckHck Complex Formation Does Not Require Hck
Autophosphorylation
GAP complex formation and
its dependence on tyrosine phosphorylation, WT and kinase-inactive Hck
were co-expressed with GAP in Sf9 insect cells. Hck proteins were
immunoprecipitated from clarified cell lysates and tested for the
presence of associated GAP by immunoblotting with the anti-GAP
monoclonal antibody. As shown in Fig. 3A, GAP was
present in the Hck immunoprecipitates from cells co-infected with WT
Hck and GAP. Surprisingly, GAP was also present in Hck
immunoprecipitates from cells co-infected with the kinase-inactive Hck
mutant and GAP, suggesting that Hck autophosphorylation is not required
for complex formation. No GAP was observed in anti-Hck
immunoprecipitates from cells expressing GAP alone. Immunoblots of the
clarified cell lysates show that Hck and GAP were expressed at
approximately equal levels in each of the cultures (Fig. 3, B and C).
Figure 3:
Association of wild-type and
kinase-inactive forms of Hck with GAP. Anti-Hck immunoprecipitates were
prepared from lysates of Sf9 cells expressing wild-type Hck (WT), kinase-inactive Hck (KE), bovine GAP (GAP), or a combination of GAP and Hck WT or KE. Hck
immunoprecipitates from Sf9 cells infected with wild-type baculovirus (AcMNPV) were included as an additional negative control. A, the presence of GAP in the Hck immunoprecipitates was
analyzed by immunoblotting with the anti-GAP monoclonal antibody. B, equivalent expression of WT and KE Hck proteins was
verified by immunoblotting aliquots of the Sf9 cell lysates with the
anti-Hck anti-serum. C, expression of GAP was verified by
immunoblotting the lysates with the anti-GAP
serum.
Deletion of the Src Homology Region and Tyr-457 of GAP
Does Not Abolish Complex Formation with Hck or Tyr Phosphorylation of
GAP
The observation that the kinase-inactive mutant of Hck is
able to associate with GAP suggests that mechanisms other than
phosphotyrosine-SH2 interaction are responsible for complex formation.
To test this hypothesis further, a mutant of GAP lacking both SH2
domains, the SH3 domain, and Tyr-457 (the major Tyr phosphorylation
site for v-Src; Ref. 27) was co-expressed with wild-type and
kinase-inactive Hck in the baculovirus system. The structure of this
mutant, known as SH, is shown in Fig. 1. Immunoprecipitates
were prepared with the Hck antiserum and probed on immunoblots with
either the N-terminal GAP antiserum or with the antiphosphotyrosine
antibody, PY20. As shown in Fig. 4A, GAP
SH
co-precipitated with both forms of Hck, indicating that the GAP Src
homology region is not required for association to occur. In addition,
this GAP mutant was readily phosphorylated on tyrosine by WT Hck (Fig. 4B), despite the fact that the major site of
phosphorylation by v-Src has been deleted (Tyr-457). Control blots
verified that GAP
SH was expressed at equal levels in the three
cultures infected with the
SH baculovirus (data not shown).
Figure 4:
GAP-Hck association does not require the
Src homology region of GAP. Anti-Hck immunoprecipitates were prepared
from lysates of Sf9 cells expressing wild-type Hck (WT),
kinase-inactive Hck (KE), a deletion mutant of GAP lacking the
Src homology region and Tyr-457 (SH), or a combination of GAP
SH and Hck WT or KE. Hck immunoprecipitates from Sf9 cells
infected with wild-type baculovirus (AcMNPV) were included as
an additional negative control. A, the presence of
SH in
the Hck immunoprecipitates was assessed by immunoblotting with the GAP
anti-serum. B, the tyrosine phosphorylation state of
SH
was determined by immunoblotting the Hck immunoprecipitates with the
antiphosphotyrosine antibody, PY20. C, expression of WT and KE
Hck proteins was verified by immunoblotting the lysates with the Hck
antibody.
The GAP Catalytic Domain Is not Required for Interaction
with Hck
To assess whether the catalytic domain of GAP is
involved in HckGAP complex formation, co-expression experiments
were conducted with a GAP mutant lacking a large C-terminal fragment
within the catalytic domain that interacts with Ras (
CAT mutant;
see Fig. 1). Immunoprecipitates were prepared with the anti-Hck
antiserum and blotted with the GAP monoclonal antibody or with
antiphosphotyrosine. As shown in Fig. 5A, the
CAT
mutant co-precipitated with both the wild-type and kinase-inactive Hck
proteins but did not bind directly to the Hck antibody, indicative of
complex formation. Antiphosphotyrosine immunoblotting of the
precipitated proteins indicates that the
CAT mutant is
phosphorylated on tyrosine (Fig. 5B). This mutant
retains the major site of tyrosine phosphorylation by v-Src (Tyr-457;
27) which may be utilized by Hck as well. Control blots shown in Fig. 5C indicate that WT and KE Hck proteins are
expressed at approximately equal levels in the co-infected cultures.
Control blots also verified that GAP
CAT was expressed at equal
levels in the three cultures infected with the
CAT baculovirus
(data not shown). This experiment suggests that the C-terminal
catalytic function of GAP is dispensable for interaction with Hck.
Figure 5:
GAP-Hck association does not require the
catalytic domain of GAP. Anti-Hck immunoprecipitates were prepared from
Sf9 cell lysates expressing wild-type Hck (WT),
kinase-inactive Hck (KE), a deletion mutant of GAP lacking
part of the catalytic domain (CAT), or a combination of
GAP
CAT and Hck WT or KE. Hck immunoprecipitates from Sf9 cells
infected with wild-type baculovirus (AcMNPV) were included as
an additional negative control. A, the presence of
CAT in
the Hck immunoprecipitates was assessed by immunoblotting with the GAP
monoclonal antibody. B, the tyrosine phosphorylation state of
CAT was determined by immunoblotting the Hck immunoprecipitates
with the antiphosphotyrosine antibody, PY20. C, expression of
WT and KE Hck proteins was verified by immunoblotting the lysates with
the Hck antibody.
Recombinant GST Fusion Proteins Containing the Hck SH3
Domain Bind to GAP,
To map the
specific domain of Hck that binds to GAP in a
phosphotyrosine-independent manner, the Hck unique N-terminal, SH3, and
SH2 domains were expressed singly or in various combinations as
glutathione S-transferase (GST) fusion proteins in E.
coli. The Hck fusion proteins or GST alone were immobilized on
glutathione-agarose beads and incubated with lysates from Sf9 cells
expressing full-length GAP, SH, and
CAT in Vitro
SH, or
CAT. Following incubation
and washing with buffer containing detergents and salt, bound proteins
were resolved by SDS-PAGE and immunoblotted with anti-GAP antibodies.
As shown in Fig. 6, A-C, full-length GAP as
well as the GAP
SH and
CAT mutants were able to bind to
fusion proteins containing the Hck SH3 domain, but not to the SH2 or
unique N-terminal domains. This result implicates the Hck SH3 domain in
association with GAP.
SH, and
CAT.
Figure 7:
Deletion of the conserved YXY
motif from the Hck SH3 domain abolishes binding to GAP, SH, and
CAT in vitro. The YDY motif was deleted from the Hck SH3
domain using PCR, and the resulting mutant SH3 domain was expressed in E. coli as a GST fusion protein either alone (SH3
YDY) or in combination with the adjacent N-terminal
domain (N-SH3
YDY). These fusion
proteins were compared to their wild-type counterparts in terms of GAP
binding activity as described in the legend to Fig. 6. The binding of
full-length GAP (A) and the GAP deletion mutants
SH (B) and
CAT (C) to the mutant and wild-type
versions of the fusion proteins are shown. Immobilized GST was included
as a negative control.
A possible target sequence for the Hck
SH3 domain is defined by GAP N-terminal amino acids 129-144,
which encompass the proline-rich sequence GFPPLPPPPPQLPTLG. This region
is shared by all of the GAP proteins observed to bind the Hck SH3
domain in vitro (Figs. 6 and 7). To determine whether this
region is a possible SH3 target, we synthesized a peptide corresponding
to this sequence and tested its ability to interfere with GAP-SH3
association using the in vitro binding assay. As shown in Fig. 8, the GAP proline-rich peptide competed for full-length GAP
and GAP deletion mutant binding to SH3 in a concentration-dependent
manner with an IC value of approximately 50
µM. An unrelated hydrophilic peptide with the sequence
KPQIAALKEETEEEV had no effect on GAP-SH3 binding under these conditions
(data not shown). These results are consistent with the hypothesis that
the N-terminal hydrophobic domain of GAP encompasses an SH3
domain-binding region.
N) lacks amino acids 25-166, which encompass the
proline-rich region described above (see Fig. 1). As shown in Fig. 6D, deletion of this proline-rich region abolished
the binding of GAP to the Hck SH3 domain, consistent with the results
of the peptide competition experiment.
Figure 9:
Association of GAP with the SH3 domains of
Src, Lck, and Fyn. To assess whether GAP binding is a unique property
of the SH3 domain of Hck, in vitro binding assays were also
conducted with immobilized SH3 domains from v-Src, Lck, and Fyn.
Immobilized GST-SH3 fusion proteins or GST alone were mixed with
lysates from Sf9 cells expressing full-length GAP (A), or the
GAP deletion mutants SH (B) and
CAT (C).
Following incubation and washing, bound GAP proteins were visualized by
immunoblotting.
SH mutant of GAP lacking
Tyr-457 was readily phosphorylated by Hck in Sf9 cells. Phosphorylation
was demonstrated on antiphosphotyrosine immunoblots of
SH in Hck
immunoprecipitates from co-infected cells (Fig. 4) and in
extracts from co-infected cells lysed directly in SDS sample buffer
(data not shown). In addition, co-expression of Hck with the wild-type,
SH, and
CAT forms of GAP resulted in nearly equal levels of
tyrosine phosphorylation of these proteins (as judged by nearly
identical levels of antiphosphotyrosine immunoreactivity when all three
were run together on the same immunoblot; data not shown). These
results are in contrast to previous studies with v-Src, which
phosphorylated the
SH GAP mutant poorly under essentially
identical experimental conditions(27) . The inability of v-Src
to phosphorylate GAP
SH may be a unique property of this
transforming kinase, since c-Src was able to phosphorylate
SH to
the same extent as Hck in the baculovirus system (data not shown).
SH mutant may occur on one or more of five
C-terminal tyrosines between amino acids 518 and 702 (see Fig. 1). Alternatively, the isolated catalytic domain of GAP may
not be phosphorylated because it lacks the N-terminal proline-rich
region and Src homology domains essential for interaction with the
tyrosine kinase. Further experiments are required to distinguish
between these possibilities. Association of GAP with Ras, p62, p190, or
other proteins has been postulated to affect the conformation of GAP (45) and may uncover sites for tyrosine phosphorylation such as
those observed following deletion of the Src homology region.
SH, and
CAT forms of GAP reveals a proline-rich
sequence which may serve as a putative SH3-binding domain
(PPLPPPPPQLPP). A synthetic peptide containing this sequence was able
to compete for Hck SH3-GAP interaction in vitro, while an
N-terminal deletion mutant of GAP lacking this region did not bind to
the Hck SH3 domain. This proline-rich sequence is similar to a region
in the protein 3BP-1, a protein identified in an SH3 screen to bind to
the Abl and Src SH3 domains (PPPLPPLV; 46, 47). The presence of a
possible proline-rich SH3-binding motif within the N-terminal region of
GAP suggests that GAP may interact with SH3-containing proteins in
addition to Src family tyrosine kinases. Such interactions could affect
GAP activity, subcellular localization, or interactions with other
proteins.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.