From the
The c-Src tyrosine kinase phosphorylates and binds to a 68-kDa
RNA-binding protein in mitotic cells. We have examined the mechanism
and functional consequence of the interaction of c-Src with this
protein, Sam 68 (Src associated in mitosis, 68 kDa). In whole cell
homogenates, Sam 68 was the predominant substrate and binding partner
of overexpressed c-Src. Mitotic, tyrosine-phosphorylated Sam 68 bound
selectively to recombinant SH2 domains with significantly different
affinities (c-Src
The c-Src tyrosine kinase is activated in mitotic cells as a
consequence of dephosphorylation of its regulatory phosphotyrosine
residue, Tyr-527 in chicken c-Src
(1, 2, 3) .
Mitotic dephosphorylation of Tyr-527 is accompanied by an increased
ability of the c-Src SH2 domain, which is otherwise occupied by
phosphorylated Tyr-527 in an intramolecular interaction, to bind to
tyrosine-phosphorylated ligands
(4) . Mitotic substrates of
c-Src might therefore be phosphorylated by activated c-Src and bind to
the disengaged SH2 domain. We and Fumagalli and colleagues
(5, 6) found such a potential mitotic target of c-Src, which we
term Sam (Src associated in mitosis) 68. Sam 68
Intracellular signal
transduction involves the transient or stable formation of highly
specific protein-protein interactions, which are mediated by protein
modules, paradigms of which are SH2 and SH3 domains
(13) . SH2
domains bind to phosphotyrosine (PTyr)-containing peptides in a
sequence-specific manner and mediate the recruitment of SH2-containing
target proteins to tyrosine-phosphorylated proteins such as activated
growth factor receptors. Phosphopeptide binding to SH2 domains can also
regulate enzymatic activity. For instance, intramolecular binding of
phosphorylated Tyr-527 to the c-Src SH2 domain represses its tyrosine
kinase activity
(4, 14, 15, 16) . SH3
domains bind to sequences containing multiple proline residues, with a
minimal consensus of P XXP
(17, 18) . They also
participate in signaling complex formation and can also modulate
enzymatic activity. For example, phosphoinositide 3-kinase is activated
by Src SH3 domain binding to its p85 subunit
(19) . The SH3
domain of c-Src is also required for the repression of Src activity by
PTyr-527-SH2 domain interaction
(20, 21, 22) .
We have set out to determine the basis of the selectivity with which
c-Src phosphorylates and binds to its potential mitotic target, Sam 68.
We have found that Src competitively selects Sam 68 as the predominant
substrate and binding partner in cell homogenates and that this can be
explained by the specificity with which Sam 68 binds to the Src SH3 and
SH2 domains. We have also found that SH3 domain binding to Sam 68
decreases its ability to bind to poly(U) in vitro, suggesting
that Src-Sam 68 interaction may have functional consequences in
vivo.
For affinity
precipitation of proteins with GST-SH2 domains, cells were lysed in 3T3
lysis buffer containing 1% SDS and then diluted 4-fold with lysis
buffer (without SDS), and DNA was sheared by passage through a 22-gauge
needle. Lysates were cleared by centrifugation in an SM24 rotor at
15,000 rpm for 20 min and precleared by incubating with GST-saturated
glutathione beads. Precleared lysate (200 µg) was incubated with 10
µg of GST or GST-SH2 domain for 40 min on ice. Complexes were
adsorbed to glutathione beads (20 µl) for 40 min, the beads were
washed three times with 3T3 lysis buffer containing 0.1% SDS, and
eluted proteins were immunoblotted and probed with anti-PTyr or
anti-Sam 68 antibodies.
Peptides used in these studies were
synthesized by Chiron Mimotopes and were biotinylated at the
amino-terminal residue. Peptides were modeled on proline-rich potential
SH3-binding motifs from human Sam 68
(7) , mouse 3BP1
(26) , and bovine p85
To examine the specificity with which c-Src phosphorylates
and associates with Sam 68 in a physiological context, homogenates of
NIH 3T3-derived c-Src overexpressor cells were incubated in the
presence of EDTA to inhibit endogenous kinase activity, allowing global
dephosphorylation of cellular proteins by endogenous phosphatases.
Reincubation of EDTA-treated homogenates with Mg
Taken together,
the results suggest that the selectivity with which Src associates with
and phosphorylates Sam 68 in mitotic cells may stem from the high
specificity of binding of Sam 68 sequences to the Src SH3 and SH2
domains. As a working model, we suggest that binding of Sam 68 to the
Src SH3 domain may enable phosphorylation of a tyrosine residue, which
then binds to c-Src SH2. Subsequent tyrosine phosphorylation of bound
Sam 68 may create SH2 binding sites for other SH2-containing proteins,
while those SH3 binding sites not engaged in binding to Src may recruit
SH3-containing proteins to the Src-Sam 68 complex. In this way, Src-Sam
68 could direct the subcellular localization or modulate the activity
of signaling proteins during the mitotic transition. This model of Sam
68 acting as a Src-elicited SH2/SH3 docking protein is consistent with
recent studies by Richard et al. (32) who found that
tyrosine-phosphorylated Sam 68 co-immunoprecipitated with Grb2 from
lysates of HeLa cells cotransfected with Sam 68, Fyn, and Grb2-encoding
plasmids and with Grb2 and phospholipase C
The ability of Src SH3 to inhibit binding of poly(U)
by Sam 68 raises the possibility that interaction of c-Src with Sam 68
modifies, either by decreasing binding ability or changing specificity,
Sam 68-RNA interactions. The consequences of such modulations in
mitotic cells might be to suppress processing/translation of
inappropriate ( e.g. G1/S-specific) messages or to prepare Sam
68 for interaction with G1-specific messages after mitosis. Since the
Src SH3 domain also binds to ribonucleoprotein K
(5, 33) , it will be interesting to determine whether
this interaction also results in a modulation of RNA binding ability.
It is clear from the results presented here and elsewhere that Sam
68 is the product of the cDNA reported to encode GAP-associated p62.
Antibodies raised against peptides or recombinant protein fragments
derived from the reported p62 cDNA sequence specifically recognize Sam
68 ( Fig. 2and Refs. 5, 8, and 32), and transcription/translation
of this clone in vitro and in vivo produces a protein
with all of the characteristics of Sam 68 ( Fig. 3and Refs. 9 and
32). However, it is also clear that Sam 68 and GAP-associated p62
differ significantly in many respects. Unlike Sam 68, GAP-associated
p62 does not bind to the Src SH3 domain ( Fig. 2and Ref. 6) or to
poly(U) beads
(8) , does not exhibit increased tyrosine
phosphorylation in mitosis (Ref. 6),
We thank Paul Polakis for the human Sam 68 cDNA, Piera
Cicchetti for bacteria expressing GST-Abl SH3, Stéphane Richard
for communication of unpublished results, Shubha Bagrodia, Douglas
Laird, and Ross Resnick for discussions, Eden Heinemann for preparation
of figures, and Paula Slocum for preparation of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Ras GTPase activating protein > p85
(amino-terminal) > Grb2
p85
(COOH-terminal)). In vitro translated Sam 68 also bound selectively to recombinant SH3
domains, with the highest affinity for the Src and p85
SH3
domains. SH3 binding was inhibited by specific Sam 68 peptides. In
vitro translated Sam 68 bound directly to immobilized poly(U), and
this was inhibited by binding of Src and p85 SH3 domains to Sam 68. The
results suggest that the selection of Sam 68 as a mitotic target by
c-Src is the result of highly specific interaction with SH2 and SH3
domains and that this interaction may modulate the RNA binding activity
of Sam 68.
(
)
is tyrosine phosphorylated in mitotic cells expressing wild
type and mutationally activated c-Src and forms a stable complex with
Src by interactions with both the SH3 and SH2 domains of Src. Peptides
derived from purified Sam 68 revealed that it was closely related to
the product of a gene, previously cloned as encoding GAP-associated p62
(7) . However, important differences in immunoreactivity,
protein and nucleic acid binding, and electrophoretic mobility have
distinguished the 62-kDa tyrosine-phosphorylated protein associated
with Ras GAP from Sam 68 (5, 6, 8, 9, and this report). Sam 68 was able
to bind to RNA homopolymers, specifically poly(U), in vitro (5) . This is consistent with the primary structure of the
p62 clone, which contains a KH domain
(10, 11) and a
region of similarity to the RGG box
(12) , both of which are
predictive of RNA-binding proteins.
Plasmids and Recombinant
Proteins
Plasmid pGEM-p68 h was created by inserting the
human p62
(7) coding region from p62R2 (supplied by P. Polakis,
Onyx Pharmaceuticals) into the EcoRI site of pGEM-3Z.
Construction of cDNAs encoding GST fusion proteins with c-Src SH3
(23) , c-Src SH2
(24) , v-Src SH3, v-Src SH3 W118R and
p85 SH3
(25) , Abl SH3
(26) , Grb2 (C) SH3, Fgr SH3,
spectrin SH3, and PLC
SH3
(27) , p85
(N,C) SH2 and GAP
(N) SH2
(28) , and Grb2 SH2
(29) have been described.
Human Crk (N) SH3 (residues 133-184) was constructed by cloning
the relevant fragment produced by polymerase chain reaction into the
BamHI- EcoRI sites of pGEX 2T. GST fusion proteins
were purified from
isopropyl-1-thio-
-D-galactopyranoside-induced
Escherichia coli lysates with glutathione-Sepharose beads.
Purified fusion proteins were eluted from the beads with 5 mM
glutathione, 50 mM Tris-Cl, pH 8, 100 mM NaCl.
Glutathione was removed by gel filtration on desalting columns, and the
protein concentrations of purified, soluble fusion proteins were
determined.
Cell Lysis, Cell Lysate Manipulations,
Immunoprecipitation, and Immunoblotting
Cell lines
overexpressing wild type c-Src (NIH(pMcsrc/foc)B), partially activated
c-Src(RF) (NIH(pR148416/pSV2neo/cos)E), and fully activated c-Src
(F527) (NIH(pcsrc527/foc/ep)B1) have been described
(5) .
Homogenization of cells and incubation of homogenates in the presence
of 5 mM EDTA was as described
(30) , except that cells
were collected by trypsinization rather than scraping, and subcellular
fractionation was not performed. After incubation for 45 min at 37
°C, homogenates (400 µl) were incubated for 15 min at room
temperature in the presence or absence of 10 mM MgCl(5 mM free Mg
)/1 mM ATP.
Homogenates were solubilized by addition of an equal volume of 2
concentrated 3T3 lysis buffer
(5) . Cell lysis,
immunoprecipitations, and immunoblotting were as previously described
(5) . An anti-Sam 68 rabbit antiserum was generated against
peptide C corresponding to the 11 carboxyl-terminal residues of the
human p62 sequence
(7) conjugated to keyhole limpet hemeocyanin
via an amino-terminal cysteine residue. Polyclonal anti-GAP antibodies
and monoclonal anti-PTyr antibody (4G10) were from UBI.
In Vitro Binding
Assays
[S]Methionine-labeled Sam 68
was produced by in vitro transcription of pGEM-p68 h with SP6
DNA polymerase (Promega) and translation of the resulting mRNA in a
rabbit reticulocyte lysate system (Promega).
[
S]Methionine metabolically labeled Sam 68 was
immunoprecipitated from lysates of NIH 3T3 cells, which had been
labeled for 12-16 h with
[
S]methionine/cysteine (ICN;
50 µCi/ml)
in methionine-depleted medium. Immunoprecipitation or affinity
precipitation of in vitro translated Sam 68 was performed in
TLB (25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM
EDTA, 0.5% Nonidet P-40, 0.1% SDS, 10 µg/ml leupeptin, and 10
µg/ml aprotinin). Prior to immunoprecipitation, lysates were
precleared by incubation with pre-immune serum and protein A-Sepharose
beads. Before precipitation with GST fusion proteins, lysates were
precleared by incubation with GST-saturated glutathione-Sepharose
beads. Precipitations were performed by incubating lysates with
antiserum (10 µl/lane) or soluble GST-fusion protein in the
presence or absence of competing peptides for 45 min at 4 °C,
followed by incubation with protein A-Sepharose or
glutathione-Sepharose beads for 30 min. Precipitates were washed
3-4 times with TLB. Precipitation with poly(U) beads (Pharmacia
Biotech Inc; AGPOLY(U)) was for 10-15 min at 4 °C. Poly(U)
precipitates were washed 3-4 times with TLB containing 1 mg/ml
heparin. Precipitates were eluted with SDS-polyacrylamide gel
electrophoresis sample buffer and subjected to SDS-polyacrylamide gel
electrophoresis (9% acrylamide) and fluorography. To confirm
precipitation of equal amounts of GST fusion proteins, gels were
Coomassie Blue stained.
(31) . Peptide 1, QTPSRQPPLPH
(Sam 68 residues 32-42); peptide 2, ATQPPPLLPPSAT (Sam 68
residues 60-72); peptide 3, RGRGAAPPPPPVPRGRG (Sam 68 residues
289-306); peptide 4, VTRGVPPPPTVRGA (Sam 68 residues
329-342); peptide 5, RIPLPPPPAPE (Sam 68 residues 354-365);
peptide 3BP1, RAPTMPPPLPPVPPQP (3BP1 residues 265-280); peptide
p85, SPPTPKPRPPRPLPV (p85
residues 83-97); peptide C,
CGAYREHPYGRY (Sam 68 residues 433-443).
/ATP
resulted in the tyrosine phosphorylation of two predominant proteins
with molecular masses of 60 and 68 kDa, revealed by anti-PTyr
immunoblotting (Fig. 1, lane 5). The 60-kDa
phosphorylated protein comigrated with c-Src, which was the predominant
PTyr-containing protein in untreated lysates ( lanes 3 and 5). The 68-kDa phosphoprotein comigrated with
tyrosine-phosphorylated Sam 68 in lysates from mitotic c-Src(RF)
overexpressor cells ( lanes 2 and 5).
Immunoprecipitation of c-Src from detergent-solubilized homogenates
demonstrated that tyrosine-phosphorylated Sam 68 co-immunoprecipitated
with c-Src after ATP treatment or with c-Src(RF) from mitotic lysates
( lanes 6-9). No other coprecipitating
PTyr-containing proteins were detected. The identity of the
coprecipitating tyrosine-phosphorylated protein as Sam 68 was confirmed
by probing a duplicate blot with a commercial anti-Sam 68 antibody
(data not shown). Therefore, Sam 68 appears to be the preferred
substrate and binding partner of dephosphorylated, activated c-Src in
total cellular homogenates, as in mitotic cells in vivo. This
implies that Sam 68 possesses highly specific recognition motifs for
phosphorylation by and for binding to c-Src and/or that high affinity
interaction of Sam 68 phosphotyrosine residues with the c-Src SH2
domain protects Sam 68 from tyrosine dephosphorylation.
Figure 1:
Src
preferentially selects Sam 68 as a substrate and binding partner in a
cell-free system. Unsynchronized ( U) or mitotic ( M)
c-Src(RF) expressor cells were directly lysed in lysis buffer.
Unsynchronized wild type ( WT) c-Src overexpressor cells were
homogenized, incubated in the presence of EDTA, and then incubated in
the presence (+) or absence () of Mg
/ATP
and solubilized with lysis buffer. Lysates ( lanes 1-5) or anti-Src immunoprecipitates ( lanes 6-9) were immunoblotted and probed with anti-PTyr
antibodies. Bound antibodies were detected by incubation with
I-protein A followed by autoradiography. The positions of
molecular mass markers (in kDa), Sam 68 (p68), and Src are
indicated.
To further
study Sam 68-Src interactions, we generated a polyclonal antiserum
against a peptide based on the Sam 68 carboxyl-terminal as predicted
from the p62 cDNA sequence (see below). This antiserum
immunoprecipitated a tyrosine-phosphorylated, 68-kDa protein from
Src(F527)-transformed NIH 3T3 cells (Fig. 2, lane 3) and recognized an anti-PTyr and GST-SH3
domain-precipitable 68-kDa protein on Western blots ( lanes 1, 3, and 6). The same antibody did not
detect any proteins in Western-blotted anti-Ras GAP immunoprecipitates,
although a coprecipitating 62-66-kDa protein (GAP-associated
p62), as well as p190, were clearly detected with an anti-PTyr antibody
( lanes 4). These results are consistent with those of
Ogawa et al. (8) who concluded that Sam 68 and
GAP-associated p62 are distinct proteins. However, we cannot exclude
the possibility that differences in immunoreactivity, SH3 binding,
electrophoretic mobility, and GAP binding in vivo are due to
differential post-translational or post-transcriptional modifications
of the same gene product.
Figure 2:
Sam 68 is distinguishable from
GAP-associated p62. Lysates from unsynchronized c-Src(F527)
overexpressor cells were immunoprecipitated with pre-immune ( lanes 2), anti-Sam 68 ( lanes 3), or anti-GAP
( lanes 4) sera or affinity precipitated with GST
( lanes 5) or GST-Src SH3 ( lanes 6).
Whole cell lysates ( WCL) ( lanes 1) and
precipitates were immunoblotted, and duplicate blots were probed with
anti-PTyr or anti-Sam 68 antibodies. Detection was by
chemiluminescence.
To analyze the specificity of binding of
mitotic, tyrosine-phosphorylated Sam 68 to individual SH2 domains,
lysates from unsynchronized or mitotic c-Src(RF) cells were incubated
with equal amounts of the SH2 domains of c-Src, Grb2, Ras GAP
(amino-terminal), and the p85 subunit of phosphoinositide 3-kinase
(amino- and COOH-terminals), and the precipitated complexes were
immunoblotted and probed with anti-PTyr or anti-p68 antibodies
(Fig. 3). Lysates from c-Src(F527) cells were also probed to
ensure that each SH2 domain was capable of binding
tyrosine-phosphorylated proteins. As expected, Sam 68 was precipitated
by the Src SH2 domain from mitotic but not from unsynchronized Src(RF)
or Src(F527) transformed cells ( lanes 7-9).
Mitotic, tyrosine-phosphorylated Sam 68 also bound to other SH2 domains
with apparent affinity Src
Ras GAP (N) > p85
(N) > Grb
2, with little detectable binding to the p85 (C) SH2 domain ( lanes 8, 11, 14, 17, and
20). The ability of mitotic, tyrosine-phosphorylated Sam 68 to
bind selectively to other SH2 domains and the presence of multiple
potential tyrosine phosphorylation sites in Sam 68
(7) suggests
that other SH2 domain-containing proteins may be recruited to the
Src-Sam 68 complex in mitotic cells. We have, however, been unable to
detect association of Sam 68 with either Ras GAP or p85 in vivo (Fig. 2).
(
)
Figure 3:
Tyrosine-phosphorylated Sam 68 from
mitotic cells selectively binds recombinant SH2 domains. Lysates from
unsynchronized ( U) or mitotic ( M) c-Src(RF) cells or
unsynchronized c-Src(F527) cells ( F) were affinity
precipitated with 10 µg of GST ( lanes 4-6)
or GST-Src SH2 ( lanes 7-9), GST-Grb2 SH2
( lanes 10-12), GST-GAP amino-terminal SH2
( lanes 13-15), GST-p85 amino-terminal SH2
( lanes 16-18), or GST-p85
COOH-terminal
SH2 ( lanes 19-21). Lysates ( lanes 1-3) and precipitates were immunoblotted, probed
with anti-PTyr or anti-Sam 68, and detected by
chemiluminescence.
In vitro transcription and translation of the human p62 cDNA produced an
[S]methionine-labeled protein of slightly faster
electrophoretic mobility than Sam 68 immunoprecipitated from
[
S]methionine metabolically labeled NIH 3T3
cells (Fig. 4, lanes 1 and 3). This
may reflect post-translational modification of Sam 68 in fibroblasts.
Transcription and translation of the human p62 cDNA produced a protein
that was specifically immunoprecipitated by anti-Sam 68 ( lanes 4 and 5) and affinity precipitated by GST-Src
SH3 ( lane 7), confirming that Sam 68 is the product
of the p62 cDNA. We have previously shown that Sam 68 is precipitated
from NIH 3T3 lysates by poly(U) beads
(5) . To determine whether
Sam 68 binds directly to poly(U), this experiment was repeated with
in vitro translated Sam 68. Specific precipitation of Sam 68
with poly(U) beads was observed. This was competed by soluble poly(U)
but not by poly(C) ( lanes 10-12), demonstrating
direct Sam 68-poly(U) interaction. Interestingly, Sam 68 was
efficiently precipitated by poly(I)-poly(C) beads (Fig. 7,
lane 11), suggesting that it may be able to bind to
double-stranded RNA.
Figure 4:
Sam 68 is the product of the p62 cDNA and
binds poly(U) directly. Sam 68 was immunoprecipitated from
[S]methionine metabolically labeled NIH 3T3
cells in the presence or absence of peptide C (40 µM)
( lanes 1 and 2). In vitro translated [
S]methionine-labeled product of
the human p62 cDNA was immunoprecipitated with anti-Sam 68 with or
without 50 µM peptide C ( lanes 4 and
5), affinity precipitated with GST ( lane 6)
or GST-Src SH3 with or without peptides C or 3 ( lanes 7-9), or affinity precipitated with poly(U) beads
with or without soluble poly(U) or poly(C) (1 mg/ml) ( lanes 10-12) or with poly(U) beads pretreated with
micrococcal nuclease ( lane 13). Lane 3 contains the input translation product (20% of amount used for
precipitations).
Figure 7:
SH3
domains inhibit binding of poly(U) to Sam 68. In vitro translated Sam 68 was precipitated with poly(U) beads ( lanes 2-10), poly(I)-poly(C) beads ( lane 11), nuclease-treated poly(U) beads ( lane 12), or glutathione beads ( lane 13) in
the presence or absence of 10 µg (unless otherwise indicated) of
the indicated GST fusion proteins. The precipitation in lane 6 was performed in the presence of 250 µM
peptide 3. Lane 1 contains 20% of input translation
product.
The specificity of the interaction between Sam
68 and SH3 domains was examined by affinity precipitating in vitro translated Sam 68 with different GST-SH3 domain fusion proteins
(Fig. 5). Sam 68 was bound most effectively by the SH3 domains of
c-Src and v-Src, but appreciable binding was also observed with the
p85 SH3 domain ( lanes 3-6, 14,
and 16). The SH3 domains of Crk (amino-terminal), Abl, and
PLC
bound Sam 68 to lesser extents, while no binding was
detectable to the SH3 domains of Fgr, spectrin, and Grb2
(COOH-terminal). Binding to the v-Src SH3 domain was abolished by point
mutation of Trp-118, which is conserved in SH3 domains. Similar results
were obtained by precipitating Sam 68 from NIH 3T3 lysates with
different GST-SH3 domains (data not shown).
Figure 5:
Sam 68 selectively binds recombinant SH3
domains. In vitro translated Sam 68 was precipitated with GST
( lane 2) or the indicated GST-SH3 fusion proteins
( lanes 3-16). 5 µg of fusion protein was
used for precipitation except where indicated (0.5 µg; lanes 3, 5, and 7). Lanes 1 and 17 contain 20% of input translation
product.
To define the SH3
binding sites within Sam 68, peptides corresponding to proline-rich,
potential SH3 binding sites in Sam 68 were synthesized. The ability of
these peptides to inhibit Sam 68 binding to the SH3 domains of Src or
p85 was assessed (Fig. 6). Peptide 3, corresponding to
residues 289-306 in Sam 68, completely inhibited Sam 68 binding
to Src SH3 ( lane 6). The same concentrations of
peptides corresponding to residues 354-365 of Sam 68 (peptide 5)
and 265-280 of 3BP1 partially competed with Sam 68 for Src SH3
binding ( lanes 8 and 9). In contrast,
peptide 3 only partially competed for binding of Sam 68 to p85
SH3. Peptide 4 from Sam 68 and residues 89-97 of p85
also
partially blocked binding ( lanes 14, 15, and
18). We conclude that Src SH3 binds primarily to a region
within residues 289-306 of Sam 68, while the p85
SH3 domain
binds to this and at least one other site with apparently lower
affinity. In similar experiments, the Ab1 SH3 domain exhibited a
binding profile similar to that of the Src SH3 domain, while the Crk
(N) and PLC
SH3 binding profiles resembled that of p85
(data
not shown). The results suggest that Sam 68 interacts in vivo with the Src SH3 domain in the region of residues 289-306
but that other SH3 binding sites in Sam 68 may mediate interaction with
other SH3 domain-containing proteins, such as p85, and possibly other
c-Src molecules.
Figure 6:
Proline-rich peptides differentially
inhibit Sam 68 binding to Src and p85 SH3 domains. In vitro translated Sam 68 was precipitated with 10 µg of GST ( lane 2), GST-Src SH3 ( lanes 3-10), or
GST-p85
SH3 ( lanes 11-18) in the presence
or absence of 100 µM of the indicated peptides. Lane 1 contains 20% of input translation
product.
To gain insight into the functional consequences of
the Src SH3-Sam 68 interaction, we examined whether Src SH3 was able to
modify the RNA binding activity of Sam 68 in vitro (Fig. 7). Affinity precipitation of in vitro translated Sam 68 by poly(U) beads was blocked in a
concentration-dependent manner by the Src SH3 domain ( lanes 2-5). This inhibition was blocked by Sam 68 peptide
3 ( lane 6), consistent with the binding data above.
The ability of other SH3 domains to inhibit poly(U) binding correlated
with their ability to bind to Sam 68 ( lanes 7-10). Coomassie Blue staining of precipitates
verified that SH3 domains did not directly bind to the poly(U) beads
(data not shown), indicating that binding of SH3 domains to Sam 68
blocks its ability to bind poly(U). It will be important to determine
whether SH3 domains and poly(U) compete for overlapping binding sites
or whether SH3 binding causes a conformation change in Sam 68 that
decreases its intrinsic poly(U) binding activity.
from v-Src-transformed
NIH 3T3 cells.
(
)
and does
not react with anti-Sam 68 antibodies (including commercially available
anti-p62 antibodies) ( Fig. 2and Ref. 8). Furthermore, Sam 68
does not appear to associate with GAP in vivo (
Fig. 2
and Ref. 8). In light of these data and until the exact
nature of GAP-associated p62 is ascertained, we suggest that Sam 68 and
GAP-associated p62 be regarded as distinct proteins and be referred to
accordingly.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.