(Received for publication, October 10, 1995; and in revised form, December 15, 1995)
From the
A necessary downstream element of Abelson murine leukemia virus (Ab-MLV)-mediated transformation is Ras, which can be activated by the phosphotyrosine-dependent association of Shc with the Grb2-mSos complex. Here we show that Shc is tyrosine-phosphorylated and associates with Grb2 in v-Abl-transformed cells, whereas Shc in NIH3T3 cells is phosphorylated solely on serine and is not Grb2-associated. In addition, Shc coprecipitates with P120 v-Abl and P70 v-Abl, which lacks the carboxyl terminus. Surprisingly, a kinase-defective mutant of P120 also binds Shc, demonstrating that Shc/v-Abl association is a phosphotyrosine-independent interaction. Glutathione S-transferase fusion proteins were used to map the interacting domains and showed that Shc from both NIH3T3 and v-Abl-transformed cells binds to the Abl SH2 domain and that P120 v-Abl binds to a region in the amino terminus of Shc. Consistent with these data, a v-Abl mutant encoding only the Gag and SH2 regions was able to bind Shc in vivo. The unique non-phosphotyrosine-mediated binding of Shc may allow direct tyrosine phosphorylation of Shc by v-Abl and subsequent activation of the Ras pathway through assembly of a signaling complex with Grb2-mSos.
Abelson murine leukemia virus (Ab-MLV) ()encodes a
single protein, v-Abl, which is sufficient for
transformation(1) . This molecule is a chimeric protein
consisting of the amino-terminal region of Moloney-MLV Gag and the SH2,
SH1, and carboxyl-terminal domains of the c-Abl non-receptor tyrosine
kinase(1) . In contrast to the tightly regulated c-Abl kinase (2, 3) , v-Abl has constitutive tyrosine kinase
activity, a feature required for transformation(1) . Signaling
via the Ras pathway is an essential part of the mechanism by which
v-Abl induces transformation (4, 5) . Expression of a
dominant negative Ras (4) or microinjection of anti-Ras
antibodies (5) causes a reversion of the Ab-MLV-transformed
phenotype. Transformation by the Bcr-Abl chimeric protein, another
oncogenic form of c-Abl, found in Philadelphia chromosome-positive
chronic myelogenous leukemia(1, 6) , is also blocked
by dominant negative Ras(4) .
The mechanism by which v-Abl activates Ras has not been elucidated. The Bcr-Abl protein may signal Ras via direct interaction with the Grb2 protein(7) , a molecule that binds the guanine nucleotide exchange factor, mSos(8, 9) . Localization of the Grb2-mSos complex to the plasma membrane activates Ras(9, 10) . Bcr-Abl-Grb2 interaction involves sequences in the Bcr domain(7, 11, 12) . This pathway is probably not used by v-Abl because the v-Abl protein does not contain a motif similar to that involved in Bcr-Abl-Grb2 interaction.
An alternative link to Ras, important in signaling by a large number of receptor tyrosine kinases including EGFR, Ins-R, PDGFR, c-Trk, c-Kit, c-ErbB2, c-ErbB3, c-Ret(13, 14, 15, 16, 17, 18) , the non-receptor tyrosine kinases, Src, Fps, FAK, Lyn, Syk(19, 20, 21) , and the oncoprotein polyoma middle T(22, 23) , involves tyrosine phosphorylation of the Shc adaptor protein. Interactions between Shc and receptor tyrosine kinases occur when the receptors are activated by interaction with ligand; the autophosphorylation sites on the receptors provide binding sites for the Shc SH2 or amino-terminal domains(16, 24, 25, 26) . Subsequent tyrosine phosphorylation of the YVNV motif in Shc by the receptors or their associated kinases creates a binding site for the Grb2 SH2 domain. Shc then associates with Grb2 which brings mSos into the complex(27, 28, 29) . Such an interaction may be involved in factor independent growth of v-Abl-expressing mast cells(30) . However, association with Grb2 and evidence of stimulation of signals downstream of Ras have not been reported in that system.
Because v-Abl lacks the Grb-2 binding site found in Bcr-Abl, we investigated the possibility that Shc protein associates with v-Abl and Grb-2 in v-Abl-transformed cells. These experiments demonstrate that Shc is tyrosine-phosphorylated in Ab-MLV-transformed fibroblasts and binds to Grb2. This interaction involves an unusual non-phosphotyrosine-dependent interaction that occurs between the v-Abl SH2 domain and the Shc amino terminus. The ability of Shc to bind Grb2 and the v-Abl SH2 simultaneously suggests a model for Shc-mediated Ras activation in Ab-MLV transformation similar to that of receptor tyrosine kinases.
To prepare GST fusion proteins, log phase Escherichia coli JM109 cells containing the pGEX-3X constructs
were grown for 3 h in the presence of 50 µg/ml ampicillin and 0.5
mM isopropyl-1-thio--D-galactopyranoside. The
cells were pelleted and lysed in ice cold RIPA buffer (10 mM sodium phosphate, pH 7.0, 150 mM sodium chloride, 0.1%
SDS, 1% Nonidet-40, 0.5% sodium deoxycholate, 2 mM EDTA, 50
mM sodium fluoride, 1 mM sodium orthovanadate, 1
mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin
(Boehringer Mannheim)) with 10 µg/ml lysozyme. After 25 min on ice,
the samples were sonicated (10
1 s) and centrifuged at 14,000
rpm for 15 min. Glutathione-Sepharose beads (Pharmacia) were added to
the bacterial lysates, and the samples were incubated for 1.5 h on a
rotating wheel at 4 °C. The beads were centrifuged and washed with
RIPA buffer. To obtain purified protein cleaved from the GST moiety,
the fusion proteins bound to GST beads were washed and resuspended in
50 mM Tris, pH 8.0, 150 mM NaCl, and 1 mM CaCl
followed by incubation with 1 unit of Factor Xa
(U. S. Biochemical Corp.).
Figure 1:
Tyrosine phosphorylation of Shc in
ANN-1 cells and coimmunoprecipitation of Grb2. Shc was
immunoprecipitated from NIH3T3 and ANN-1 cells, subjected to SDS-PAGE,
and transferred to a polyvinylidene difluoride membrane as described
under ``Experimental Procedures.'' A, Western blot
of immunoprecipitated Shc was probed with -Shc antibody. The p46
form of Shc could not be separated from the IgG heavy chain. p66 Shc is
visible in the ANN-1/
-Shc lane upon longer exposure (data not
shown). B, Western blot from A was stripped and
reprobed with
-phosphotyrosine antibody. C, lower portion
of A was removed and probed with
-Grb2 antibody. The
positions of the different Shc forms are noted with arrows. Control, RGG. * indicates IgG heavy
chain.
Figure 2:
Phosphoamino acid analysis of
[P]orthophosphate-labeled Shc. NIH3T3 and ANN-1
cells were labeled overnight with
[
P]orthophosphate in phosphate-free DMEM and 5%
dialyzed fetal calf serum. Cells were lysed, immunoprecipitated with
-Shc antibody, and the precipitates were used for SDS-PAGE. A, the gel was transferred to a polyvinylidene difluoride
membrane, and autoradiography was performed. B, the
autoradiogram and membrane were aligned, and the portion containing
both the p46 Shc and p52 Shc bands was excised for phosphoamino acid
analysis as described under ``Experimental Procedures.'' A
PhosphorImager was used to examine the thin layer plate. The migration
points of the cold phosphoamino acid standards and inorganic phosphate
(P
) are indicated.
To determine if the tyrosine-phosphorylated Shc was able to bind Grb2, the lower portion of the blot shown in Fig. 1A was probed with anti-Grb2 antibody (Fig. 1C). Shc from ANN-1 cells binds Grb2, in contrast to Shc precipitated from NIH3T3 cells which does not. Therefore, the presence of v-Abl causes the association of Shc and Grb2, most likely through the phosphorylation of Tyr-317 of Shc. This pattern is consistent with Shc-mediated Ras activation as seen with other tyrosine kinases(16, 18, 20, 29) .
Figure 3:
In vivo association of v-Abl and
Grb2 with Shc. 70wt (P70) and ANN-1 (P120) cells were lysed and
immunoprecipitated for Western analysis as described under
``Experimental Procedures.'' A, v-Abl was
precipitated with -Abl antibody from 70wt and ANN-1 cells, and the
Western blot was probed with
-Abl antibody. P70 appears as a
doublet due to a glycosylated isoform(32) . B, the
blot from A was stripped and reprobed with
-Shc antibody. C, Shc was immunoprecipitated with
-Shc antibody from
70wt and ANN-1 cells. The Western blot of the precipitates was probed
with
-Shc antibody. D, the blot in C was
stripped and probed with
-Abl. E, the lower portion of
the C blot was probed with
-Grb2 antibody.
Abl, H548 antibody; control (A and B), UPC10 antibody, C-E, RGG. Shc, v-Abl, and Grb2
bands are indicated by arrows. * indicates Ig heavy
chain.
To determine if the carboxyl terminus of v-Abl is required for association with Shc, lysate from cells transformed with the Ab-MLV P70, which encodes a v-Abl protein containing the Gag, SH2, and SH1 domains(32) , was immunoprecipitated with anti-Abl antibodies and probed with anti-Shc antibody (Fig. 3B). P70, like P120, was found to bind Shc. Immunoprecipitation of Shc also coprecipitated P70 (Fig. 3D). Shc in P70 v-Abl-transformed cells is also tyrosine-phosphorylated (data not shown) and coprecipitates with Grb2 (Fig. 3E). Therefore, the presence of the v-Abl carboxyl terminus is not essential for interaction with Shc or Shc phosphorylation.
Most interactions
of Shc with tyrosine kinases have shown that binding of Shc requires a
tyrosine-phosphorylated
motif(14, 17, 24, 25, 28) .
To assess the contribution of v-Abl kinase activity to v-Abl/Shc
association, NIH3T3 cells expressing the mutant
Ab-MLV-P120k, which encodes a kinase-defective P120
v-Abl, were utilized. P120k
contains an Asp to Asn
amino acid change in the kinase domain rendering it kinase-defective.
Cells expressing this protein do not contain increased levels of
phosphotyrosine (Fig. 4, A and C) and
immunoprecipitated P120k
has no detectable kinase
activity in vitro (data not shown). P120k
was immunoprecipitated from cells expressing the mutant virus and
analyzed on Western blots for Shc coprecipitation (Fig. 4, B and D). Immunoprecipitation of P120k
showed that Shc still bound to v-Abl. Therefore, Shc binding is
independent of v-Abl kinase activity.
Figure 4:
Coimmunoprecipitation of
120k and Shc. NIH3T3 cells expressing
P120k
(P120k
) and cells
expressing wild type P120 (ANN-1) were lysed, and
immunoprecipitates were analyzed by Western blotting. A,
equivalent amounts of total cell lysate from P120k
and ANN-1 cells were analyzed by Western blotting with
-phosphotyrosine antibody. B, P120k
and
wt P120 were precipitated from P120k
and ANN-1 cells,
respectively, with
-Abl antibody. Western analysis was performed
using
-Abl antibody. C, the blot in B was
stripped and reprobed with
-phosphotyrosine antibody. D,
the blot was stripped again and probed with
-Shc antibody.
-Abl, H548 antibody; control,
UPC10.
Figure 5:
Mapping the v-Abl binding site for Shc. A, GST fusion proteins of v-Abl domains were used to
precipitate lysates from NIH3T3 and ANN-1 cells as described under
``Experimental Procedures.'' Precipitates were analyzed by
Western blot with -Shc antibody. Equivalent amounts of total cell
lysates are included on the far left. Stripping and reprobing
of the blot with
-GST antibody revealed equivalent amounts of
GST-SH2SH1 and GST-SH1, but twice as much GST-SH2 (data not shown). B, the lower portion of A was probed with
-Grb2
antibody. C, GagSH2 was immunoprecipitated from NIH3T3 cells
with
-Abl antibody and analyzed on a Western blot probed with
-Abl antibody. Pr65 is the pr65 Gag precursor protein
encoded by Mo-MLV.
Abl, H548 antibody. D, the
blot from C was stripped and reprobed with
-Shc antibody.
Upon longer exposure, p46 Shc and p66 Shc are also visible (not shown). E, ANN-1 cell lysates were precipitated with equivalent
amounts of the indicated GST fusion proteins and analyzed by Western
blotting with
-Shc antibody. F, full-length Shc (0.5
µg) cleaved from bacterial GST fusion protein was precipitated with
either GST or GST-Abl SH2 fusion proteins and analyzed on a Western
blot with
-Shc antibody.
A mutant Ab-MLV was made to test whether the Abl SH2 domain is sufficient for interaction in vivo. The GagSH2 virus, which consists of the Ab-MLV Gag-coding region and the Abl SH2-coding region followed by a stop codon, was expressed in NIH3T3 cells. Immunoprecipitates of the GagSH2 protein were analyzed by Western blotting with anti-Shc antibodies, revealing that Shc coprecipitated with the GagSH2 protein (Fig. 5, C and D). The Abl SH2 domain is therefore sufficient for binding of Shc both in vivo and in vitro. To examine whether the binding is direct or requires an intermediary for complex formation, the ability of the Abl SH2 to bind purified Shc was tested. Affinity-purified GST-Shc was treated with Factor Xa to release a full-length Shc protein. The GST-Abl SH2 was able to precipitate the Shc protein as observed by Western analysis with anti-Shc antibodies (Fig. 5F) despite the bacterial origin of both proteins.
Western blot analysis of GST-Abl SH2 precipitates from NIH3T3 and ANN-1 cells with anti-Grb2 antibody showed that Grb2 was precipitated with the GST-Abl SH2 in ANN-1 lysates but not in NIH3T3 lysates (Fig. 5B). This result presumably reflects the association of Grb2 with tyrosine-phosphorylated Shc in ANN-1 cells. In NIH3T3 cells, Shc and Grb2 are not associated, and, therefore, Grb2 would not be precipitated by the GST-Abl SH2 protein. Accordingly, these data suggest that binding of Shc to the Abl SH2 domain does not interfere with binding of Grb2 to Shc at tyrosine 317 and that Shc possesses independent binding sites for Grb2 and v-Abl. The potential for a v-Abl-Shc-Grb2 complex to form in Ab-MLV-transformed cells is suggestive of the Shc-mediated Ras activation described for receptor protein tyrosine kinases.
Figure 6:
Mapping the Shc binding site for v-Abl. A and B, GST fusions of different regions of Shc were
used to precipitate ANN-1 cell lysates as described above. Western
analysis of the precipitates was done using -Abl antibody.
Approximately equal amounts of GST fusion proteins were used in each
precipitation with the exception of GST-NT1 and NT2 which had
30-40% less fusion protein than the GST-NT precipitation (data
not shown).
-Abl, 24-21 antibody. C, GST
and GST-Shc NT were used to precipitate 0.5 µg of Abl SH2SH1 domain
cleaved from the bacterially generated fusion as described under
``Experimental Procedures.'' The precipitates were analyzed
by Western blotting.
Abl,
19-84.
Our results show that Shc is tyrosine-phosphorylated and associated with Grb2 in v-Abl-transformed cells. This association could lead to activation of Ras, an event required for v-Abl-mediated transformation. In receptor activation models, association of Shc with an activated receptor enables the receptor or a related kinase to phosphorylate Tyr-317, the Grb2 binding site on Shc(28) . This modification allows the Shc-Grb2-mSos complex to assemble at the membrane in proximity to Ras(9, 10, 29) . The binding we have documented may allow v-Abl or an associated kinase to directly phosphorylate Shc, stimulating the formation of a Shc-Grb2-Sos complex. The Gag domain of v-Abl may also provide a plasma membrane-localized platform on which the Shc-Grb2-Sos complex can assemble. Localization of this complex is a critical event for mSos function because addition of a membrane localization signal to Sos is in itself sufficient to activate Ras(10) .
Some tyrosine kinases can bypass Shc and activate Ras by binding the Grb2-mSos complex directly through the Grb2 SH2 domain(8, 9, 27) . Bcr-Abl accomplishes this by binding Grb2 to a phosphotyrosine-containing motif within the Bcr portion of the protein (7, 11, 12) . However, v-Abl lacks a similar Grb2 binding site. Another possible site for direct Grb2 interaction with v-Abl is located within the carboxyl terminus(45) . The Grb2 SH3 domains bind a proline-rich motif in the Abl carboxyl terminus in vitro. The carboxyl terminus of v-Abl also contains proline-rich motifs that bind Crk and Nck(45, 46) , proteins that can associate with the Ras activators, C3G and mSos(47, 48, 49) . However, the efficient transformation of NIH3T3 cells by P70(32) , which lacks these proline-rich motifs, precludes any essential role for these interactions. Tyrosine phosphorylation of Shc and its association with Grb2 could allow Ras signaling in the absence of the Nck, Crk, and Grb2 binding sites.
The v-Abl/Shc interaction
we have documented occurs in a phosphotyrosine independent manner and
contrasts to the conventional view of SH2-mediated interactions which
involve recognition of a phosphotyrosine residue in the context of
three residues carboxyl-terminal to the tyrosine(50) . Tyrosine
phosphorylation of v-Abl is not required because both the
P120k and the GagSH2 proteins bind Shc. In addition,
the GST-Abl SH2 fusion binds Shc and the GST-Shc NT binds Abl SH2SH1 in vitro; these proteins are produced in E. coli and
do not contain phosphotyrosine (51, 52) . The
possibility that tyrosine phosphorylations on Shc in vivo mediate the interaction is excluded because Shc from NIH3T3 cells
binds the GST-Abl SH2 fusion and phosphoamino acid analysis of Shc
recovered from those cells shows no phosphotyrosine. Shc also binds to
P120k
and GagSH2 despite the absence of tyrosine
phosphorylation observed on Western analysis (data not shown).
Recently, Owen-Lynch et al.(30) described the interaction of Shc with a v-Abl protein encoded by a temperature-sensitive (ts) strain of Ab-MLV at both the permissive and nonpermissive temperature. Previous descriptions of the ts strain used note the persistence of phosphotyrosine on v-Abl protein at the nonpermissive temperature(53) . Our own experiments with another ts Ab-MLV yielded similar data (not shown). The persistence of phosphotyrosine at the nonpermissive temperature suggests that these ts systems may not be practical for assessing the phosphotyrosine independence of protein-protein interactions.
Although many
SH2-mediated interactions involve phosphotyrosine, the number observed
that occur independent of this modification is growing.
Phosphotyrosine-independent association between the Abl SH2 and Bcr has
been observed(54, 55) . Two serine-rich regions of Bcr
are important for this binding, but the specific motifs involved have
not been identified. SH2 domains from other proteins including
phospholipase C, Src, and GTPase activating protein also bind to
Bcr in this manner(54) . The SH2 domain of the protein-tyrosine
phosphatase SH-PTP2 binds its own catalytic domain in the absence of
phosphotyrosine(56) . Another instance of
phosphotyrosine-independent binding involves interactions of the Src
and Fyn SH2 domains with Raf, a protein which is phosphorylated on
serine residues(57) . It is possible that the Abl/Shc
interaction is mediated by phosphoserine; however, it has not been
determined whether the GST fusion proteins are phosphorylated
correctly. Serine and threonine phosphorylation has been observed on
bacterially produced GST fusion proteins(51) .
The interaction of v-Abl with the GST-Shc NT1 protein indicates residues 1-85 of p52 Shc are sufficient for binding. p46 Shc, which also binds v-Abl, possesses only residues 46-85 of that region. Therefore, the essential binding site must lie within the shared sequence of both p52 and p46. The region is also of interest because it contains the recently described phosphotyrosine binding domain(58) . This domain binds to NPXpY motifs, including those found on polyoma middle T, EGFR, and Trk(23, 25) . However, this property of the Shc amino terminus is not involved in v-Abl binding because the interaction is phosphotyrosine independent. Additionally, the GST-Shc NT1 and NT2 fusion proteins precipitate v-Abl, whereas similar GST fusions cannot bind NPXpY targets without the minimal 46-238 amino acid region(59) .
Another interaction observed in the Shc amino terminus is the protein kinase C-dependent binding of the PEST-phosphatase to p52 Shc(42) . In addition to inducing Shc/Grb2 association, the v-Abl interaction could also regulate the Shc signaling complex. The PEST-phosphatase binds to Shc but does not alter tyrosine phosphorylation of Shc or its association with Grb2(42) . Presumably, the PEST-phosphatase dephosphorylates other proteins in the Shc signaling complex. By binding to Shc, v-Abl could phosphorylate additional proteins in the complex. Furthermore, because the v-Abl and c-Abl SH2 domains are identical, Shc may be involved in cytoplasmic interactions that are important for normal c-Abl function.