(Received for publication, October 27, 1994; and in revised form, January 20, 1995)
From the
Insulin activates the ras signaling pathway and
promotes hematopoietic cell proliferation. One possible mediator in
such signaling is the vav proto-oncogene product
(p95), which is specifically expressed in cells
of hematopoietic origin and contains domains typical of guanine
nucleotide exchange factors as well as Src homology 2 and Src homology
3 domains. We studied the tyrosine phosphorylation of p95
in hematopoietic cells expressing insulin receptors.
Immunoblotting experiments with an antiphosphotyrosine monoclonal
antibody disclosed that insulin induces rapid and transient tyrosine
phosphorylation of p95
in the human U-266
myeloma cell line. These findings were confirmed by immunoprecipitation
experiments performed with
P-labeled cells and
phosphoamino acid analysis of the bands corresponding to
p95
. Similarly, insulin-dependent tyrosine
phosphorylation of p95
was observed in the human
IM-9 and mouse J558L hematopoietic cell lines. Furthermore, insulin
treatment of cells led to the association of the Src homology 2 domain
of p95
with the activated
-subunit of the
insulin receptor in vitro. Altogether, these data suggest that
p95
is a substrate for the insulin receptor
tyrosine kinase and may be involved in an insulin signaling pathway
linking receptor-generated signals to Ras or other GTP-binding proteins
in cells of hematopoietic origin.
The vav proto-oncogene product
(p95) is a tyrosine kinase substrate that is
specifically expressed in cells of hematopoietic origin and has regions
of homology to bcr, dbl, and the yeast CDC24 guanine
exchange factor as well as one SH2 (
)and two SH3
domains(1, 2, 3, 4, 5, 6, 7, 8) .
p95
undergoes rapid tyrosine phosphorylation in
response to a variety of stimuli in different cell types: in T-cells
after activation of the T-cell antigen receptor or interleukin-2
stimulation (7, 8, 9) ; in B-cells after
activation of IgM receptors(10) ; in activated mast cells after
stimulation of the IgE receptor(8) ; in stem cell factor
responsive cells after stimulation of the c-kit receptor(11) ; and in hematopoietic cell lines in response
to interferon-
, -
, and -
(12) . Although vav was predicted to function as a guanine nucleotide exchange factor
for Rho-like proteins, it was recently demonstrated that it exhibits
guanine nucleotide exchange activity toward ras during T-cell
activation(13) , in NIH-3T3 cells transfected with the vav gene(14) , and in antigen receptor-triggered B-cells (15) .
Insulin exhibits mitogenic effects in a variety of
hematopoietic cells, including myeloid(16) , multiple
myeloma(17) , and lymphoblastoid (18) cell lines. After
insulin treatment of cells, the -subunit of the insulin receptor
tyrosine kinase is activated, resulting in tyrosine phosphorylation of
several cellular substrates (19) . A major substrate for the
insulin receptor tyrosine kinase is IRS-1(19) , which acts as a
docking protein for the SH2 domains of several proteins involved in
insulin signaling, including the p85 regulatory subunit of
phosphatidylinositol
3-kinase(20, 21, 22, 23) , the
phosphotyrosine phosphatase Syp(24) , the oncogenic protein
Nck(25) , and the adaptor protein
Grb-2(26, 27, 28) . In this study, we
examined the effect of insulin on the phosphorylation status of
p95
. We report that p95
is tyrosine-phosphorylated in response to insulin in several
hematopoietic cell lines expressing the insulin receptor. We also
demonstrate that p95
associates via its SH2
domain with the
-subunit of the insulin receptor in
vitro, providing evidence of direct interaction of
p95
with this receptor tyrosine kinase.
Figure 1:
Insulin-dependent
tyrosine phosphorylation of p95. U-266 cells
were stimulated with insulin for the indicated times at 37 °C. Cell
lysates were immunoprecipitated with either nonimmune rabbit
immunoglobulin (lane1) or an antibody against
p95
as indicated (lanes 2-5). A, shown is the antiphosphotyrosine (anti-Ptyr)
immunoblot. The tyrosine-phosphorylated form of p95
is indicated. B, the same blot was reprobed with an
antibody against p95
to establish that equal
amounts of p95
were present in all
lanes.
Figure 4:
Association of the SH2 domain of
p95 with the activated
-subunit of the
insulin receptor detected in in vitro kinase assays. A, cells (8
10
/lane) were treated with
insulin for the indicated time points, and cell lysates were incubated
with either glutathione S-transferase (GST) alone or
GST-vavSH2 bound to glutathione-Sepharose beads or were
immunoprecipitated (IP) with an antibody against the
-subunit of the insulin receptor (aIR) as indicated and
subjected to an in vitro kinase assay. B, shown are
the results from phosphoamino acid analysis of the autophosphorylated
insulin receptor associating with vavSH2 (lane1) or immunoprecipitated by an anti-insulin receptor
antibody (lane2). PSer, phosphoserine; PThreo, phosphothreonine; PTyr,
phosphotyrosine.
Figure 2:
Analysis of the phosphoamino acid content
of p95 before and after insulin stimulation. A,
P-labeled U-266 cells (8
10
/lane) were incubated in the presence or absence of
insulin (Ins) for 10 min at 37 °C as indicated. Cell
lysates were immunoprecipitated with an antibody against
p95
(aVav) or control nonimmune rabbit
IgG (RIgG) as indicated. B, shown are the results
from phosphoamino acid analysis of p95
prior to
and after insulin treatment. PSer, phosphoserine; PThreo, phosphothreonine; PTyr,
phosphotyrosine.
Figure 3:
p95 associates via
its SH2 domain with the
-subunit of the insulin receptor.
Serum-starved IM-9 cells (8
10
/lane) were treated
with insulin for the indicated times at 37 °C. The cells were lysed
in denaturing RIPA buffer, and cell lysates were incubated with either
a GST-vavSH2 fusion protein or glutathione S-transferase (GST) alone bound to
glutathione-Sepharose beads or were immunoprecipitated (IP)
with an antibody against a region of the C terminus of the
-subunit of the insulin receptor (aIR) as indicated.
Proteins were separated by SDS-PAGE and immunoblotted with an antibody
against the
-subunit of the insulin
receptor.
Figure 5:
Tyrosine phosphorylation of
p95 in 32D myeloid cells transfected with the
-subunit of the insulin receptor. A, serum-starved cells
were incubated in the presence or absence of insulin (Ins) for
10 min at 37 °C, and cell lysates were immunoprecipitated with an
antibody against p95
(aVav) or control
nonimmune rabbit IgG (RIgG) as indicated and immunoblotted
with antiphosphotyrosine. B, serum-starved cells were treated
with insulin as indicated and lysed in denaturing RIPA buffer, and cell
lysates were incubated with either glutathione S-transferase (GST) alone or GST-vavSH2 bound to
glutathione-Sepharose beads as indicated and immunoblotted with
antiphosphotyrosine (aPTyr). C, the same blot shown
in B was stripped and reblotted with an antibody against the
-subunit of the insulin receptor (aIR).
The mechanisms of insulin signal transduction have been
extensively studied(19) . After insulin binds to its receptor,
there is activation of the intrinsic tyrosine kinase activity present
in the intracellular portion of the -subunit(19) .
Activation of the insulin receptor tyrosine kinase leads to tyrosine
phosphorylation of the principal substrate, IRS-1. IRS-1 acts as an
SH2-docking protein for several signaling proteins, including the p85
regulatory subunit of phosphatidylinositol
3-kinase(20, 21, 22, 23) , the
tyrosine phosphatase Syp (24) , the oncogenic protein
Nck(25) , and the adaptor protein Grb-2, which links the
guanine nucleotide factor mSos to p21
(26, 27, 28) . Another substrate for
insulin-dependent tyrosine kinase activity is the oncogenic protein
Shc, which also interacts with the adaptor protein
Grb-2(26, 32, 33) . In myeloid hematopoietic
cell lines, insulin induces tyrosine phosphorylation of the
interleukin-4-induced phosphotyrosine substrate (4PS)(16) , a
functional homologue of IRS-1 that is essential for the mitogenic
effects of insulin and interleukin-4(34) .
In this study, we
examined the tyrosine phosphorylation status of p95 in
response to insulin treatment of the hematopoietic cell lines U-266,
IM-9, and J558L, which express functional insulin receptors. We found
that p95
is tyrosine-phosphorylated in a rapid and
transient manner in response to insulin, as determined by
antiphosphotyrosine immunoblotting and
P labeling
experiments. We subsequently sought to determine whether p95
associates with the
-subunit of insulin receptor. The
insulin receptor could not be coimmunoprecipitated by the
anti-p95
antibody, probably due to low stoichiometry of
association between these two proteins. Glutathione S-transferase binding experiments, however, demonstrated that
the SH2 domain of vav associates with the activated insulin
receptor tyrosine kinase in vitro in an insulin-dependent
manner. Similar results were obtained when the 32D myeloid cell line
transfected with a full-length cDNA for the
-subunit of the
insulin receptor was studied. These findings are of considerable
interest as they suggest that p95
can associate directly
with the insulin receptor tyrosine kinase without the requirement of
docking proteins of the IRS family. Thus, vav seems to be
another signaling protein, in addition to Shc, that is not regulated by
IRS-signaling proteins, but instead may be activated by direct
interaction with the insulin receptor tyrosine kinase.
Our data
suggest that p95 is involved in the signal transduction
of insulin in certain cells of hematopoietic origin. The biological
significance of insulin-dependent tyrosine phosphorylation of
p95
, however, remains to be determined. Previous reports
demonstrated that p95
acts as a guanine nucleotide
exchange factor for ras(12, 13, 14, 15) ,
despite its predicted function as a guanine nucleotide exchange factor
for members of the Rho family. These studies also demonstrated that
tyrosine phosphorylation of vav is required for its guanine
exchange function(12, 13, 14, 15) .
In a more recent report, however, Bustelo et al.(35) failed to confirm that vav acts as a guanine
exchange factor for ras, but instead reported that it
cooperates with ras to induce cell transformation. Another
report also demonstrated that while transformation of NIH-3T3 cells
with the ras guanine exchange factor GRF/CDC25 induces
elevated ras-GTP and transcriptional activation from ras-responsive DNA elements, transformation with vav does not(36) . However, similarly to ras- and
GRF-transformed cells, vav-transformed cells exhibit
constitutive activation of mitogen-activated protein
kinases(36) . Based on all these reports and our data, it is
tempting to hypothesize that insulin-dependent tyrosine phosphorylation
of p95
is involved in a pathway regulating the activation
of ras in hematopoietic cells or in a pathway acting
synergistically with ras to induce mitogenesis. The
relationship of such pathways with known signals leading to
insulin-dependent activation of ras (such as the pathway
involving the adaptor protein Grb-2 and the Shc protein) is unclear at
this time. Interestingly, a recent report has provided evidence
suggesting the existence of a complex of Shc-Grb-2-vav in T
lymphocytes in vivo(37) . It remains to be determined
if such a complex is formed in insulin-responsive cells and, if so, how
it functions to regulate ras activation. It is also possible
that p95
has other signaling functions in addition to the
regulation of GTP-binding proteins. This has been suggested by the
finding that the C-terminal SH3 domain of vav forms a stable
association with the heterogeneous ribonucleoprotein K, which is
implicated in the regulation of the c-myc gene(38) .
This finding raises the possibility of direct transmission of
cell-surface receptor signals through vav activation to the
nucleus to regulate gene expression(38) . Future studies should
provide valuable information on the importance of such functions of vav in the signal transduction of insulin.