(Received for publication, November 3, 1994)
From the
The phosphorylation state of pp125 focal adhesion kinase in
response to insulin was examined in parental and transfected Rat-1
fibroblasts expressing both wild-type (HIRc cells) and mutant human
insulin receptor cDNAs lacking the C-terminal twin tyrosine
phosphorylation sites (YF2 cells) or a deletion mutant lacking the
distal 43 amino acids of the -subunit (
CT cells). In HIRc
cells insulin stimulated the tyrosine dephosphorylation of
pp125
, whereas IGF-I did not. In contrast, the
tyrosine phosphorylation state of pp125
was
unchanged in the parental Rat-1 fibroblasts and the YF2 or
CT
mutant cell lines in response to insulin. Analysis of the supernatants
revealed that pp125
was only one component of
the major M
120-130-kDa phosphotyrosine band
seen in HIRc cells. We conclude that: 1) in contrast to other growth
factors, insulin stimulates the dephosphorylation of
pp125
; 2) the presence of the insulin receptor
C-terminal tyrosines 1328 and 1334 is required for the
insulin-stimulated tyrosine dephosphorylation of
pp125
, suggesting a possible SH2
domain-dependent interaction; 3) insulin may modulate integrin-mediated
signaling through pp125
by altering the
phosphorylation state of pp125
.
Insulin stimulates autophosphorylation of its receptor, resulting in activation of the intrinsic tyrosine kinase activity of the receptor and the phosphorylation of its major intracellular substrates leading to signal transduction(1, 2) . A number of substrates have been identified, of which IRS-1 and SHC have been the most extensively characterized(1, 2, 3) . Phosphorylation of these molecules, particularly IRS-1, in response to insulin enables the recruitment of numerous secondary signaling molecules that contain SH2 domains. These include the p85 subunit of phosphatidylinositol 3-kinase, GRB2, and the SH2 domain containing phosphatase Syp (SHPTP2, PTP1D, PTP2C)(1, 2) .
Much of the research on insulin receptor signaling has focused on the identification of proteins that undergo increases in tyrosine phosphorylation as a result of receptor activation. In comparison, relatively little attention has been paid to the role of tyrosine dephosphorylation in insulin action. Protein tyrosine phosphatases may act as transducers or modulators of signaling pathways. Recently, we and others (4, 5) have shown that inhibition of the tyrosine phosphatase Syp blocks insulin stimulation of mitogenesis and MAP kinase activity, indicating that Syp-mediated tyrosine dephosphorylation positively participates in the signal transduction cascade leading to cell growth.
The purpose of this study was to
identify phosphoproteins that undergo tyrosine dephosphorylation in
response to insulin. This led to the identification of
pp125 as a target protein for insulin-stimulated
tyrosine dephosphorylation.
Figure 1:
Insulin-stimulated tyrosine
dephosphorylation of an M 120-130-kDa
protein in HIRc cells. Whole cell lysates of HIRc cells stimulated with
insulin were analyzed by anti-phosphotyrosine immunoblotting as
described under ``Experimental Procedures.'' PanelA, dose response of insulin-stimulated dephosphorylation
of the M
120-130-kDa proteins. Serum-starved
HIRc cells were stimulated with varying concentrations of insulin for
10 min as indicated. Immunoblots were developed using anti-rabbit-HRP,
luminol, and chemiluminography. PanelB shows a time
course of the insulin-stimulated dephosphorylation of the M
120-130-kDa
proteins.
Figure 2:
Effects of IGF-1 on the phosphorylation of
pp125 and other M
120-130-kDa
proteins. Serum-starved HIRc cells were exposed to insulin and IGF-1 as
indicated for 15 min. pp125
was precipitated
using antibody 2A7, and the immunoprecipitates (leftpanel) and supernatants (rightpanel)
were analyzed by anti-phosphotyrosine immunoblotting and
autoluminography as described under ``Experimental
Procedures.''
Figure 3:
Insulin-stimulated dephosphorylation of
pp125 in transfected and untransfected Rat-1 fibroblasts.
The cell lines indicated were stimulated with 0.1 µM insulin for 10 min, and p125 focal adhesion kinase was
immunoprecipitated using antibody 2A7 as described under
``Experimental Procedures.'' The immunoprecipitates were
subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting
with anti-phosphotyrosine antibodies. An autoluminograph is shown. HirC, Rat-1 cells transfected with wild-type human insulin
receptor; Rat1, parental Rat-1 fibroblasts; YF2,
cells transfected with insulin receptor lacking the C-terminal tyrosine
phosphorylation sites;
CT, Rat-1 cells transfected with a
deletion mutant lacking the C-terminal 43 amino acids of the
-subunit.
Activation of the insulin receptor after ligand binding results in the tyrosine phosphorylation of multiple endogenous substrates, and much effort has been devoted to the characterization of such substrates (1, 2) . The major endogenous phosphoprotein observed after insulin stimulation is IRS-1, which undergoes phosphorylation on multiple residues and recruits other signaling molecules that contain SH2 domains(2) . Apart from the primary action of tyrosine phosphorylation, tyrosine dephosphorylation may also play a critical role in the propagation of the insulin signal. Such dephosphorylation could either inhibit signal transmission or play a positive transducing role. Recently, we and others have shown that the SH2 domain containing phosphatase Syp exerts a positive role in insulin-stimulated mitogenesis, indicating a necessary role for a tyrosine dephosphorylation event in signal transduction(4, 5) . Syp may be activated by binding to IRS-1 or the insulin receptor(15, 16) . Activation of Syp or other tyrosine phosphatases (17) would lead to the dephosphorylation of susceptible tyrosine phosphoproteins.
The M 120-130-kDa phosphoprotein is the major
tyrosine phosphoprotein found in quiescent serum-starved monolayer HIRc
cells. We have demonstrated that a component of this protein band is
subject to insulin-stimulated tyrosine dephosphorylation in a time and
dose-dependent manner. Using specific
antibodies(10, 18) , we have identified the relevant
phosphoprotein within the 120-130-kDa region as
pp125
. Thus, immunoprecipitation studies showed that
pp125
is tyrosine-phosphorylated in unstimulated cells,
and insulin stimulation results in marked tyrosine dephosphorylation.
The time course of pp125
dephosphorylation is delayed
relative to insulin receptor kinase activation and is maximal 10 min
after peak insulin receptor and IRS-1 tyrosine phosphorylation. After
immunoprecipitation of pp125
, analysis of the
supernatants revealed that the other phosphoproteins in this region are
not dephosphorylated in response to insulin. The decrease in the level
of tyrosine phosphorylation of pp125
may result from the
activation of a tyrosine phosphatase or possibly the inhibition of a
tyrosine kinase in response to insulin.
pp125 was
first identified as a phosphotyrosine protein in chicken embryo
fibroblasts transformed with v-src(10) . Subsequent
cDNA cloning revealed that pp125
, which was a substrate
for pp60
, was itself a novel tyrosine kinase (12, 19, 20, 21, 22) that
colocalizes with components of focal adhesions(23) , tensin,
vinculin, and talin. pp125
differs from other receptor
and nonreceptor tyrosine kinases in that it contains a conserved
catalytic domain flanked by large N- and C-terminal domains that lack
sequence similarity to other tyrosine kinases(11) . In
addition, it does not contain any sequence determinants for membrane
association. Overexpression of activated c-src results in a
large increase in pp125
tyrosine phosphorylation, which
is blocked by the expression of kinase negative
c-src(11) . Hence, pp125
may contribute
to transformation of cells by v-src. Dephosphorylation of
pp125
would prevent complex formation with and activation
of pp60
.
Both IRS-1 and the insulin receptor bind to
the SH2 domain of Syp and activate it in
vitro(4, 15, 16) . Microinjection and
transfection studies indicate a positive role for Syp in
insulin-stimulated mitogenesis(4, 5) . Potentially,
activation of Syp in response to insulin could result in the
dephosphorylation of substrates including pp125. However,
we were unable to demonstrate the presence of Syp in pp125
immunoprecipitates from HIR cells, and a Syp-GST fusion protein
containing a Syp SH2 domain did not precipitate pp125
,
although these do not rule out a role for Syp in the dephosphorylation
of pp125
. Alternatively, or additionally, there may be
another tyrosine phosphatase mediating this effect(17) .
Overexpression of a dominant negative Syp protein increases tyrosine
phosphorylation of a 120-kDa protein in response to insulin, which
binds to the SH2 domain of Syp and is not related to
pp125
(5) .
In the cell lines overexpressing
the C-terminal insulin receptor mutants, insulin-stimulated IRS-1
phosphorylation is intact, and hence Syp activation is
intact(6, 7, 8) . Taken together, this raises
the possibility that a distinct phosphotyrosine phosphatase exists,
which is activated in response to insulin and requires the presence of
an intact -subunit C terminus for activation. Alternatively, the
association of the insulin receptor with Syp could provide a unique
localization of Syp not provided by IRS-1, which would imply that both
activation and cellular localization are important for the functional
effects of Syp.
Recently, a cDNA for a new tyrosine phosphatase
designated PTPD1 has been cloned(24) . PTPD1 has an M of 130,000 and associates with and is
phosphorylated by Src kinase(24) . Src kinase associates with
pp125
(13, 25) as well, suggesting that
PTPD1 and pp125
may colocalize. Several stimuli lead to
the phosphorylation of 120-130-kDa proteins(21) . These
include: clustering of B1 integrins by cell
adhesion(26, 27, 28) ; stimulation by
vasopressin(29) , bombesin(30, 31) ,
platelet-derived growth factor (32) ,
endothelin(29, 33) , and lysophosphatidic
acid(33, 34, 35, 36) , and
transformation of cells by v-src(21, 24) .
These stimuli result in the phosphorylation and activation of focal
adhesion
kinase(18, 22, 24, 28, 31, 37) .
In contrast, we have demonstrated that pp125
is
dephosphorylated in response to insulin. The functional significance of
this observation is unclear but suggests that insulin may regulate the
adhesive ability of cells and consequently modulate integrin-mediated
cell adhesion and migration.
Although the biologic role of
pp125 dephosphorylation remains unknown, the fact that
CT and YF2 cells do not mediate pp125
dephosphorylation provides some clues. Both cell lines exhibit
enhanced mitogenesis in response to
insulin(6, 7, 8) . Although speculative,
since insulin-stimulated mitogenesis is enhanced in
CT cells and
YF2 cells, it is possible that dephosphorylation of pp125
serves to restrain entry of cells into the growth cycle. This
would tend to retard insulin's mitogenic effects, consistent with
the fact that the stronger mitogenic growth factor IGF-1 did not cause
pp125
dephosphorylation. It is also interesting to note
that of the two C-terminal phosphorylation sites in the insulin
receptor
-subunit, only the tyrosine corresponding to tyrosine
1334 is conserved in the IGF-1 receptor, while tyrosine 1328 is
replaced by a phenylalanine. This is the first demonstration of a
signaling difference between the insulin and IGF-1 receptors. This
difference may arise from the phosphorylation of tyrosine 1328 of the
insulin receptor, which may lead to an interaction with an SH2 domain
containing protein. Interestingly, tyrosine 1328 is located adjacent to
serine 1327, which is a major site of phosphorylation by protein kinase
C(38, 39) . Phosphorylation of serine 1327 could
regulate the interaction of tyrosine 1328 with its putative target, a
hypothesis that deserves further investigation.
In summary, we have
demonstrated that when Rat-1 cells transfected with human insulin
receptors are stimulated with insulin, pp125 focal adhesion kinase is
dephosphorylated by about 50%. This process is specific for insulin,
does not occur with IGF-I, and requires the presence of the C-terminal
tyrosine phosphorylation sites of the insulin receptor -subunit.
The exact sites on pp125 focal adhesion kinase, which are
dephosphorylated in response to insulin, are unknown, although it has a
number of consensus tyrosine phosphorylation sites for SH2 domain
binding(40) .