(Received for publication, October 5, 1994; and in revised form, December 20, 1994)
From the
Insulin receptor substrate-1 (IRS-1) is tyrosine-phosphorylated in response to insulin resulting in association with and activation of phosphatidylinositol 3-kinase (PI 3-kinase), thereby initiating some of the effects of insulin. We have recently shown that the insulin-like effects of growth hormone (GH) in adipocytes can be inhibited by the selective PI 3-kinase inhibitor wortmannin (Ridderstråle, M., and Tornqvist, H.(1994) Biochem. Biophys. Res. Commun. 203, 306-310), suggesting a similar role for PI 3-kinase in GH action. Here we show that IRS-1 is tyrosine-phosphorylated in a time- and dose-dependent manner in response to GH in primary rat adipocytes. This phosphorylation coincided with the extent of interaction between IRS-1 and the 85-kDa subunit of PI 3-kinase as evidenced by coimmunoprecipitation. Stimulation with 23 nM GH increased the PI 3-kinase activity associated with IRS-1 4-fold. Our data suggest that GH-induced tyrosine phosphorylation of IRS-1 and the subsequent docking of PI 3-kinase are important postreceptor events in GH action. The mechanism for the phosphorylation of IRS-1 induced by GH is unknown, but involvement of JAK2, the only known GH receptor-associated tyrosine kinase, seems possible.
Major advances have been made in recent years in elucidating the
components of the intracellular signaling chains of different hormones,
reaching from their membrane receptors to intranuclear events
controlling transcription, or to intracellular target proteins
controlling cellular metabolism. Growth hormone (GH) ()exerts pleiotropic actions on the growth, differentiation,
and metabolism of cells and constitutes a good example of a hormone
presumably utilizing a wide array of intracellular proteins in its
signaling chains. The initial event in GH action involves binding and
dimerization of its membrane receptor(1, 2) . The GH
receptor belongs to the cytokine receptor family, characterized by
homologies in the extracellular domains and lack of intrinsic tyrosine
kinase activity(3) . Instead, activation of receptor-associated
cytosolic tyrosine kinases belonging to the Janus kinase family, in the
case of GH the JAK2 tyrosine kinase(4) , is an emerging theme
for this family of receptors(5) . In addition, a number of
intracellular key proteins have been suggested to be involved in GH
signaling further downstream of these or other, as yet unidentified,
receptor-proximal events(6) .
We have recently shown that
the insulin-like effects of GH in isolated rat adipocytes can be
blocked by wortmannin, a selective inhibitor of phosphatidylinositol
3-kinase (PI 3-kinase)(7) . A number of other hormones and
growth factors such as insulin, insulin-like growth factor-1 (IGF-I),
platelet-derived growth factor, and different cytokines have been shown
to stimulate the activity of PI 3-kinase(8) . PI 3-kinase is a
dual specificity lipid and serine kinase (9) consisting of a
regulatory 85-kDa subunit (p85) containing two Src homology 2 (SH2)
domains and a catalytic 110-kDa subunit
(p110)(10, 11) . The role of its lipid kinase
products, phosphatidylinositol 3-phosphate, phosphatidylinositol
3,4-bisphosphate, and phosphatidylinositol
3,4,5-trisphosphate(12) , as intracellular second messengers is
not known, but regulation of intracellular protein trafficking (13) and/or activation of the -isoform of protein kinase C (14) have been suggested. The protein kinase activity of PI
3-kinase can phosphorylate p85 on serine resulting in a feed-back
inhibition the catalytic activity of p110(9) .
Insulin stimulation of glucose uptake and counteraction of lipolysis in adipocytes is believed to be mediated by activation of PI 3-kinase (15, 16, 17, 18, 19) . Insulin binding results in activation and autophosphorylation of the insulin receptor tyrosine kinase on specific tyrosines(20, 21) . This is followed by tyrosine phosphorylation of a major exogenous substrate for the receptor called the insulin receptor substrate-1 (IRS-1) (22, 23, 24) on multiple tyrosines in YXXM/YMXM motifs(25) , thus providing specific binding sites for the SH2-domains of p85 and subsequent activation of p110(26) . Furthermore, recent data suggest that PI 3-kinase can serine phosphorylate IRS-1(19) . In addition to insulin, IGF-I has been shown to utilize IRS-1 in intact cells and interleukin 4 (IL-4) has been shown to utilize the functionally related but immunologically distinct protein 4PS in myeloid cell lines(27, 28, 29, 30) . Tyrosine phosphorylation of IRS-1/4PS results in the association of several other proteins (Grb2, SH-PTP2, and Nck) through their SH2 domains(31) .
To further explore the possible role of PI 3-kinase in GH action, we precipitated PI 3-kinase from GH-treated rat adipocytes and looked for associated tyrosine-phosphorylated proteins. Two major high molecular weight proteins were found. One of these was identified as IRS-1 by immunoblotting. IRS-1 was tyrosine-phosphorylated and bound to the p85 subunit of PI 3-kinase in a dose- and time dependent manner in response to GH.
Adipocytes were prepared essentially according to Rodbell (32) with modifications (7, 33) from 36-day-old Sprague-Dawley rats (B&, Stockholm, Sweden) fasted overnight prior to the experiments. After incubation with GH the cells, 4 ml of a 10% cell suspension (400 µl packed cell volume) for each condition, were homogenized in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 100 µM orthovanadate, 0.1 mM diisopropyl fluorophosphate, 1 µg/ml pepstatin A, and 10 µg/ml each of antipain and leupeptin. In some experiments cells were stimulated with GH, IGF-I, or insulin in the presence or absence of a neutralizing monoclonal anti-IGF-I antibody (recombinant human GH, human insulin, IGF-I, and anti-IGF-I (mAb 41) were generously supplied by Novo Nordisk, Bagsvaerd, Denmark). Lysates free from fat and cellular debris were supplemented with Triton X-100 to 1% (w/v) and solubilized for 1 h at 4 °C. Insoluble material was removed by centrifugation and the lysates incubated with either anti-IRS-1 antibody (0.7 µg/ml) or anti-p85 (PI 3-kinase) antisera (1:1000) from Upstate Biotechnology Inc. (Lake Placid, NY). Immunoprecipitates were collected by adding 25 µl of Protein A-Sepharose 4B (Pharmacia, Uppsala, Sweden), washed three times in 20 mM Tris-HCl, pH 7.6, containing 137 mM NaCl, dissolved in SDS sample buffer, run on 7% SDS-PAGE gels, and transferred to Immobilon-P membranes. For Western blotting a polyclonal anti-phosphotyrosine antibody (a generous gift from Dr. L. Rönnstrand, Ludwig Institute, Uppsala, Sweden) was used at 1 µg/ml, the anti-IRS-1 antibody at 0.7 µg/ml, and the anti-p85 antisera at a 1:1000 dilution. Western blotting, stripping the blots, and reblotting were performed using enhanced chemiluminescence according to the manufacturer's instructions (Amersham).
For in vitro phosphatidylinositol kinase assays, Protein
A-Sepharose collected immunoprecipitates were washed twice with lysis
buffer supplemented with Nonidet P-40 to 1% (w/v), three times with 10
mM Tris-HCl, pH 7.4, and 145 mM NaCl, and twice with
assay buffer containing 40 mM Tris-HCl, pH 7.4, 5 mM MgCl, and 0.5 mM EGTA, and finally
resuspended in 50 µl assay buffer. The assay was started by
addition of 50 µl of phosphorylation mix containing 0.2 mg/ml
phosphatidylinositol (a gift from Dr L. Krabisch, Lund, Sweden), 0.01
mg/ml phosphatidylserine (Sigma), and 0.2 mM [
-
P]ATP (1 µCi; Amersham) in assay
buffer. Reactions were terminated after 10 min at 30 °C by addition
of 200 µl of 1 M HCl and 400 µl of methanol:chloroform
(1:1). The organic phase was recovered, and lipids were dried down and
dissolved in 20 µl of chloroform:methanol (95:5), spotted onto
Silica Gel 60 plates (Merck, Darmstadt, Germany), and developed in
chloroform:methanol:H
O:ammonium hydroxide (25%)
(45:35:7.5:2.8). Phosphatidylinositol 4-phosphate, which comigrates
with 3-phosphorylated phosphatidylinositol, was used as standard.
P incorporated into phosphatidylinositol was visualized
and quantified by Fujix Bas 2000 System.
Isolated rat adipocytes were incubated in absence of hormones
for 3 h in order to restore responsiveness to the insulin-like effects
of GH(34) . The cells were then resuspended in fresh medium and
treated with GH for varying times and at varying concentrations. Whole
cell lysates were subjected to immunoprecipitation with antibodies
against either p85 (p85) or IRS-1 (
IRS-1). The
immunoprecipitates were analyzed by Western blotting with
anti-phosphotyrosine antibody (
PY) or with the respective
immunoprecipitating antibodies, or assayed for phosphatidylinositol
kinase activity.
IRS-1 was phosphorylated on tyrosine in response to
GH (Fig. 1, lane2, upperpanel) as revealed by PY immunoblotting of
IRS-1 immunoprecipitates. Furthermore, when reblotting with
p85, p85 was found to coimmunoprecipitate with IRS-1 in response
to GH (Fig. 1, lane2, lowerpanel). Immunoprecipitation with
p85 showed
coimmunoprecipitation of two high molecular weight
tyrosine-phosphorylated proteins (Fig. 1, lane4, upperpanel), whereas neither p85 (Fig. 1, lanes3 and 4) nor p110
(data not shown) were phosphorylated on tyrosine in response to GH. One
of the p85-associated proteins migrated to the same position as IRS-1
and was, upon reblotting, recognized by
IRS-1 (Fig. 1, lane4, middle panel). The other
tyrosine-phosphorylated protein, tentatively called pp180, migrated
above the position for IRS-1 and was not recognized by
IRS-1.
Apparently,
p85 immunoprecipitation brought down more of the
tyrosine-phosphorylated material corresponding to IRS-1 than
precipitated with
IRS-1 (lane 2 versuslane4). This is probably due to incomplete precipitation of
IRS-1 with
IRS-1 (data not shown). Yet, the presence of other
tyrosine-phosphorylated proteins in the
p85 precipitate cannot be
excluded. To further explore the significance of our findings, the
IRS-1 immunoprecipitates were assayed for association of PI
3-kinase activity. Fig. 2shows that GH stimulated a 4-fold
increase (mean of 3 experiments) in phosphatidylinositol kinase
activity associated with the
IRS-1 immunoprecipitate. Whether this
reflects activation of the kinase upon association with IRS-1 or
translocation of active kinase is not known (cf. (35) ). Insulin-induced docking of PI 3-kinase to IRS-1 has
been shown to result in activation of the kinase(26) .
Figure 1:
Tyrosine-phosphorylated IRS-1
associates with PI 3-kinase in response to GH. Isolated rat adipocytes
were prepared and treated as under ``Experimental
Procedures.'' Cells were either treated (+) or not
treated(-) with 23 nM GH for 10 min. Whole cell lysates
were subjected to immunoprecipitation with antibody against the insulin
receptor substrate-1 (IRS-1; lanes1 and 2) or the p85 subunit of phosphatidylinositol
3-kinase (
p85; lanes3 and 4).
Proteins in the immune complexes were separated by SDS-PAGE (7%) and
transferred to an Immobilon-P membrane. The membrane was Western
blotted with an anti-phosphotyrosine antibody (
PY; upperpanel), stripped, and reblotted with either
IRS-1 (middlepanel) or
p85 (lowerpanel). Immunoreactive bands were visualized by enhanced
chemiluminescence. The migration of molecular weight markers are
indicated to the right (
1000). Results are
representative of at least three individual
experiments.
Figure 2:
Increased PI 3-kinase activity in
anti-IRS-1 immunoprecipitates from GH-treated cells. Isolated rat
adipocytes were prepared and treated as described under
``Experimental Procedures.'' Cells were either treated
(+) or not treated(-) with GH (23 nM) for 10 min.
Whole cell lysates were subjected to immunoprecipitation with antibody
against the insulin receptor substrate-1. The precipitates were washed
and immediately used for an in vitro phosphatidylinositol
kinase assay as described under ``Experimental Procedures.''
The conversion of phosphatidylinositol to phosphatidylinositol
phosphate in the presence of [-
P]ATP was
analyzed by TLC. PIP indicates the migration of a
phosphatidylinositol 4-phosphate standard. The origin, ori,
contains an irrelevant residual amount of [
P]ATP
that was not removed by organic extraction. Results are representative
of three individual experiments.
Having demonstrated that GH induces tyrosine phosphorylation of
IRS-1 and association of PI 3-kinase to IRS-1, we proceeded by
investigating the time course and dose dependence of these events (Fig. 3). Increased tyrosine phosphorylation of IRS-1 (Fig. 3A, upperpanel) as well as the
appearance of p85 in the IRS-1 immunoprecipitates (Fig. 3A, lowerpanel) were seen
within 2 min of GH stimulation reaching a maximum at 5-10 min.
Since GH effects have been demonstrated over a wide range of
concentrations(36) , it was interesting to observe the dose
dependence of the effects induced by GH found in the present
investigation. Tyrosine phosphorylation of IRS-1 (Fig. 3B, upperpanel) and the
subsequent association of p85 in response to GH appeared at 0.23
nM, reaching a maximum at 23 nM, and then to decrease
at 230 nM. In addition, the dose-dependent appearance of
tyrosine-phosphorylated IRS-1 and pp180 (Fig. 3C, upperpanel) and the association of the IRS-1 protein
with p85 (Fig. 3C, middlepanel) in
the
p85 immunoprecipitates followed the same dose dependence.
Similar GH dose-response curves (ED
1-2
nM) have been observed for the insulin-like effects of GH (36) and for the tyrosine phosphorylation of the GH receptor in
response to GH in adipocytes. (
)The observed decrease at
higher concentrations is likely to reflect the need of receptor
dimerization for signaling(37) .
Figure 3:
GH stimulates the tyrosine phosphorylation
of IRS-1 and its association with PI 3-kinase in a dose- and
time-dependent manner. Isolated rat adipocytes were prepared and
treated as described under ``Experimental Procedures.'' The
cells were stimulated either with 23 nM GH for 0-20 min (A) or with 0-230 nM GH for 10 min (B and C). Whole cell lysates were subjected to
immunoprecipitation with an antibody against the insulin receptor
substrate 1 (IRS-1, A and B) or the p85 subunit
of phosphatidylinositol 3-kinase (
p85, C). Proteins in
the immune complexes were separated by SDS-PAGE (7%) and transferred to
Immobilon-P membranes. The membranes were Western blotted with an
anti-phosphotyrosine antibody (
PY; upperpanels), stripped, and reblotted with either
IRS-1 (middlepanels) or
p85 (lowerpanels). Immunoreactive bands were visualized by enhanced
chemiluminescence. The migration of molecular weight markers are
indicated to the right (
1000). Results are
representative of at least three individual
experiments.
From these results we conclude that GH stimulation of adipocytes results in a rapid and dose-dependent tyrosine phosphorylation of IRS-1 and association of the p85 subunit of PI 3-kinase under conditions when the hormone exerts its acute insulin-like effects. Previous data obtained with the selective PI 3-kinase inhibitor wortmannin supports this view(7) . Our results have the general implication that stimulation through receptors of the cytokine receptor family, like the GH receptor, may result in tyrosine phosphorylation of IRS-1 and subsequent association of PI 3-kinase in primary cells. This has also been shown for IL-4 stimulation of 32D myeloid progenitor cells overexpressing IRS-1(38) . On the other hand, Gold et al.(39) could not demonstrate that stimulation of hematopoietic cell lines with other cytokines resulted in phosphorylation of IRS-1 or related proteins associating with PI 3-kinase.
It is not known which tyrosine kinase is responsible for
the GH-induced phosphorylation of IRS-1. For insulin it has been
proposed that the insulin receptor tyrosine 960 has a function in
enabling the receptor kinase to phosphorylate IRS-1(40) .
Tyrosine 960 is positioned in a sequence motif, NPXY, which is
also found in the IL-4 and IGF-I receptors(38) . This motif
has, however, not been found in the GH receptor. A role for the
insulin- or IGF-I receptor kinases seems unlikely, since GH did not
induce detectable tyrosine phosphorylation, i.e. receptor
tyrosine kinase activation, in the 95-kDa region corresponding to the
-subunits of these receptors, as evidenced by SDS-PAGE of
PY-immunoprecipitated solubilized membrane proteins from
P-labeled adipocytes, in comparison to the effect of
insulin.
It has also been shown that primary rat adipocytes
do not express IGF-I receptors capable of binding IGF-I on the cell
surface(41) . It is also possible that GH might induce local
production of IGF-I from the adipocytes, which by binding to IGF-I
receptors or, at higher concentrations, to insulin receptors could
result in the observed phosphorylation of IRS-1. This possibility was
investigated by stimulating adipocytes with GH, IGF-I, or insulin in
presence or absence of a neutralizing anti-IGF-I antibody (
IGF-I).
As expected, IGF-I stimulated both lipogenesis, measured as
incorporation of [
H]glucose into adipocyte
triglycerides(42) , and tyrosine phosphorylation of IRS-1 in a
dose-dependent manner (data not shown). Half-maximal stimulation
(
20 nM) by IGF-I was 100-1000 times higher than that
reported for insulin(43) , indicating that IGF-I acts through
the insulin receptor and not the IGF-I receptor. The presence of
IGF-I (5.4 µg/ml) clearly inhibited the effect of IGF-I (10
nM) but had no effect on GH- or insulin-induced IRS-1 tyrosine
phosphorylation (Fig. 4). An irrelevant monoclonal mouse
antibody used as a control at the same concentration as
IGF-I had
no effect on either hormonal effects (data not shown). We conclude that
GH-induced tyrosine phosphorylation of IRS-1 and subsequent association
with PI 3-kinase is not mediated through local IGF-I production and
activation of the IGF-I- or insulin receptors.
Figure 4:
Neutralizing IGF-I antibody does not block
GH-stimulated tyrosine phosphorylation of IRS-1 and pp180. Isolated rat
adipocytes were prepared and treated as described under
``Experimental Procedures.'' The cells (2 ml, 10% suspension)
were stimulated with 23 nM GH, 10 nM IGF-I, 1 nM insulin or vehicle for 10 min in the absence or presence of 5.4
µg/ml monoclonal IGF-I antibody (IGF-I). Whole cell lysates
were subjected to immunoprecipitation with an antibody to the p85
subunit of phosphatidylinositol 3-kinase (
p85). Proteins in the
immune complexes were separated by SDS-PAGE (7%) and transferred to
Immobilon-P membranes. The membranes were Western blotted with an
anti-phosphotyrosine antibody (
PY; upperpanel),
stripped, and reblotted with either an antibody against the insulin
receptor substrate 1 (
IRS-1) (middlepanel) or
p85 (lowerpanel). Immunoreactive bands were
visualized by enhanced chemiluminescence. The migration of molecular
weight markers are indicated to the right (
1000).
Results are representative of at least three individual
experiments.
The kinase
responsible for the IL-4-stimulated 4PS or IRS-1 phosphorylation has
not been identified, but it is expected to be a kinase related to the
Janus kinase family proteins or the Src homology protein
Fyn(31) . A role for JAK2, which is tyrosine-phosphorylated in
response to GH in 3T3-F442A preadipocytes (4) and
adipocytes, in the phosphorylation of IRS-1 therefore seems
possible.
In addition to PI 3-kinase, several other SH2 domain-containing proteins (Grb2, SH-PTP2, and Nck) have been shown to associate with IRS-1 phosphorylated by the insulin receptor(31) . GH-induced tyrosine phosphorylation of IRS-1 might turn out to be an important tool in the investigation of specificity in the signals initiated by IRS-1 phosphorylation. It has for example been shown that GH stimulation results in activation of MAP kinase and S6 kinase activity as well as induction of c-Fos and c-Jun(44, 45, 46, 47) . Our findings suggest that GH-induced tyrosine phosphorylation of IRS-1 might be positioned upstream of such events. Identification of the sites on IRS-1 phosphorylated in response to GH as well as the identity of the kinase responsible for these actions will be of significant importance.
In addition to the tyrosine-phosphorylated IRS-1,
tyrosine-phosphorylated pp180 was coimmunoprecipitated by p85 in
response to GH (Fig. 1) as well as IGF-I and insulin (Fig. 4). The identity of pp180 is not known, but it might be
functionally related to IRS-1 since it is tyrosine-phosphorylated and
associates with p85 in response to these hormones. Interestingly,
insulin, IGF-I, and IL-4 stimulation of myeloid cell lines results in
tyrosine phosphorylation of a protein with relative molecular weight
and function similar to that of IRS-1 called 4PS(30) . Two
recent reports on transgenic mice lacking IRS-1 expression indicates
that a protein migrating above the position for IRS-1 and designated
IRS-2 exists in adipocytes, liver, and muscle cells with functions
similar to those of IRS-1(48, 49) . The tissue
distribution of these proteins has not yet been established, but the
pp180 protein found here might be identical or related to 4PS or IRS-2.