From the Department of Medicine,
§ Department of Pharmacology, and ¶ Kaplan Cancer
Center, New York University Medical Center,
New York, New York 10016
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ABSTRACT |
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A physiologically relevant response to insulin,
stimulation of prolactin promoter activity in GH4 pituitary cells, was
used as an assay to study the specificity of protein-tyrosine
phosphatase function. Receptor-like protein-tyrosine phosphatase (RPTP
) blocks the effect of insulin to increase prolactin gene
expression but potentiates the effects of epidermal growth factor and
cAMP on prolactin promoter activity. RPTP
was the only
protein-tyrosine phosphatase tested that did this. Thus, the effect of
RPTP
on prolactin-chloramphenicol acetyltransferase (CAT) promoter
activity is specific by two criteria.
A number of potential RPTP targets were ruled out by finding
(a) that they are not affected or (b) that they
are not on the pathway to insulin-increased prolactin-CAT activity. The
negative effect of RPTP
on insulin activation of the prolactin
promoter is not due to reduced phosphorylation or kinase activity of
the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not
mitogen-activated protein kinase dependent. Experiments with inhibitors
of phosphatidylinositol 3-kinase suggest that insulin-increased
prolactin-CAT expression is phosphatidylinositol 3-kinase-independent. These results suggest that RPTP
may be a
physiological regulator of insulin action.
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INTRODUCTION |
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Prolactin production is abnormal in humans with diabetes (1), and studies with animal and cell culture models of diabetes suggest that a decrease in steady-state prolactin mRNA levels secondary to decreased prolactin gene expression accounts for these findings (2, 3). The GH4 pituitary tumor cells have proven invaluable for studies of prolactin gene regulation by hormones and growth factors. Physiological insulin concentrations increase the level of prolactin and prolactin mRNA production approximately 7-10-fold in GH3 cells (4). Runoff transcription experiments indicate that the predominant effect of insulin is on the transcription of the prolactin gene. The >10-fold insulin stimulation of prolactin promoter/chloramphenicol acetyltransferase (CAT)1 reporter plasmid expression in GH4 cells confirms these results (5).
The effects of insulin are mediated through the insulin receptor that is a tyrosine kinase. Autophosphorylation of the insulin receptor upon ligand binding activates its kinase activity. The insulin receptor then phosphorylates IRS-1 at multiple sites. This activates IRS-1 for interaction with several signaling systems that have been proposed to mediate the diverse effects of insulin (6). Recently, the adapter protein Shc has also been shown to associate with the insulin receptor through interaction of the PTB domain of Shc with phosphotyrosine 960 of the insulin receptor. Thus, Shc may also act as a mediator of insulin signaling (50).
The Ras/MAP kinase pathway appears to mediate the responses of several growth factors acting through tyrosine kinases, including insulin (7). The phosphorylation of IRS-1 by the insulin receptor promotes the association of Grb2 with IRS-1 (7). This may activate Ras by recruiting SOS, a GTP/GDP exchange protein. Several studies indicate that the activation of Ras is essential for some insulin-mediated effects (8, 9). Activated Ras then recruits Raf to the plasma membrane, apparently in association with other proteins (10). This activates the kinase activity of Raf and the phosphorylation of MAP kinase kinase (MEK-1), MAP kinase, p90rsk, and nuclear transcription factors such as Elk-1 (11). Other studies indicate that phosphorylation of Shc protein and the ensuing Grb2 recruitment may be an important mechanism of Ras activation (12). A second insulin response pathway is activated by the association of phosphorylated IRS-1 with the SH2 domain of the 85-kDa subunit of PI 3-kinase. This association was shown to activate the enzyme. GTP-Ras was also shown to associate with and activate the catalytic subunit of PI 3-kinase. PI 3-kinase may participate in the increase in glucose transport in response to insulin (13) and in insulin-stimulated mitogenesis (14). However, the mechanism for these effects of PI 3-kinase is unknown.
Since tyrosine phosphorylation is the critical first step in signaling by insulin and other hormones and growth factors, dephosphorylation of phosphotyrosine must be of considerable physiological importance. The protein-tyrosine phosphatases (PTPases) can be divided into two classes: the receptor-like, membrane-spanning PTPases and the cytosolic PTPases. Receptor-like PTPases are characterized by an extracellular domain of variable length, a single membrane-spanning domain, and one or two catalytic domains on the intracellular portion of the molecule (15). Several candidate ligands for receptor-like PTPases have been defined, although none has so far been shown to modulate PTPase function (16, 17). The cytosolic PTPases have only a single PTPase domain and variable N- and C-terminal extensions. It has been postulated that these sequences target the cytosolic PTPases to specific intracellular locations (18).
These studies demonstrate a specific effect of RPTP on a
physiological response to insulin, activation of prolactin promoter activity. The RPTP
effect is specific by two criteria. First, no
other PTPases tested have the same effect. Second, the response to EGF
or elevated cAMP levels is not inhibited (and is actually enhanced) by
RPTP
expression.
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EXPERIMENTAL PROCEDURES |
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Materials-- [32P]ATP (3000 Ci/mmol) and [14C]chloramphenicol (50 mCi/mmol) were obtained from ICN Biochemicals Corp. [32P]H3PO4 was from DuPont. Fast CAT®, reagents for fluorescent assay of CAT activity were purchased from Molecular Probes (Eugene, OR). Acetyl-CoA, myelin basic protein, and silica gel plates for thin layer chromatography were obtained from Sigma. Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose (DMEM) was from Life Technologies, Inc., and iron supplemented calf serum was obtained from Hyclone Laboratories. LY294002 and PD98059 were from Calbiochem. Wortmannin was the gift of T. Payne (Sandoz, Basel, Switzerland). Rapamycin was provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, NCI, National Institutes of Health. All other reagents were of the highest purity available and were obtained from Sigma, Pierce, Behring Diagnostics, Bio-Rad, Eastman Kodak Co., Fisher, or Boehringer Mannheim.
Antibodies--
Antibodies to MAP kinase (anti Erk-1 and anti
Erk-2), LAR, SHP1, SHP2, and horseradish peroxidase-conjugated goat
anti-mouse and donkey anti-goat secondary antibodies were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to the T-cell
PTPase was purchased from Oncogene Sciences. Antibodies directed
against phosphotyrosine and Shc were the generous gift of Dr. J. Schlessinger (New York University Medical Center, New York). Anti-human
insulin receptor antibody was supplied by Dr. K. Siddle
(Cambridge, United Kingdom). Antibody to IRS-1 was the
generous gift of M. White (Brigham and Women's Hospital, Boston, MA).
Antibody against human influenza virus hemagglutinin was purchased from
Boehringer Mannheim. Antisera against RPTP and RPTP
were
described previously (19, 20).
Plasmids--
The construction of pPrl-CAT plasmids containing
173/+75 of prolactin 5'-flanking DNA was described (4). The human
insulin expression vector, pRT3HIR2, was the gift of Dr. J. Whittaker (Stony Brook, NY). The T-cell PTPase expression plasmids PTP TC and PTP
TC-M (21) were generously contributed by Dr. N. Tonks (Cold Spring
Harbor Laboratories) The expression plasmids for RPTP
(22), RPTP
(20), and SHP1 (previously known as PTP1C) have been previously
described. The expression vector for SHP2 was provided by Dr. B. Neel
(Beth Israel Hospital, Boston, MA) (23). The expression vector
for the protein-tyrosine phosphatase LAR was the generous gift of Dr.
B. Goldstein (Jefferson Medical College, Philadelphia, PA) (24).
The HA-tagged MAP kinase expression plasmid was from Dr. E. Skolnik
(New York University Medical Center). The dominant negative Raf,
pRSV-Raf-C4, was from Ulf Rapp (NCI-Frederick Cancer Research and
Development Center), while the dominant negative Ras,
Rasn17, was the generous gift of Dr. L. Feig (Tufts
University, Boston, MA).
Analysis of Prolactin Promoter Responsiveness Using Transient Transfection-- Electroporation experiments and CAT assays were performed as described (25). GH4 cells were harvested with an EDTA solution, and 20-40 × 106 cells were used for each electroporation. Trypan blue exclusion before electroporation ranged from 95 to 99%. The voltage of the electroporation was 1550 V. This gives trypan blue exclusion of 70-80% after electroporation. The transformed cells were then plated in multiwell dishes (Falcon Plastics) at 5 × 106 cells/9-cm2 tissue culture well in DMEM with 10% hormone-depleted serum. Cells were refed at 24 h with DMEM with 10% hormone depleted serum with or without insulin (1 µg/ml bovine insulin; Calbiochem), EGF (40 ng/ml recombinant human EGF; R & D Systems), or cAMP (0.1 mM, 8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphate; Sigma). After 48 h, the flasks were washed three times with normal saline and frozen. The cells were harvested, and CAT activity was assayed as described previously (26) except that in later experiments [14C]chloramphenicol was replaced with BODIPY chloramphenicol (Molecular Probes, Eugene, OR), and fluorescence intensity was measured using a FluorImager 575 (Molecular Dynamics, Sunnyvale, CA) with ImageQuant software.
Transfection efficiency was controlled for using an RSV-Immunoprecipitation and Western Immunoblot Analysis-- GH4 cells were harvested in a lysis buffer consisting of 50 mM HEPES, pH 7.5, 1% Triton X-100, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1 mM Na3VO4, 50 mM Na4P2O7, 1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride/HCl, and 10 µg/ml aprotinin. Protein was determined using the Bradford reagent (Bio-Rad). Immunoprecipitations were performed for 2-16 h at 4 °C in this buffer using 200 µg of protein. Samples were then incubated for an additional 2 h with protein A-agarose (1.5 mg/immunoprecipitation) and washed extensively. The immunoprecipitates were then dissolved in Laemmli sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis using 10% gels. The proteins were then transferred to nitrocellulose membranes (Micron Separations) and immunoblotted using enhanced chemiluminescence (Pierce).
MAP Kinase Assay--
GH4 cells, transfected with HA-MAP kinase,
were treated with hormones for various times or were left untreated as
controls. They were then washed and frozen at 70 C. The kinase assay
was performed with HA-MAP kinase immunoprecipitated with 10 µl of anti-HA. The assay was performed in a buffer containing 10 mM HEPES, pH 7.4, 50 µM
[32P]ATP, 25 µM ATP, 10 mM
MgCl2, 1 mM dithiothreitol, and 0.1 µg of
myelin basic protein (27). The supernatant was electrophoresed on a
10% SDS-polyacrylamide gel.
32P Labeling and Immunoprecipitation of Insulin
Signaling Molecules--
GH4 cells in 9-cm2 tissue culture
wells were incubated for 2 h with phosphate-free DMEM containing
10% dialyzed, charcoal-treated calf serum. They were then washed and
incubated for 2 h in phosphate-free DMEM containing 10% dialyzed,
charcoal-treated calf serum with 0.5 mCi/ml
[32P]H3PO4. Insulin was then
added, and the incubation was continued for 1 h. The cells were
rapidly chilled, washed three times with ice-cold saline, and frozen at
70 °C. The cells were then harvested, and immunoprecipitation was
performed as described above.
Assay of Insulin Receptor Autophosphorylation-- The assay of the autocatalytic activity of the insulin receptor was performed as described by Wilden et al. (28). GH4 cells were harvested in lysis buffer (see above), and insulin receptor was partially purified by affinity chromatography on wheat germ-agarose. The insulin receptor binding activity in the eluants was then assayed. Kinase assays were performed using equal amounts of insulin receptor binding activity. Reactions (50 µl) contained 10 mM HEPES, pH 7.4, 5 mM MnCl2, 5 mM MgCl2, and 0.1% Triton X-100. Reactions were incubated with or without insulin for 10 min at 22 °C, and [32P]ATP (40 µCi/assay) was then added for an additional 20 min. The reaction was stopped by the addition of 2 × Laemmli sample buffer, and the 32P-labeled proteins were analyzed by SDS-polyacrylamide gel electrophoresis.
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RESULTS |
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Inhibition of Prolactin-CAT Expression by RPTP--
GH4 cells
were transfected with the prolactin-CAT reporter plasmid without or
with increasing amounts of an expression vector for RPTP
. The cells
were refed after 24 h, and 1 µg/ml insulin, 40 ng/ml EGF, or 0.1 mM cAMP was added to the cultures for an additional 24 h. The cells were harvested, and CAT activity was assayed to determine
the effect of RPTP
on hormone-increased prolactin-CAT expression.
Insulin increases prolactin-CAT expression almost 20-fold in GH4 cells
transfected with only prolactin-CAT and insulin receptor (Fig.
1A). This stimulation is
dramatically decreased by co-transfection of small amounts of an
expression vector for RPTP
. Half-maximal inhibition of
insulin-increased prolactin-CAT expression is achieved using only 0.5 µg of expression vector for RPTP
, and maximal inhibition of >90%
is achieved with 15 µg or more of RPTP
. Maximal inhibition is
achieved without a significant reduction in basal prolactin-CAT
expression (1.30 ± 0.5% acetylation/10 µg of protein/h
versus 1.35 ± 0.5% acetylation/10 µg of protein/h
in control cells), although basal prolactin-CAT transcription decreased
with the highest amounts of expression vector for RPTP
(0.91 ± 0.14% acetylation/10 µg of protein/h using 50 µg and more of
RPTP
expression vector). In contrast to its inhibitory effect on
insulin-increased prolactin promoter activity, the increase in
prolactin-CAT expression in EGF- and cAMP-treated cells is augmented by
expression of RPTP
. EGF increased prolactin-CAT expression 13-fold
in control transfections, but this was increased to 36-fold when 15 µg of RPTP
expression vector were included. Likewise, cAMP
increased prolactin-CAT 12-fold in control transfections but 20-fold
with 15 µg of RPTP
. Further, the increase in prolactin-CAT
expression due to EGF or cAMP was never significantly below that seen
in control cultures, even using amounts of RPTP
expression vector
that totally abolish the effect of insulin (50 and 150 µg; data not
shown). Thus, under conditions where identical amounts of enzyme are
produced, RPTP
expression specifically inactivates the insulin
signaling pathway, while it enhances EGF and cAMP signaling.
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The Effect of RPTP Is Specific Both for Insulin and for
RPTP
--
Several other PTPases were found to be without effect on
insulin-increased prolactin-CAT expression. The receptor-like PTPases are classified by the structure of their extracellular domain. RPTP
is a type IV PTPase with a short glycosylated extracellular domain.
RPTP
is a receptor-type PTPase of class II and is characterized by
one Ig- and several fibronectin type III-like repeats in its extracellular domain. LAR is a receptor-like PTPase of type II that has
three Ig-like repeats and nine fibronectin III-like repeats in its
extracellular domain (29). PTP TC, PTP TC-M, SHP1, and SHP2 are
cytosolic phosphatases. The T cell PTPase PTP-TC is a 48-kDa protein
that is found exclusively in the particulate fraction of cellular
lysates. Truncation of this PTPase with trypsin yields a 37-kDa protein
with constitutive PTPase activity. We have used a plasmid PTP TC-M that
has a premature stop codon inserted into the T cell PTPase cDNA so
that it produces only this 37-kDa, constitutively active form of T-cell
PTPase (21). SHP1 and SHP2 are cytosolic PTPases that have two SH2
domains in their N-terminal regions. These SH2 domains restrict the
potential substrates for these phosphatases. The expression of the
phosphatases used was checked by Western blotting (Fig.
2A). This technique does not
allow the comparison of actual amounts of protein expressed; however,
the relative difference in amount of the PTPase in control
versus transfected cultures can be determined. All of the
phosphatases except the truncated form of the T cell phosphatase, PTP
TC-M, were found to be significantly overexpressed in transfected
cells, and the level of overexpression was similar in all cases. The truncated T cell PTPase was not detected. This probably results from
the removal of the epitope for the monoclonal antibody that was used
for detection of this protein, since expression of this protein
significantly affected EGF-increased prolactin-CAT expression (see
below; Fig. 2B).
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Insulin Receptor Phosphorylation in RPTP- and
RPTP
-overexpressing Cells--
Ligand binding to the insulin
receptor results in autophosphorylation of several tyrosines on the
insulin receptor
-subunit. Some of these phosphorylations activate
the receptor kinase, while others have been shown to be important as
binding sites for signaling molecules. It was possible that RPTP
rapidly dephosphorylated the insulin receptor and interfered with its
signaling. Experiments done to test this hypothesis are shown in Fig.
4. The magnitude of insulin stimulation
of tyrosine phosphorylation of transfected human insulin receptor in
control cells and in cells also transfected with RPTP
was determined
by immunoprecipitation and Western blotting with antibody to
phosphotyrosine (Fig. 4A). A 6-min insulin treatment results
in a large increase in tyrosine phosphorylation of the insulin receptor
in cells transfected with the human insulin receptor alone (control).
However, there is no significant loss of insulin-increased human
insulin receptor phosphorylation in cells transiently transfected with
both the human insulin receptor and with RPTP
. These results are
confirmed in stably transformed cells (Fig. 4B). An antibody that recognizes both the endogenous rat insulin receptor and the transfected human insulin receptor was used for these experiments. Neither the clone overexpressing RPTP
nor the clone overexpressing RPTP
shows any loss of insulin-increased tyrosine phosphorylation of
the insulin receptor. Further, experiments using lysates from cells
metabolically labeled with
[32P]H3PO4 did not demonstrate
any differences among control GH4 cells and clones expressing RPTP
or RPTP
in insulin-increased incorporation of 32P into
insulin receptor (data not shown). Thus, expression of RPTP
does not
indirectly reduce the level of Ser/Thr phosphorylation of the insulin
receptor.
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Insulin Receptor Kinase activity in RPTP- and
RPTP
-overexpressing Cell Lines--
These results suggested that
RPTP
did not reduce the general level of tyrosine phosphorylation of
the insulin receptor. However, dephosphorylation of a phosphotyrosine
critical to the activation of insulin receptor tyrosine kinase cannot
be ruled out by these experiments. Therefore, the catalytic activity of
the insulin receptor was directly tested. Insulin increases the
autocatalytic activity of the insulin receptor 4.2-fold in GH4 cells
(Fig. 4C). Insulin increased the kinase activity of its
receptor 4.3-fold in cells overexpressing RPTP
and 3.6-fold in cells
overexpressing RPTP
.
IRS-1 and Shc Phosphorylation in RPTP- and
RPTP
-overexpressing Cell Lines--
The previous experiments
suggest that RPTP
does not decrease levels of insulin receptor
kinase activity and that RPTP
does not reduce the increase in
insulin receptor phosphorylation in insulin-treated cells. However, it
is possible that RPTP
specifically dephosphorylates a tyrosine on
the insulin receptor that impairs its ability to transmit signal to
downstream events. Such a condition could occur if dephosphorylation by
RPTP
inactivates a substrate binding site. Therefore, the effect of
RPTP
expression on phosphorylation of insulin receptor substrates
was examined. Two substrates of the insulin receptor kinase, IRS-1 and
Shc, have been shown to be intermediates for some of the actions of
insulin (6). Insulin increases the phosphorylation of IRS-1 3 ± 0.25-fold in GH4 cells (Fig.
5A). Levels of IRS-1
phosphorylation in GH4 cells stably expressing RPTP
were increased
3.5 ± 0.4-fold by insulin, while in RPTP
-overexpressing cells
IRS-1 phosphorylation was increased 3.2 ± 0.15-fold by insulin.
Thus, IRS-1 phosphorylation does not appear to be affected by
overexpressing RPTP
or RPTP
.
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Dominant Negative Inhibition of Ras and Raf in GH Cells-- Dominant negative inhibitors of Ras were used to determine whether the Ras/Raf/MAP kinase signaling pathway might be involved in insulin signaling to prolactin-CAT expression. GH4 cells were cotransfected with the prolactin-CAT reporter and the human insulin receptor. Some electroporations also contained either p21S17N-Ras, a dominant negative Ras (30), or Raf-C4, which competes with wild type Raf for binding to Ras and Ras-related proteins and inhibits the downstream effects of these molecules (31). Insulin increased prolactin-CAT expression 10-fold in control cells (Fig. 6). Cotransfection of p21 S17N-Ras had no effect on insulin-increased prolactin gene expression, which was 8-9-fold at 10 µg of cotransfected inhibitor (Fig. 6A). Prolactin-CAT expression increased by cAMP was also not affected. It was 11-fold in control cells and 8-9-fold in p21 S17N-Ras-transfected cells. This does not result from failure of the p21 S17N-Ras to be expressed in GH4 cells, since EGF-increased prolactin-CAT expression was strongly inhibited by p21 S17N-Ras expression. EGF increased prolactin-CAT expression 7-fold in control GH4 cells, whereas in p21 S17N-Ras cotransfected cells EGF did not significantly increase prolactin-CAT expression.
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Effect of Insulin on MAP Kinase Activity in GH4 Cells Expressing
Inhibitors--
Insulin increases MAP kinase activity in a
Ras/Raf-dependent manner in several cell lines. MAP kinase
activity in cells treated for 6 min with insulin or EGF or left
untreated as controls was determined (Fig. 6B). Insulin
increased MAP kinase activity 10-fold in control cells. This is not
affected by cotransfection with dominant negative Ras. However, MAP
kinase activity was increased only 4-fold in insulin-treated cells that
were cotransfected with Raf-C4. Thus, dominant negative Raf reduces the
effect of insulin by 60%. Cotransfection with RPTP reduces the
effect of insulin approximately 75%. The effect of EGF is reduced 40%
by dominant negative Raf and 33% by dominant negative Ras, but it is
not affected by overexpression of RPTP
.
Inhibition of MEK with PD098059--
These experiments suggested
that a Ras-independent activation of MAP kinase by insulin might
mediate the increase in prolactin gene expression in insulin-treated
cells. The experiment shown in Fig. 7
eliminates this possibility. Incubation of GH4 cells with the inhibitor
of MEK, PD098059, for 2 h before the addition of hormones
eliminates the ability of insulin and cAMP to activate MAP kinase and
reduces the activation of MAP kinase by EGF from 80- to 30-fold (Fig.
7A). Prolactin-CAT expression in response to these hormones
is not significantly affected by PD098059 under the same conditions
(Fig. 7B). Thus, insulin increased prolactin-CAT expression
is MAP kinase-independent. Numerous other negative results support this
conclusion. First, Raf kinase assays performed in control GH4 cells,
A23 (RPTP-expressing) cells, and in K5 (RPTP
-expressing) cells
show the same level of insulin and EGF activation of both c-Raf-1 and
B-Raf kinase (data not shown). Second, a dominant negative MEK and
dominant negative MAP kinase do not inhibit prolactin gene
transcription (data not shown). Third, constitutively active MEK does
not activate prolactin gene transcription (data not shown). Fourth, a
MAP kinase phosphatase does not block the action of insulin, and
phosphatase-inactive MAP kinase phosphatase does not increase prolactin
gene expression (data not shown).
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Effect of Inhibition of PI 3-Kinase and p70S6 Kinase on Prolactin-CAT Expression-- LY294002 and wortmannin inhibit PI 3-kinase activity and insulin-stimulated glucose transport in 3T3-L1 adipocytes. Therefore, LY 294002 was used at a concentration of 50 µM and wortmannin was used at a concentration of 20 µM to determine the effect of insulin, EGF, and cAMP in cells where PI 3-kinase was inhibited. Insulin increases prolactin-CAT expression 11-fold over untreated cells with or without LY294002. However, LY294002 alone causes an increase in prolactin-CAT expression over untreated cells. Thus, it is difficult to determine if the effect of LY294002 is inhibition of the insulin response or whether LY294002 and insulin are both affecting the same pathway to increase prolactin-CAT expression. The effect of EGF is doubled by LY294002 from 6- to 14-fold, and the effect of cAMP is tripled from 7- to over 20-fold the levels seen in untreated cells in the same experiment. In cells treated with 20 µM wortmannin, insulin increased prolactin-CAT expression was only 25% (2.5-fold) of the insulin stimulation seen in control cells (11-fold; Fig. 8). Both EGF- and cAMP-increased prolactin-CAT expression were increased by wortmannin treatment (Fig. 8). Thus, the effects of EGF and cAMP are independent of PI 3-kinase, while insulin-increased prolactin-CAT expression may be PI 3-kinase-dependent.
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DISCUSSION |
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Stimulation of prolactin gene transcription is a well established
and physiologically relevant response to insulin (32, 35). We have used
the responsiveness of the prolactin promoter to insulin and other
factors as a defined assay to study the effect of PTPases on insulin
signaling. The data presented document strikingly specific effects of
RPTP on insulin-dependent gene transcription. First,
overexpression of RPTP
inhibits the insulin-mediated increase in
prolactin promoter activity. These results are observed both in
transiently and stably transfected cells. Second, the overexpression of
RPTP
does not reduce either EGF- or cAMP-increased prolactin promoter activity, but, in transient expression experiments, RPTP
expression augmented the increase in prolactin-CAT expression due to
EGF and cAMP. This demonstrates that the effect of RPTP
overexpression does not involve a general inhibition of growth factor
or second messenger responses. Finally, none of the other phosphatases
tested inhibited insulin-increased transcription from the prolactin
promoter.
Others have reported studies on the important role of the LAR PTPase as a potential negative regulator of insulin signaling (36). By contrast, we did not observe any effect of LAR expression on the regulation of the prolactin promoter by insulin in GH4 cells (Fig. 2B). Thus, our data may seem to conflict with the reports on the control of insulin signaling by LAR. However, (a) distinct PTPases may regulate different insulin signaling pathways, (b) distinct PTPases may contribute to regulation of insulin signaling in different cell types, (c) more than one PTPase may act together within the same cell, or (d) different PTPases may control insulin signaling with varying degrees of specificity (see below).
Recently, Moller et al. (37) showed that substratum
detachment of insulin-treated, insulin receptor-expressing BHK cells was reduced in cells that also expressed either RPTP or RPTP
. This was interpreted as evidence that expression of either RPTP
or
RPTP
blocked insulin signaling. However, these data might reflect an
effect of RPTP
or RPTP
expression on cell adhesion unrelated to a
physiological insulin response, since the effect of other factors was
not tested. In our assay system, the expression of RPTP
has no
effect on basal or on EGF- or cAMP-increased prolactin-CAT transcription. This makes it clear that the effect of RPTP
on prolactin promoter activity is probably mediated through a direct effect on the insulin signaling pathway. This is further supported by
the RPTP
inhibition of MAP kinase activation by insulin.
The insulin signaling pathway to gene expression is not currently
defined. However, a number of signaling molecules have been identified
that may also participate in signaling to gene transcription. Therefore, experiments were performed to determine whether expression of RPTP alters the response of these molecules to insulin.
Phosphorylation of the insulin receptor was substantially the same in
control GH4 cells and in GH4 cells expressing RPTP, either
transiently (Fig. 4A) or stably (Fig. 4B).
Previously published studies (37) suggested that insulin receptor was
hypophosphorylated in RPTP
-overexpressing cells. However, in that
study, both the insulin receptor and RPTP
were highly overexpressed
using the 293 expression system. It is possible that, under such
conditions, less specific events contribute to the phenomena observed.
This is suggested by the fact that transient insulin receptor
expression induced ligand-independent phosphorylation of the
receptor and that PTPase co-expression also reduced these levels of
basal phosphorylation of the receptor. Nonspecificity is also suggested
by the fact that, in the latter study, similar degrees of insulin
receptor dephosphorylation were induced by CD45 and RPTP
, yet only
the RPTP
protein abolished the antiadhesive effect of insulin (37).
Data from in vivo studies suggest that precise control of
phosphorylation of the insulin receptor regulatory region is important (38). Since there are multiple sites of insulin receptor
phosphorylation, it remains possible that critical tyrosines on the
insulin receptor are specifically targeted by RPTP, whose
dephosphorylation might have gone undetected in the experiment shown in
Fig. 4, A and B. However, it is unlikely that
RPTP
blocks insulin action through selective dephosphorylation of
insulin receptor tyrosines, since the kinase activity of the insulin
receptor was not significantly changed by the expression of RPTP
(Fig. 4C). Likewise, no effect of RPTP
was seen when
insulin-increased phosphorylation of IRS-1 and Shc was examined.
However, these studies cannot eliminate an effect on specific
phosphorylation sites of these molecules. Further, it is also possible
that Shc and IRS-1 are not in the insulin signaling pathway to
prolactin. Recently, a new insulin signaling intermediary, IRS-2, was
found in IRS-1 knockout mice (39). Thus, it remains possible that this
or another, as yet unknown, intermediary mediates insulin signaling to
prolactin gene expression.
The process that RPTP influences probably occurs in association with
or close to the cell membrane. The small GTPases such as Ras, Rac, and
Rap are membrane-anchored and might be targets for inactivation by
RPTP
, or RPTP
might block activation of an insulin-responsive
GTPase. This could then result in inactivation of downstream effectors
such as Raf/MEK/MAP kinase, PI 3-kinase, or p70S6 kinase.
Therefore, experiments were done to determine if any of these signaling
pathways were essential for the response to insulin and were altered in
RPTP
overexpressing cells.
The experiments using dominant negative Ras and Raf indicate that EGF-increased prolactin-CAT expression is Ras-mediated, while insulin-increased prolactin-CAT expression is dependent on a Ras-like GTPase (Fig. 6). The activity of MAP kinase in hormone-treated GH4 cells expressing these inhibitors paralleled the inhibition of insulin- and EGF-increased prolactin-CAT activity. This suggested that insulin and EGF might increase prolactin-CAT expression in a MAP kinase-dependent manner. However, experiments with PD098059 (Fig. 7) demonstrate that MAP kinase does not mediate the increase in prolactin-CAT expression in insulin-treated cells, and numerous other experiments supported this conclusion.
Ras was also shown to activate PI 3-kinase (40) and, indirectly,
p70S6 kinase (41). The experiments with rapamycin (Fig. 8),
an inhibitor of p70S6 kinase phosphorylation (42),
demonstrate that insulin-increased prolactin-CAT expression is not
mediated by p70S6 kinase. However, wortmannin, an
irreversible inhibitor of PI 3-kinase, blocked insulin-increased
prolactin-CAT expression without affecting basal levels of
prolactin-CAT gene transcription or its increase by EGF or cAMP. This
could suggest that insulin mediates its effect by activating PI
3-kinase. However, the concentration of wortmannin needed to inhibit
insulin-increased prolactin-CAT expression is 100-1000-fold higher
than that reported to be necessary for PI 3-kinase inhibition, and
lower concentrations of wortmannin do not inhibit insulin-increased
prolactin-CAT expression (data not shown). This makes it unlikely that
the wortmannin inhibition of insulin-increased prolactin-CAT expression
is due to inhibition of PI 3-kinase. The failure of LY294002, a
reversible inhibitor of PI 3-kinase (13), to reduce insulin-increased
prolactin-CAT expression and other experiments with constitutively
activated p110 or with p85 (data not shown) supports this conclusion.
Recently, wortmannin was shown to inhibit phosphatidyl 4-kinase (43) at concentrations 100-1000-fold higher than necessary to inhibit PI
3-kinase. Experiments are presently focusing on whether insulin increases inositol -4-PO4 and/or inositol-3-PO4
in RPTP-expressing cell lines.
The identification of PTPases that inactivate insulin signaling will
provide important insights into regulation of insulin action and may
provide clues for new treatment modalities. PTPases that are potential
regulators of insulin responsiveness would be expected to be
(a) specific to insulin responses and (b) widely distributed and expressed in insulin-responsive tissues. Experiments in vivo and in vitro have implicated numerous
protein tyrosine phosphatases in the control of insulin signaling.
These include, in addition to RPTP, the membrane-spanning PTPases,
LAR and CD45, and the cytosolic PTPases, PTP1B, and SHP2 (23, 24, 36, 44-46). However, many of these do not have the characteristics expected of a phosphatase that specifically down-regulates the insulin
response pathway. LAR is widely expressed and has been shown to
dephosphorylate the insulin receptor in vitro (24), and LAR
expression blocks insulin signaling (36). However, these effects are
not specific for the insulin receptor (24) and the insulin signaling
pathway (47). The transmembrane PTPase CD45 (leukocyte common antigen)
was shown to block phosphorylation of the platelet-derived growth
factor receptor and the insulin growth factor-1 receptor and to block
their downstream effects (23), and it is able to dephosphorylate both
the insulin and EGF receptors in vitro (44). But this enzyme
is not specific and is confined to cells of hematopoietic origin. PTP1B
was shown to dephosphorylate the insulin receptor (24) in
vitro, and microinjected PTP1B blocks insulin-induced oocyte
maturation (48). However, the effect of PTP1B was not specific, since
it also blocked maturation induced by progesterone and
maturation-promoting factor. SHP2 is ubiquitously expressed and
dephosphorylates IRS-1, if not the insulin receptor (45), but SHP2
positively regulates effects of insulin (46). By contrast, the data
presented here suggest RPTP
may serve as a negative regulator for at
least some insulin signaling pathways. It specifically blocks the
insulin signaling pathway leading to prolactin gene expression but does
not inhibit the effects of EGF or cAMP. Further, it is the only PTPase
from among those that were tested that blocks the insulin response pathway. Finally, it is widely expressed including in adipose and
muscle cells (49).2
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ACKNOWLEDGEMENTS |
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We thank L. Feig, V. Narrayanan, T. Payne, U. Rapp, J. Schlessinger, E. Skolnik, K. Siddle, N. Tonks, M. White, and J. Whittaker for plasmids and antibodies used in these studies.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK43365 (to F. M. S.) and R29CA68365 (to J. S.) and grants from the Juvenile Diabetes Foundation International (to F. M. S.) and from Sugen Inc. (to J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Medicine,
TH 450, NYU Medical Center, 550 First Ave., New York, NY 10016. Tel.:
212-263-7927; Fax: 212-263-7701.
1 The abbreviations used are: CAT, chloramphenicol acetyltransferase; EGF, epidermal growth factor; MAP, mitogen-activated protein; DMEM, Dulbecco's modified Eagle's medium; RPTP, receptor-like protein-tyrosine phosphatase; 8-CPT-cAMP, 8-(chlorophenylthio)-3',5'-cyclic AMP; PI 3-kinase, phosphatidylinositol 3-kinase; IRS, insulin receptor substrate; MEK, MAP kinase kinase; PTPase, protein-tyrosine phosphatase; HA, hemagglutinin; RSV, Rous sarcoma virus; Prl, prolactin.
2 J. Sap, unpublished observations.
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REFERENCES |
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