(Received for publication, December 28, 1995; and in revised form, February 9, 1996)
From the
Okadaic acid has been described previously as being a negative
regulator of insulin signaling, as it inhibits insulin stimulation of
glucose transport. In addition, this drug induces on insulin receptor
substrate-1 (IRS-1) a decrease in tyrosine phosphorylation,
concomitantly with an increase in serine/threonine phosphorylation. The
present work was aimed at the identification of the serine/threonine
residues that, upon phosphorylation, might be involved in modulating
insulin signaling. To this end, we studied double-point mutants of
IRS-1, in which serines 612/632 and 662/731 were replaced with alanine.
These are four plausible sites of phosphorylation by mitogen-activated
protein kinases and are in the immediate proximity of tyrosine
residues, which are potential sites of interaction with the
phosphatidylinositol 3-kinase Src homology 2 domains. Using transient
expression in 293 EBNA cells, we demonstrate that serines 612, 632,
662, and 731 and mitogen-activated protein kinases are not involved in
the okadaic acid effect on IRS-1. Rather, these serines appear to play
a role in modulating basal and insulin-stimulated IRS-1 tyrosine
phosphorylation, association of IRS-1 with p85, and
phosphatidylinositol 3-kinase activity in the IRS-1p85 immune
complex, since mutation of these sites enhances these events. Our
findings suggest the existence of an IRS-1 desensitization mechanism
resulting from serine/threonine phosphorylation, occurring at least on
serines 612, 632, 662, and 731.
Insulin exerts its biological effects by binding to its specific
tyrosine kinase receptor on the cell surface. Thereafter, the activated
receptor tyrosine kinase leads to its autophosphorylation and
phosphorylation of cellular proteins(1, 2) . A major
substrate of the insulin receptor kinase is the insulin receptor
substrate-1 (IRS-1). ()This cytosolic protein was first
identified as a 185-kDa phosphoprotein in anti-phosphotyrosine
immunoprecipitates from insulin-stimulated Fao hepatoma
cells(3) , and subsequently cloned from rat liver(4) .
IRS-1 is expressed in most cells and appears to play an important role
in insulin signal transmission(5, 6, 7) .
After insulin stimulation, IRS-1 is phosphorylated on several tyrosine
residues, most of them being located in YXXM or YMXM
motifs(4, 8) . This tyrosine phosphorylation step is
crucial, since it allows Src homology 2 domain-containing proteins to
interact with IRS-1, leading to the activation of several intracellular
processes and resulting in the final effects of insulin. A growing list
of proteins interacting with IRS-1 is appearing. Among these are growth
factor receptor-bound protein 2, Grb2(9) , the tyrosine
phosphatase SH-PTP2(10, 11) , Nck (12) ,
p85
and p85
, the two isoforms of the PI 3-kinase regulatory
subunit(13, 14, 15) , and
p55
(16) .
In addition to tyrosine
phosphorylation, IRS-1 also undergoes serine/threonine phosphorylation.
Indeed, in SDS-PAGE, IRS-1 usually migrates with an electrophoretic
mobility corresponding to 165-185 kDa, while its expected
molecular mass is only 131 kDa. It is believed that this discrepancy is
due essentially to serine/threonine phosphorylation of the protein in
the basal state. Insulin leads to increased IRS-1 serine
phosphorylation and, to a lesser extent, threonine
phosphorylation(5) . These phosphorylation events occur on
multiple sites on the protein and are probably due to several
serine/threonine kinases activated during insulin action. Recently, our
laboratory has demonstrated that okadaic acid, an inhibitor of
serine/threonine phosphatases, is also able to increase IRS-1
serine/threonine phosphorylation(17) . Moreover, after okadaic
acid treatment, IRS-1 immunoprecipitated from 3T3-L1 adipocytes was
less able to be tyrosine phosphorylated by the activated insulin
receptor in a cell-free system. In vitro reconstitution
experiments also showed a reduced PI 3-kinase activity associated with
IRS-1 from okadaic acid-treated cells, compared to PI 3-kinase activity
associated with IRS-1 from nontreated cells. These results provide an
explanation for the observation that okadaic acid induces an
insulin-resistant state. Indeed, a previous study from our laboratory
showed an inhibitory effect of okadaic acid on insulin-induced glucose
transport in isolated soleus muscle and 3T3-L1 adipocytes(18) .
In addition, in the presence of the phosphatase inhibitor, decreased
insulin-stimulated IRS-1 tyrosine phosphorylation and decreased PI
3-kinase activity immunoprecipitated with antibodies to phosphotyrosine
were observed. These effects of okadaic acid could be due to the
increased IRS-1 serine/threonine phosphorylation seen with the drug.
Very recently, tumor necrosis factor- was found to cause a similar
decrease in insulin-induced tyrosine phosphorylation of
IRS-1(19) . Like okadaic acid, tumor necrosis factor-
induces insulin resistance and leads to the phosphorylation of IRS-1 on
serine/threonine residues(19, 20, 21) . Taken
together, these data point to an important role of IRS-1
serine/threonine phosphorylation in the modulation of insulin
signaling.
In the present study, we attempt to determine the serine and/or threonine residues implicated in the regulation of IRS-1 tyrosine phosphorylation and associated PI 3-kinase activity. We focused on the residues that might be phosphorylated upon okadaic acid treatment of cells. The primary sequence of IRS-1 contains at least 35 potential sites for serine/threonine phosphorylation by several cytosolic kinases, including cAMP-dependent protein kinase A, protein kinase C, casein kinase II, and MAP kinases(4, 22) . The approach we used was to construct IRS-1 proteins mutated on serine residues. We chose to mutate four potential phosphorylation sites for MAP kinases, serine 612, 632, 662, and 731, for the following reasons. (i) MAP kinases are activated by okadaic acid(23) . (ii) These four serine residues, located in the YMXMSP sequence, are adjacent to potential tyrosine phosphorylation sites for the activated insulin receptor. (iii) Finally, these four tyrosine residues reside in potential binding sites for the Src homology 2 domains of the p85 subunit of PI 3-kinase, and three of them (608, 628, and 658) have been shown to participate in such an interaction(11, 24) . In brief, we have replaced the four serine residues with alanine to obtain two double mutants, IRS-1 S612A/S632A and S662A/S731A. Both mutants, as well as wild-type IRS-1, were expressed in 293 EBNA cells, and we compared the effects of insulin and okadaic acid on the different IRS-1 proteins in intact cells with respect to: (i) their phosphorylation state (serine/threonine and tyrosine), (ii) their ability to associate the p85 of PI 3-kinase, and (iii) their associated PI 3-kinase activity.
Figure 1:
Immunoprecipitation
of [ S]methionine and
[
S]cysteine-labeled wild-type IRS-1, IRS-1
S612A/S632A, and IRS-1 S662A/S731A, expressed in 293 EBNA cells. 293
EBNA cells, transfected with pCEP containing cDNAs encoding the
different IRS-1 proteins or with pCEP vector alone, were labeled
overnight with Tran
S-label. After solubilization, IRS-1
proteins were immunoprecipitated with a specific antibody to rat IRS-1.
The samples were subjected to a 7.5% SDS-PAGE under reducing
conditions. An autoradiogram of the gel is
shown.
Figure 2:
Effect of okadaic acid on wild-type IRS-1,
IRS-1 S612A/S632A, and IRS-1 S662A/S731A electrophoretic mobility. 293
EBNA cells transfected with the plasmids expressing the different IRS-1
proteins were treated or not for 40 min with 2 µM okadaic
acid and then stimulated or not with insulin (10M) for 5 min. Proteins were solubilized, separated on a
7.5% SDS-PAGE, and transferred to an Immobilon membrane. Rat IRS-1
proteins were revealed by specific antibodies and
I-protein A. A representative autoradiogram of three
independent experiments is shown.
Figure 3:
PI 3-kinase activity associated with
wild-type IRS-1, IRS-1 S612A/S632A, and IRS-1 S662A/S731A. 293 EBNA
cells expressing the different IRS-1 proteins were treated without or
with 2 µM okadaic acid (oka) for 40 min and/or
insulin (10M) for 5 min. Cells were
solubilized, and the lysates were immunoprecipitated with antibodies to
rat IRS-1. The PI 3-kinase activity was determined as described
previously(15) . A, upper panel, histogram of
one representative PI 3-kinase assay, done in triplicate; lower
panel, Western blotting with antibodies to IRS-1 of 100 µg of
proteins from the lysates, to evaluate the expression level of the
IRS-1 proteins in each condition. B, histogram of the basal
and insulin-stimulated PI 3-kinase activity. The results are expressed
as percentages of basal and insulin-stimulated PI 3-kinase activity
associated with wild-type IRS-1 and represent the mean ± S.E. of
three independent experiments done in triplicate. *, p <
0.005.
A statistical comparison of the basal and insulin-stimulated PI 3-kinase activities associated with both IRS-1 mutants and with wild-type IRS-1 was performed (Fig. 3B). For IRS-1 S612A/S632A, we found that the basal associated PI 3-kinase activity was increased approximately 30% over the basal activity obtained with wild-type IRS-1, while the insulin-stimulated activity was not significantly different from that of wild-type IRS-1. Moreover, for this mutant the increment between insulin-stimulated and basal conditions was slightly, but significantly, decreased from 360% over basal for wild-type IRS-1 to 310% for IRS-1 S612A/S632A (p < 0.005).
With IRS-1 S662A/S731A, we observed an increase in the basal associated PI 3-kinase activity, as well as a significant increase in the insulin-stimulated one, which reached 80 and 130% over basal and insulin-stimulated activities obtained with wild-type IRS-1, respectively. As opposed to what we found for IRS-1 S612A/S632A, the fold stimulation was significantly increased, from 360% for wild-type IRS-1 to 460% for IRS-1 S662A/S731A (p < 0.005). This indicates that the increase in the PI 3-kinase activity we observed was not only due to an increase in the basal activity, but also to an increase in insulin's effect. In conclusion, serine residues 612, 632, 662, and 731 are able to modulate the PI 3-kinase activity associated with IRS-1 in the absence as well as in the presence of insulin.
Figure 4:
In vivo tyrosine phosphorylation
and association with p85 of wild-type IRS-1, IRS-1 S612A/S632A, and
IRS-1 S662A/S731A. 293 EBNA cells expressing the different IRS-1
proteins were treated without or with okadaic acid (2 µM)
for 40 min and/or insulin (10M) for 5 min.
After cell solubilization and immunoprecipitation of the proteins with
a specific antibody to rat IRS-1, the samples were subjected to
SDS-PAGE, followed by transfer to an Immobilon membrane. The blot was
probed with either antibodies to phosphotyrosine (panel A) or
to p85 (panel B). On panel C, the
anti-phosphotyrosine antibodies were removed and the membrane was
reprobed with an antibody to IRS-1. The experiment shown is
representative of six independent
experiments.
We also compared in the same experiment the insulin-induced tyrosine phosphorylation of wild-type IRS-1 versus that of the two mutants, and the effect of okadaic acid on this tyrosine phosphorylation (Fig. 4, panel A). Similar to what we observed for the insulin-stimulated association of IRS-1 with p85, okadaic acid strongly inhibited insulin-induced tyrosine phosphorylation of wild-type IRS-1 and IRS-1 S612A/S632A (lanes 3 and 6). Compared to wild-type IRS-1, in insulin-stimulated conditions, a slight increment in phosphotyrosine was seen with IRS-1 S612A/S632A, but it was not statistically significant (compare lane 5 and lane 2). In contrast, the tyrosine phosphorylation of IRS-1 S662A/S731A was significantly increased (p < 0.025; compare lane 8 and lane 2), which is compatible with the increase in its association with p85 (Fig. 4, panel B). We also observed for this mutant an increased residual phosphotyrosine content in okadaic acid-treated cells, compared to that seen for wild-type IRS-1 and IRS-1 S612A/S632A (compare lane 9 to lanes 3 and 6). Nevertheless, the decrease in insulin-induced tyrosine phosphorylation of IRS-1 S662A/S731A was approximately the same as that obtained with wild-type IRS-1 and IRS-1 S612A/S632A (70% versus 80%), indicating that okadaic acid exerts the same effect on the three IRS-1 proteins.
In the experiment shown, the basal tyrosine phosphorylation, as well as the association with p85, was too weak to allow the detection of possible differences between the three IRS-1 proteins (panels A and B, lanes 1, 4, and 7). When the complete series of experiments was analyzed, the enhanced basal associated PI 3-kinase activity seen with IRS-1 S662A/S731A and, to a lesser extent, with IRS-1 S612A/S632A (Fig. 3) could not be correlated either with an increased basal tyrosine phosphorylation or with an increased basal association with p85. This could be due, at least in part, to variations in the basal level of tyrosine phosphorylation and association with p85 of the IRS-1 proteins seen in the different experiments.
Figure 5:
Effect of the MEK inhibitor, PD 098059, on
the electrophoretic mobility of wild-type IRS-1, IRS-1 S612A/S632A, and
IRS-1 S662A/S731A induced by okadaic acid. 293 EBNA cells expressing
the different IRS-1 proteins were treated or not with okadaic acid (2
µM) for 40 min after an 18-h incubation in the presence or
absence of PD 098059 (50 µM). The cells were lysed, 100
µg of proteins were separated on SDS-PAGE and transferred to an
Immobilon membrane. IRS-1 (panel A) and p42 MAP kinase (panel B) were revealed by antibodies to IRS-1 or to p42 MAP
kinase, respectively, and I-protein A. The experiment
shown is representative of three independent
experiments.
Okadaic acid, a serine/threonine phosphatase inhibitor, is able to promote an insulin-resistant state in intact cells(18, 29) . In addition, okadaic acid leads to an increase in serine/threonine phosphorylation of IRS-1(17) . These results suggest that both phenomena observed in the presence of okadaic acid, i.e. IRS-1 serine/threonine phosphorylation and altered insulin action could be causally linked. In the present study, we wished to identify the serine/threonine residues of IRS-1 implicated in this process. Therefore, we decided to mutate the four serine residues located in YMXMSP motifs and we constructed the IRS-1 mutants, IRS-1 S612A/S632A and IRS-1 S662A/S731A, in which the serine residues had been replaced with alanine residues two at a time. These mutants were expressed in 293 EBNA cells, and we compared their phosphorylation state and some of their biological properties to those seen with wild-type IRS-1.
It was described previously that okadaic acid induces a slower electrophoretic migration of IRS-1 on SDS-PAGE(17) . This is thought to be due to the increased level of IRS-1 serine/threonine phosphorylation induced by okadaic acid, compared to the one seen in basal conditions. We found that upon okadaic acid treatment our IRS-1 mutants displayed the same decrease in electrophoretic mobility as wild-type IRS-1. This result suggests that IRS-1 mutants are still phosphorylated and, consequently, that the mutated serine residues are not involved, at least in a major fashion, in the okadaic acid-induced phosphorylation of IRS-1. However, we cannot exclude the possibility that the mutated serine residues are actually phosphorylated in wild-type IRS-1. Indeed, it could be possible that the decreased electrophoretic mobility seen with IRS-1 S612A/S632A and IRS-1 S662A/S731A was still maintained, because it might be due to other IRS-1 residues phosphorylated after okadaic acid treatment.
Analysis of the insulin-stimulated PI 3-kinase activity, tyrosine phosphorylation, and association with p85 of IRS-1 mutants led us to conclude that the potential serine phosphorylation sites removed in our IRS-1 mutants, i.e. serines 612, 632, 662, and 731, are not implicated in the insulin-resistant state induced by okadaic acid. Thus, for both mutants we found a total inhibition of these insulin-stimulated effects, comparable to that obtained with wild-type IRS-1. Finally, the specific inhibitor of MEK, PD 098059, which completely inhibited the activation of p42 MAP kinase induced by okadaic acid in 293 EBNA cells, was totally unable to reverse the effect of okadaic acid on the electrophoretic mobility of wild-type IRS-1 as well as of IRS-1 S612A/S632A and IRS-1 S662A/S731A. This demonstrates that okadaic acid-induced serine/threonine phosphorylation of IRS-1 does not involve the MAP kinase pathway. Taking our results together, we conclude that one or several serine/threonine kinase(s), distinct from the MAP kinases and MEK, are activated by okadaic acid and are responsible for the phosphorylation of IRS-1 under these conditions.
Interestingly, regarding the basal and insulin-induced PI 3-kinase activity associated with IRS-1 mutants, independently of okadaic acid action, we found that, compared to wild-type IRS-1, IRS-1 S612A/S632A had an increased basal PI 3-kinase activity and IRS-1 S662A/S731A showed a markedly increased basal and insulin-stimulated PI 3-kinase activity. For IRS-1 S662A/S731A, we found in addition an increase in its insulin-stimulated association with p85. This observation was correlated with an enhanced tyrosine phosphorylation of IRS-1 S662A/S731A in response to insulin. At least the following two non-mutually exclusive mechanisms could explain this regulatory effect of serine phosphorylation: (i) inhibition of IRS-1 tyrosine phosphorylation and/or (ii) a blockade of the subsequent binding to the p85 Src homology 2 domains. Our results seen with IRS-1 S662A/S731A in insulin-stimulated cells are compatible with the first hypothesis, since the tyrosine phosphorylation content of this mutant appeared to be increased. This increase occurs probably only on tyrosine 658 and/or 727, which are close to serines 662 and 731. Thus, our result indicates that both tyrosine residues are major phosphorylation sites in intact cells, since we were able to detect an increase in the total amount of IRS-1 S662A/S731A tyrosine phosphorylation. However, it is possible that by itself the increase in IRS-1 tyrosine phosphorylation does not account for the enhanced association with p85 and PI 3-kinase activity. Indeed, these effects could also be due, at least in part, to the fact that p85 could interact more efficiently at least with tyrosines 658 and 727 of the IRS-1 mutant, since the phosphorylated serines 662 and 731 are absent.
We did not detect striking modulations of tyrosine phosphorylation and association with p85 for both IRS-1 mutants, in the basal state. However, we would expect that the same mechanisms as those proposed for insulin-stimulated conditions are responsible for the increase seen in basal PI 3-kinase activity associated with both IRS-1 mutants, compared to wild-type IRS-1. In such a scenario, we have to assume that part of the serine residues are phosphorylated by serine/threonine kinase(s) in the absence of insulin. This is compatible with the continuous serine/threonine phosphorylation of IRS-1 in the basal state(5) . This phosphorylation would occur at least on serine 662 and 731, since the effect on PI 3-kinase activity seen with IRS-1 S662A/S731A is more pronounced than the one seen with IRS-1 S612A/S632A. The differences observed between both IRS-1 mutants after insulin stimulation could be due to a preferential phosphorylation of serine 662/serine 731 by the serine/threonine kinase(s). While we have no conclusive evidence, we favor the idea that the serine/threonine kinase involved in the above discussed phenomena is likely to correspond to a MAP kinase, given the fact that the serine residues in question are comprised in consensus sequences for phosphorylation by MAP kinases.
In summary, in the present work, we were interested in studying the mechanisms participating in the modulation of insulin signaling. We hypothesized that MAP kinase phosphorylation sites, located next to tyrosine phosphorylation sites in YMXM motifs, serines 612, 632, 662, and 731, were obvious candidates for such a role. The two key contributions of the present study are as follows. (i) These potential MAP kinase phosphorylation sites, and probably more generally the MAP kinase pathway, do not appear to participate actively in the okadaic acid-induced changes in IRS-1, and hence in the insulin-resistant state provoked by okadaic acid. (ii) However, these potential MAP kinase phosphorylation sites are likely to be important negative regulators for PI 3-kinase activity associated with IRS-1. Phosphorylation of these sites would continuously desensitize the IRS-1 pathway, and interestingly this desensitization could be enhanced by insulin itself.
To the best of
our knowledge, our data are the first to demonstrate a serine IRS-1
phosphorylation event acting as a negative signal in insulin signaling,
this event being different from the okadaic acid action on IRS-1. It
remains to be determined which serine/threonine kinases are implicated
in the okadaic acid-induced insulin resistant state. Moreover, it would
be interesting to see if other serine/threonine residues are involved
in the desensitization mechanism of IRS-1 with respect to the PI
3-kinase pathway, and if the binding of other proteins to IRS-1, for
example PTP2C and p55, is also controlled by
serine/threonine phosphorylation of IRS-1.