Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action - PubMed (original) (raw)

Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action

H Eldar-Finkelman et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. Expression of wild-type GSK-3 or an "unregulated" mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Phosphorylation of IRS-1 by GSK-3. (A) Recombinant IRS-1 (designated as IRS-1) or IRS-1 immunoprecipitated from CHO cells overexpressing IRS-1 (designated as IP-IRS-1) was incubated with recombinant rabbit GSK-3 in the presence of 50 mM Tris (pH 7.3), 10 mM magnesium acetate, 0.01% 2-mercaptoethanol, and 50 μM [γ-32P]ATP (0.25 mCi/ml) in a final volume of 30 μl at 30°C for 15 min. Reactions were stopped by addition of Laemmli sample buffer, subjected to SDS/PAGE, and autoradiographed. (B) Phosphoamino acid composition of each type IRS-1 phosphorylated by GSK-3 was determined as described. The migration positions of phosphorylated tyrosine (P-Tyr), threonine (P-Thr), and serine (P-Ser) are indicated. (C) IRS-1, c-jun, and p9CREB peptide (see Materials and Methods), 10 pmol of each, were phosphorylated by GSK-3 under the conditions described in A for the indicated times. Reactions with IRS-1 or c-jun as the substrates were stopped by addition of Laemmli sample buffer, subjected to SDS/PAGE, and autoradiographed; radioactive bands were cut from the gels and counted for radioactivity. The reaction mixtures containing p9CREB was spotted on phosphocellulose paper and handled as described. Phosphate incorporation into each substrate is presented.

Figure 2

Figure 2

Phosphorylation of IRS-1 by GSK-3 inhibits IR tyrosine kinase activity. (A) Recombinant IRS-1 (0.1 μg) was subjected to sequential phosphorylation reactions, the first involved serine phosphorylation by GSK-3 using unlabeled ATP, and the second involved tyrosine phosphorylation catalyzed by purified IR, again using unlabeled ATP. The reactions were stopped by addition of Laemmli buffer, boiled, and subjected to SDS/PAGE. To detect IR-catalyzed tyrosine phosphorylation of the substrate IRS-1, immunoblot analysis was performed using monoclonal antibodies to phosphotyrosine. Experimental detail are provided in Material and Methods. The composition of the various reactions, including controls are presented in the upper panel of the figure. (B) The same as in A except that 10 μg histone H2B was added together with IR and [γ-32P]ATP. In this instance the phosphorylation of H2B was determined by autoradiography.

Figure 3

Figure 3

Expression of GSK-3 in CHO/IR/IRS-1 cells. Cells were transiently transfected with pCMV4 expression plasmids encoding WT GSK-3β or its S9A mutant as described. GSK-3 expression levels (Upper) were determined by Western blot analysis of cell lysates using polyclonal antibodies to GSK-3β. GSK-3 was immunoprecipitated from cell lysates and kinase activities were assayed in the immunoprecipitate complex using a peptide substrate P-GS1 (Lower). Kinase activities are presented as percent of control (cells transfected with vector alone), and are mean ± SEM from three experiments.

Figure 4

Figure 4

Phosphorylation of IRS-1 by GSK-3 in intact CHO/IR/IRS-1 cells. Transfected cells were metabolically labeled with [32P]orthophosphoric acid for 4 h, and IRS-1 was immunoprecipitated from the cell lysates. Samples were (A) analyzed by gel electrophoresis followed by autoradiography or (B) transferred to polyvinylidene difluoride membrane followed by acid digestion. Phosphoamino acid analysis was performed on the digested samples as described. The migration positions of phosphorylated tyrosine (P-Tyr), threonine (P-Thr), and serine (P-Ser) are indicated.

Figure 5

Figure 5

Insulin-induced tyrosine phosphorylation of IRS-1 and IR in GSK-3-expressing cells. (A) CHO/IR/IRS-1 cells expressing WT GSK-3 or the S9A mutant were starved for 4 hr followed by stimulation with insulin (100 nM) for 15 min. Equal amount of protein from cell lysates were subjected to gel electrophoresis, transferred to polyvinylidene difluoride membrane, and immunoblotted with monoclonal antibodies to phosphotyrosine. Tyrosine phosphorylated IRS-1 and the β subunit of IR are indicated. (B) Image density of tyrosine phosphorylated IRS-1 and the β subunit of IR from A. Results are presented as percent of control cells and are the mean ± SE from three independent experiments.

Similar articles

Cited by

References

    1. Myers M G J, White M F. Annu Rev Pharmacol Toxicol. 1996;36:615–658. - PubMed
    1. Czech M P. Annu Rev Nutr. 1995;15:441–471. - PubMed
    1. Saltiel A R. Am J Physiol. 1996;33:E375–E385. - PubMed
    1. Myers M G J, Backer J M, Sun X J, Shoelson S, Hu P, Schlessinger J, Yoakim M, Schaffhausen B, White M F. Proc Natl Acad Sci USA. 1992;89:10350–10354. - PMC - PubMed
    1. Skolnik E Y, Batzer A, Li N, Lee C H, Lowenstein E, Mohammadi M, Margolis B, Schlessinger J. Science. 1993;260:1953–1955. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources