Roles of mTOR and JNK in serine phosphorylation, translocation, and degradation of IRS-1

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Abstract

In 3T3-L1 adipocytes, insulin or anisomycin stimulated phosphorylation of IRS-1 at Ser307 and Ser636/639, both of which were partially reduced by the mTOR inhibitor, rapamycin, or the JNK inhibitor, SP600125, and were further inhibited by a combination of them. Interestingly, anisomycin-induced p70S6K phosphorylation was reduced by SP600125, while insulin-induced p70S6K phosphorylation was not. Furthermore, unlike insulin, anisomycin failed to elicit translocation or degradation of IRS-1. These results indicate that mTOR and JNK play roles in phosphorylating IRS-1 serine residues, and that insulin and anisomycin are different in terms of the relationship of activation between mTOR and JNK, and the effects on IRS-1 localization and stability.

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Materials and methods

Materials. The Lilly Research Laboratories (Indianapolis, IN) kindly provided porcine insulin. Anti-IRS-1 and anti-phospho-Ser307IRS-1 antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-Ser636/639IRS-1, anti-phospho-Thr389p70S6K, and anti-phospho-Ser63c-Jun antibodies were purchased from Cell Signaling Technology (Beverly, MA). HRP-conjugated anti-immunoglobulin G secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rapamycin was

Roles of mTOR and JNK in phosphorylation of IRS-1 at Ser307 and Ser636/639 induced by insulin or anisomycin

To evaluate the roles of mTOR and JNK in Ser phosphorylation of IRS-1 induced by either insulin or anisomycin, we first examined the effects of the specific mTOR inhibitor, rapamycin [14], or JNK inhibitor, SP600125 [15], on phosphorylation of IRS-1 at Ser307 and Ser636/639 as well as phosphorylation of p70S6K at Thr389, which depends upon activation of mTOR pathway [16] and phosphorylation of c-Jun at Ser63, which is the direct target of JNK [15] (Fig. 1).

Insulin stimulation for 1 h induced

Discussion

In this study, we investigated the roles of mTOR and JNK in Ser phosphorylation, translocation, and degradation of IRS-1 in 3T3-L1 adipocytes stimulated either by insulin or anisomycin (Fig. 5). Despite the similar extent of phosphorylation of the same residues, Ser307 and Ser636/639, insulin and anisomycin were different in terms of the relationship of activation between mTOR and JNK, while both of them seem to cooperate in phosphorylating the serine residues. Insulin and anisomycin were also

Acknowledgments

This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (to T.H.).

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Abbreviations: IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; mTOR, mammalian target of rapamycin; PI 3-kinase, phosphatidylinositol 3-kinase; TSC, tuberous sclerosis complex; LDM, low-density microsomes; p70S6K, p70 S6 kinase.

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