Abstract
The ability of glycogen synthase kinase-3 (GSK-3) to phosphorylate insulin receptor substrate-1 (IRS-1) is a potential inhibitory mechanism for insulin resistance in type 2 diabetes. However, the serine site(s) phosphorylated by GSK-3 within IRS-1 had not been yet identified. Using an N-terminal deleted IRS-1 mutant and two IRS-1 fragments, PTB-1 1-320 and PTB-2 1-350, we localized GSK-3 phosphorylation site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the GSK-3 consensus motif (SXXXS) within PTB-2 or IRS-1, to alanine abolished their phosphorylation by GSK-3. This suggested that Ser332 is a GSK-3 phosphorylation site and that Ser336 serves as the "priming" site typically required for GSK-3 action. Indeed, dephosphorylation of IRS-1 prevented GSK-3 phosphorylation. Furthermore, the phosphorylated peptide derived from the IRS-1 sequence was readily phosphorylated by GSK-3, in contrast to the nonphosphorylated peptide, which was not phosphorylated by the enzyme. When IRS-1 mutants S332A(IRS-1), S336A(IRS-1), or S332A/336A(IRS-1) were expressed in Chinese hamster ovary cells overexpressing insulin receptors, their insulin-induced tyrosine phosphorylation levels increased compared with that of wild-type (WT) IRS-1. This effect was stronger in the double mutant S332A/336A(IRS-1) and led to enhanced insulin-mediated activation of protein kinase B. Finally, immunoblot analysis with polyclonal antibody directed against IRS-1 phosphorylated at Ser332 confirmed IRS-1 phosphorylation in cultured cells. Moreover, treatment with the GSK-3 inhibitor lithium reduced Ser332 phosphorylation, whereas overexpression of GSK-3 enhanced this phosphorylation. In summary, our studies identify Ser332 as the GSK-3 phosphorylation target in IRS-1, indicating its physiological relevance and demonstrating its novel inhibitory role in insulin signaling.
Highlights
Insulin resistance, commonly defined as the inability of peripheral tissues to respond normally to physiological concentrations of insulin, is considered one of the earliest changes associated with the onset of type 2 diabetes [1,2,3]
We showed previously that glycogen synthase kinase-3 (GSK-3) promotes the phosphorylation of insulin receptor substrate-1 (IRS-1) exclusively on serine residues, which, in turn, attenuated insulin signaling in cultured cells [14]
GSK-3 phosphorylated PTB-2, but not PTB-1. These results indicated that GSK-3 phosphorylation site(s) reside within amino acids 320 –350 and support our speculation that serines 332, 336, and 340 are the sites involved in GSK-3 phosphorylation
Summary
Commonly defined as the inability of peripheral tissues to respond normally to physiological concentrations of insulin, is considered one of the earliest changes associated with the onset of type 2 diabetes [1,2,3]. We showed previously that GSK-3 promotes the phosphorylation of IRS-1 exclusively on serine residues, which, in turn, attenuated insulin signaling in cultured cells [14]. We hypothesized that hyperactive GSK-3 may enhance serine phosphorylation of IRS-1, decreasing its ability to be tyrosyl-phosphorylated The specific IRS-1 serine sites phosphorylated by GSK-3 were not identified. We used deletion and mutation techniques, together with specific phosphoantibody to identify serine site(s) within. We show that GSK-3 phosphorylates Ser332 of IRS-1 and that mutation at this site enhances insulin-induced tyrosine phosphorylation of IRS-1. We demonstrate the physiological relevance of Ser332 phosphorylation with specific phospho-IRS-1 antibody
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