Modulation of Insulin Receptor Substrate-1 Tyrosine Phosphorylation and Function by Mitogen-activated Protein Kinase

Serine and threonine phosphorylation of IRS-1 (insulin receptor substrate-1) has been reported to decrease its ability to be tyrosine-phosphorylated by the insulin receptor. Insulin itself may negatively regulate tyrosine phosphorylation of IRS-1 through a PI3K (phosphoinositide 3-kinase)-dependent feedback pathway. In the present study, we examined the regulation and role of IRS-1 serine phosphorylation in the modulation of IRS-1 tyrosine phosphorylation in physiologically relevant cells, namely freshly isolated primary adipocytes. We show that insulin-stimulated phosphorylation of Ser312 and Ser616 in IRS-1 was relatively slow, with maximal phosphorylation achieved after 20 and 5 min respectively. The effect of insulin on phosphorylation of both these sites required the activation of PI3K and the MAPKs (mitogen-activated protein kinases) ERK1/2 (extracellular-signal-regulated kinase 1 and 2), but not the activation of mTOR (mammalian target of rapamycin)/p70S6 kinase, JNK (c-Jun N-terminal kinase) or p38MAPK. Although inhibition of PI3K and ERK1/2 both substantially decreased insulin-stimulated phosphorylation of Ser312 and Ser616, only wortmannin enhanced insulin-stimulated tyrosine phosphorylation of IRS-1. Furthermore, inhibition of mTOR/p70S6 kinase, JNK or p38MAPK had no effect on insulin-stimulated IRS-1 tyrosine phosphorylation. The differential effect of inhibition of ERK1/2 on insulin-stimulated IRS-1 phosphorylation of Ser312/Ser616 and tyrosine indicates that these events are independent of each other and that phosphorylation of Ser312/Ser616 is not responsible for the negative regulation of IRS-1 tyrosine phosphorylation mediated by PI3K in primary adipocytes.

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Modulation of human insulin receptor substrate-1 tyrosine phosphorylation by protein kinase Cdelta

Biochemical Journal ◽

10.1042/bj20031493 ◽

2004 ◽

Vol 378 (1) ◽

pp. 105-116 ◽

Cited By ~ 62

Author(s):

Michael W. GREENE ◽

Nick MORRICE ◽

Robert S. GAROFALO ◽

Richard A. ROTH

Keyword(s):

Fatty Acid ◽

Protein Kinase ◽

Insulin Receptor ◽

Tyrosine Phosphorylation ◽

Inhibitory Effect ◽

Insulin Receptor Substrate ◽

Triple Mutant ◽

Insulin Receptor Substrate 1

Non-esterified fatty acid (free fatty acid)-induced activation of the novel PKC (protein kinase C) isoenzymes PKCδ and PKCθ correlates with insulin resistance, including decreased insulin-stimulated IRS-1 (insulin receptor substrate-1) tyrosine phosphorylation and phosphoinositide 3-kinase activation, although the mechanism(s) for this resistance is not known. In the present study, we have explored the possibility of a novel PKC, PKCδ, to modulate directly the ability of the insulin receptor kinase to tyrosine-phosphorylate IRS-1. We have found that expression of either constitutively active PKCδ or wild-type PKCδ followed by phorbol ester activation both inhibit insulin-stimulated IRS-1 tyrosine phosphorylation in vivo. Activated PKCδ was also found to inhibit the IRS-1 tyrosine phosphorylation in vitro by purified insulin receptor using recombinant full-length human IRS-1 and a partial IRS-1–glutathione S-transferase-fusion protein as substrates. This inhibition in vitro was not observed with a non-IRS-1 substrate, indicating that it was not the result of a general decrease in the intrinsic kinase activity of the receptor. Consistent with the hypothesis that PKCδ acts directly on IRS-1, we show that IRS-1 can be phosphorylated by PKCδ on at least 18 sites. The importance of three of the PKCδ phosphorylation sites in IRS-1 was shown in vitro by a 75–80% decrease in the incorporation of phosphate into an IRS-1 triple mutant in which Ser-307, Ser-323 and Ser-574 were replaced by Ala. More importantly, the mutation of these three sites completely abrogated the inhibitory effect of PKCδ on IRS-1 tyrosine phosphorylation in vitro. These results indicate that PKCδ modulates the ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by direct phosphorylation of the IRS-1 molecule.

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Protein Kinase C-ζ-induced Phosphorylation of Ser318in Insulin Receptor Substrate-1 (IRS-1) Attenuates the Interaction with the Insulin Receptor and the Tyrosine Phosphorylation of IRS-1

Journal of Biological Chemistry ◽

10.1074/jbc.m402477200 ◽

2004 ◽

Vol 279 (24) ◽

pp. 25157-25163 ◽

Cited By ~ 87

Author(s):

Klaus Moeschel ◽

Alexander Beck ◽

Cora Weigert ◽

Reiner Lammers ◽

Hubert Kalbacher ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Insulin Receptor ◽

Tyrosine Phosphorylation ◽

Insulin Receptor Substrate ◽

Insulin Receptor Substrate 1 ◽

Kinase C

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Stimulation of protein synthesis, eukaryotic translation initiation factor 4E phosphorylation, and PHAS-I phosphorylation by insulin requires insulin receptor substrate 1 and phosphatidylinositol 3-kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2857 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2857-2864 ◽

Cited By ~ 145

Author(s):

R Mèndez ◽

M G Myers ◽

M F White ◽

R E Rhoads

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

Insulin Receptor ◽

Translation Initiation Factor ◽

Mitogen Activated Protein Kinase ◽

Insulin Receptor Substrate ◽

Phosphatidylinositol 3 Kinase ◽

Mitogen Activated Protein ◽

Stimulation Of ◽

Phosphatidylinositol 3

Insulin rapidly stimulates protein synthesis in a wide variety of tissues. This stimulation is associated with phosphorylation of several translational initiation and elongation factors, but little is known about the signaling pathways to these events. To study these pathways, we have used a myeloid progenitor cell line (32D) which is dependent on interleukin 3 but insensitive to insulin because of the very low levels of insulin receptor (IR) and the complete lack of insulin receptor substrate (IRS)-signaling proteins (IRS-1 and IRS-2). Expression of more IR permits partial stimulation of mitogen-activated protein kinase by insulin, and expression of IRS-1 alone mediates insulin stimulation of the 70-kDa S6 kinase (pp70S6K) by the endogenous IR. However, expression of both IR and IRS-1 is required for stimulation of protein synthesis. Moreover, this effect requires activation of phosphatidylinositol 3-kinase (PI3K), as determined by wortmannin inhibition and the use of an IRS-1 variant lacking all Tyr residues except those which activate PI3K. Stimulation of general protein synthesis does not involve activation by IRS-1 of GRB-2-SOS-p21ras or SH-PTP2, since IRS-1 variants lacking the SH2-binding Tyr residues for these proteins are fully active. Nor does it involve pp70S6K, since rapamycin, while strongly inhibiting the synthesis of a small subset of growth-regulated proteins, only slightly inhibits total protein synthesis. Recruitment of mRNAs to the ribosome is enhanced by phosphorylation of eIF4E, the cap-binding protein, and PHAS-I, a protein that specifically binds eIF4E. The behavior of cell lines containing IRS-1 variants and inhibition by wortmannin and rapamycin indicate that the phosphorylation of both proteins requires IRS-1-mediated stimulation of PI3K and pp70S6K but not mitogen-activated protein kinase or SH-PTP2.

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