Reciprocal feedback regulation of insulin receptor and insulin receptor substrate tyrosine phosphorylation by phosphoinositide 3-kinase in primary adipocytes.
Signalling by the insulin receptor substrate (IRS) proteins is critically dependent on the tyrosine phosphorylation of specific binding sites that recruit Src homology 2 (SH2)-domain-containing proteins, such as the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase), the tyrosine phosphatase SHP-2 and the adapter protein Grb2. Here we show that stimulation by insulin of freshly isolated primary adipocytes resulted in the expected rapid tyrosine phosphorylation of the insulin receptor, IRS-1 and IRS-3. Inhibition of PI 3-kinase enhanced the insulin-stimulated phosphorylation of IRS-1 on (i) Tyr(612) and Tyr(941) (p85 binding sites), concomitant with an increased association of the p85 subunit of PI 3-kinase; (ii) Tyr(896) (a Grb2 binding site); and (iii) Tyr(1229) (an SHP-2 binding site), although little or no binding of SHP-2 to IRS-1 was detectable under any conditions. In contrast, inhibition of PI 3-kinase led to a decrease in insulin-stimulated p85 binding to IRS-3, but had no effect on SHP-2 binding. Furthermore, insulin-induced insulin receptor tyrosine phosphorylation, phosphorylation of Tyr(1158) and insulin receptor tyrosine kinase activity were all reduced by inhibition of PI 3-kinase at later time points (>or=20 min). The results demonstrate that, in primary adipocytes, PI 3-kinase feedback control of signalling by the insulin receptor and IRS proteins is multifaceted and reciprocal, illustrating the complexity of predicting the net flux of the insulin signal(s) through the IRS proteins.
Hers
Ingeborg
I
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK. i.hers@bris.ac.uk
Bell
Christopher J
CJ
Poole
Alastair W
AW
Jiang
Donyang
D
Denton
Richard M
RM
Schaefer
Erik
E
Tavaré
Jeremy M
JM
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004822:Epididymis / Q000166:cytology; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011972:Receptor, Insulin / Q000378:metabolism*; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; C526292:IRS1 protein, human; C491585:IRS3P protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; C108872:Irs3 protein, mouse; C491584:Irs3 protein, rat; D009025:Morpholines; D010750:Phosphoproteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; C516727:Ptpn11 protein, rat; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D000077191:Wortmannin