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) Tyr612 and Tyr941 (p85 binding sites), concomitant with an increased association of the p85 subunit of PI 3-kinase; (ii) Tyr896 (a Grb2 binding site); and (iii) Tyr1229 (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 Tyr1158 and insulin receptor tyrosine kinase activity were all reduced by inhibition of PI 3-kinase at later time points (20min). 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.
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December 2002
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Research Article|
December 15 2002
Reciprocal feedback regulation of insulin receptor and insulin receptor substrate tyrosine phosphorylation by phosphoinositide 3-kinase in primary adipocytes
Ingeborg HERS;
Ingeborg HERS
1
∗Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
1To whom correspondence should be addressed (e-mail i.hers@bris.ac.uk).
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Christopher J. BELL;
Christopher J. BELL
∗Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
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Alastair W. POOLE;
Alastair W. POOLE
†Department of Pharmacology, School of Medical Sciences, University Walk, Bristol BS8 1TD, U.K.,
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Donyang JIANG;
Donyang JIANG
‡Hopkinton Division of BioSource International, 3 Avenue D, Hopkinton, MA 01748, U.S.A.
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Richard M. DENTON;
Richard M. DENTON
∗Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
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Erik SCHAEFER;
Erik SCHAEFER
‡Hopkinton Division of BioSource International, 3 Avenue D, Hopkinton, MA 01748, U.S.A.
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Jeremy M. TAVARÉ
Jeremy M. TAVARÉ
∗Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
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Publisher: Portland Press Ltd
Received:
June 11 2002
Revision Received:
August 16 2002
Accepted:
September 09 2002
Accepted Manuscript online:
September 09 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 368 (3): 875–884.
Article history
Received:
June 11 2002
Revision Received:
August 16 2002
Accepted:
September 09 2002
Accepted Manuscript online:
September 09 2002
Citation
Ingeborg HERS, Christopher J. BELL, Alastair W. POOLE, Donyang JIANG, Richard M. DENTON, Erik SCHAEFER, Jeremy M. TAVARÉ; Reciprocal feedback regulation of insulin receptor and insulin receptor substrate tyrosine phosphorylation by phosphoinositide 3-kinase in primary adipocytes. Biochem J 15 December 2002; 368 (3): 875–884. doi: https://doi.org/10.1042/bj20020903
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