Serine residues 1177/78/82 of the insulin receptor are required for substrate phosphorylation but not autophosphorylation

Bossenmaier, B; Strack, V; Stoyanov, B; Krützfeldt, J; Beck, A; Lehmann, R; Kellerer, M; Klein, H; Ullrich, A; Lammers, R; Häring, H U (2000). Serine residues 1177/78/82 of the insulin receptor are required for substrate phosphorylation but not autophosphorylation. Diabetes, 49(6), pp. 889-95. Alexandria, Va.: American Diabetes Association 10.2337/diabetes.49.6.889

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Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of Gynaecology, Paediatrics and Endocrinology (DFKE) > Clinic of Endocrinology, Diabetology and Clinical Nutrition

UniBE Contributor:

Krützfeldt, Jan

ISSN:

0012-1797

ISBN:

10866039

Publisher:

American Diabetes Association

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 15:06

Last Modified:

04 May 2014 23:20

Publisher DOI:

10.2337/diabetes.49.6.889

PubMed ID:

10866039

Web of Science ID:

000087314100002

URI:

https://boris.unibe.ch/id/eprint/28602 (FactScience: 121997)

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