Association of Insulin Receptor Substrate 1 (IRS-1) Y895 with Grb-2 Mediates the Insulin Signaling Involved in IRS-1-Deficient Brown Adipocyte Mitogenesis

Departamento de Bioquímica y Biología Molecular, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain.
Molecular and Cellular Biology (Impact Factor: 4.78). 05/2001; 21(7):2269-80. DOI: 10.1128/MCB.21.7.2269-2280.2001
Source: PubMed


We have recently generated immortalized fetal brown adipocyte cell lines from insulin receptor substrate 1 (IRS-1) knockout mice and demonstrated an impairment in insulin-induced lipid synthesis as compared to wild-type cell lines. In this study, we investigated the consequences of IRS-1 deficiency on mitogenesis in response to insulin. The lack of IRS-1 resulted in the inability of insulin-stimulated IRS-1-deficient brown adipocytes to increase DNA synthesis and enter into S/G2/M phases of the cell cycle. These cells showed a severe impairment in activating mitogen-activated protein kinase kinase (MEK1/2) and p42-p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. IRS-1-deficient cells also lacked tyrosine phosphorylation of SHC and showed no SHC-Grb-2 association in response to insulin. The mitogenic response to insulin could be partially restored by enhancing IRS-2 tyrosine phosphorylation and its association with Grb-2 by inhibition of phosphatidylinositol 3-kinase activity through a feedback mechanism. Reconstitution of IRS-1-deficient brown adipocytes with wild-type IRS-1 restored insulin-induced IRS-1 and SHC tyrosine phosphorylation and IRS-1-Grb-2, IRS-1-SHC, and SHC-Grb-2 associations, leading to the activation of MAPK and enhancement of DNA synthesis. Reconstitution of IRS-1-deficient brown adipocytes with the IRS-1 mutant Tyr895Phe, which lacks IRS-1-Grb-2 binding, restored SHC-IRS-1 association and SHC-Grb-2 association. However, the lack of IRS-1-Grb-2 association impaired MAPK activation and DNA synthesis in insulin-stimulated mutant cells. These data provide strong evidence for an essential role of IRS-1 and its direct association with Grb-2 in the insulin signaling pathway leading to MAPK activation and mitogenesis in brown adipocytes.

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    • "The IRS complex consists of IRS 1–4, with each substrate playing a significant role in the body; however, animal studies have shown that a vast majority of insulin actions signal through IRS-1 and IRS-2 [47]. The amino acid sequence of IRS-1 possesses a unique signaling mechanism of tyrosine phosphorylation sites and serine phosphorylation sites [23,28-31,42,44,46,48] to regulate cellular actions. Phosphorylation of various tyrosine sites (Y99, Y1150, Y1151) and several serine sites (Ser265, Ser302, Ser325, Ser358) increase the downstream signaling mediated by IRS-1. "
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    • "acterized of these are SH2 domain proteins that bind to phosphotyrosines in specific sequence motifs on the IRS proteins . These SH2 proteins fall into two major categories : adaptor molecules , such as the regulatory subunit of PI 3 - kinase , Grb2 , which associates with SOS to activate the Ras - MAP kinase pathway ( Baltensperger et al . 1993 ; Valverde et al . 2001 ) , CrkII ( Karas et al . 2001 ) and Nck2 ( Tu et al . 2001 ) , and enzymes , such as the phosphotyrosine phosphatase SHP2 ( Rocchi et al . 1996 ) and the tyrosine kinase Fyn ( Sun et al . 1996 ) . The IRS proteins also interact with proteins that do not contain SH2 domains , including the calcium ATPases SERCA 1 and 2 ( Algenstaedt et "
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