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Molecular and Cellular Biology, April 2001, p. 2269-2280, Vol. 21, No. 7
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.7.2269-2280.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Angela M. Valverde,1 Cecilia Mur,1 Sebastián Pons,2,dagger Alberto M. Alvarez,3 Morris F. White,2 C. Ronald Kahn,2 and Manuel Benito1,*

Departamento de Bioquímica y Biología Molecular, Centro Mixto CSIC/UCM, Facultad de Farmacia,1 and Centro de Citometría de Flujo y Microscopía Confocal,3 Universidad Complutense, 28040 Madrid, Spain, and Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts2

Received 13 April 2000/Returned for modification 18 October 2000/Accepted 3 January 2001

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.

* Corresponding author. Mailing address: Departamento de Bioquímica y Biología Molecular, Centro Mixto CSIC/UCM, Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain. Phone: 34-91-3941777. Fax: 34-91-3941779. E-mail: benito{at}eucmax.sim.ucm.es.

dagger Present address: Instituto de Neurobiología Cajal, Consejo Superior de Investigaciones Científicas, 28002 Madrid, Spain.

Molecular and Cellular Biology, April 2001, p. 2269-2280, Vol. 21, No. 7
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.7.2269-2280.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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