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Molecular and Cellular Biology, April 2001, p. 2269-2280, Vol. 21, No. 7
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.
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

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