The Pleckstrin Homology Domain in Insulin Receptor Substrate-1 Sensitizes Insulin Signaling

doi:10.1074/jbc.270.20.11715

The Pleckstrin Homology Domain in Insulin Receptor Substrate-1 Sensitizes Insulin Signaling (*)

From the (1) Research Division, Joslin Diabetes Center and Division of Medical Sciences and the
(2) Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02215 and the
(3) Laboratory of Cell and Molecular Biology, National Institutes of Health, Bethesda, Maryland 20892

^§§To whom correspondence should be addressed:
Research Division, 1 Joslin Pl., Boston, MA 02215.
Tel.: 617-732-2578; Fax: 617-732-2593; E-mail: whitemor{at}joslab.harvard.edu.

Abstract

The NH₂ terminus of insulin receptor substrate-1 (IRS-1) contains a pleckstrin homology (PH) domain. We deleted the PH domain in IRS-1 (IRS-1) and expressed the mutant in Chinese hamster ovary and 32D cells. During insulin stimulation, IRS-1 is poorly tyrosine-phosphorylated in CHO cells, but undergoes serine/threonine phosphorylation. Similarly, IRS-1 fails to undergo insulin-stimulated tyrosine phosphorylation in 32D cells, which uncouples the activation of phosphatidylinositol 3′-kinase and p70 from the endogenous insulin receptors. Overexpression of the insulin receptor in 32D cells, however, restores tyrosine phosphorylation of IRS-1 and rescues insulin responses including mitogenesis. Thus, while the PH domain is not required for the engagement of downstream signals, it is one of the elements in the NH₂ terminus of IRS-1 that is needed for a sensitive coupling to insulin receptors, especially at ordinary receptor levels found in most cells and tissues.

Footnotes

↵^§ Supported in part by the Albert J. Ryan Foundation at Harvard Medical School and National Institutes of Health National Research Service Award Training Grant DK 07260.
↵^¶ Established Investigator of the American Heart Association and a recipient of Harvard Medical School Funds for Discovery.
↵** Fellow of the Juvenile Diabetes Foundation.
↵* This work was supported in part by National Institutes of Health Grants DK 38712, DK 43808 (to M. F. W.), and CA 46595 (to J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

IRS-1

insulin receptor substrate-1

SH2

Src homology-2

PI

phosphatidylinositol

CHO

Chinese hamster ovary

IL

interleukin

PAGE

polyacrylamide gel electrophoresis

IH

IRS homology region

PH

pleckstrin homology region.
↵²X. J. Sun, L. M. Wang, Y. Zhang, L. Yenush, M. G. Myers, Jr., E. Glasheen, S. Pons, G. Wolf, S. E. Shoelson, W. S. Lane, J. H. Pierce, and M. F. White, submitted for publication.
³M. G. Myers, Jr., Y. Zhang, E. Glasheen, T. Grammer, L. Yenush, L. M. Wang, X. J. Sun, J. Blenis, J. H. Pierce, and M. F. White, manuscript in preparation.
↵⁴M. G. Myers, Jr., J. H. Pierce, and M. F. White, unpublished data.