Tyrosine Phosphorylation of Insulin Receptor Substrate-1 in Vivo Depends upon the Presence of Its Pleckstrin Homology Region

doi:10.1074/jbc.270.30.18083

Tyrosine Phosphorylation of Insulin Receptor Substrate-1 in Vivo Depends upon the Presence of Its Pleckstrin Homology Region (*)

From the (1) Department of Chemical Immunology, the Weizmann Institute of Science, Rehovot 76100, Israel and the
(2) Diabetes Branch, National Institutes of Health, Bethesda, Maryland 20982

^§ Incumbent of the Philip Harris and Gerald Ronson Career Development Chair in Diabetes Research. To whom correspondence should be addressed:
Dept. of Chemical Immunology, Weizmann Institute of Science, P. O. Box 26, Rehovot 76100, Israel
. Fax: 972-8-342-380; E-Mail: Lizick{at}weizmann.weizmann.ac.il.

Abstract

To characterize the structural basis for the interactions between the insulin receptor (IR) and its major substrate, insulin receptor substrate-1 (IRS-1), a segment of the NH₂-terminal region of IRS-1 (Pro⁵-Pro) was deleted. This region contains the first four conserved boxes of a pleckstrin homology (PH) domain, located at the NH₂-terminal part of IRS-1. COS-7 cells were then cotransfected with the genes coding for IR and a wild-type (WT) or a mutated form of IRS-1. IRS-1 underwent significantly reduced insulin-dependent tyrosine phosphorylation compared with WT IRS-1. The reduced in vivo tyrosine phosphorylation of IRS-1 was accompanied by reduced association between IRS-1 and its downstream effector p85 regulatory subunit of phosphatidylinositol-3 kinase. In contrast, both WT IRS-1 and IRS-1 underwent comparable insulin-dependent tyrosine phosphorylation in vitro when incubated with partially purified insulin receptor kinase. These findings suggest that the overall structure of IRS-1 is not altered by deletion of its PH domain and that the PH domain is not the main site for protein-protein interactions between the insulin receptor and IRS-1, at least in vitro. In conclusion, the PH region might facilitate in vivo binding of IRS-1 to membrane phospholipids or other cellular constituents in close proximity to the IR, whereas the actual interactions with the IR are presumably mediated through other domains of the IRS-1 molecule. This could account for the fact that partial deletion of the PH domain selectively impairs the in vivo interactions between the insulin receptor and IRS-1, whereas their in vitro interactions remain unaffected.

Footnotes

↵* 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:

IR

insulin receptor

IRS-1

insulin receptor substrate-1

IRS-1

IRS-1 with a segment of the NH₂-terminal region (Pro⁵-Pro) deleted

PH

pleckstrin homologue

WT

wild-type

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

CHO

Chinese hamster ovary.
- Received May 8, 1995.