Reconstitution of Phosphoinositide 3-Kinase-dependent Insulin Signaling in a Cell-free System

Haruhiko Murata,Richard C Hresko,Mike Mueckler

doi:10.1074/jbc.m302934200

Abstract

Early insulin signaling events were examined in a novel cell-free assay utilizing subcellular fractions derived from 3T3-L1 adipocytes. The following cellular processes were observed in vitro in a manner dependent on insulin, time of incubation, and exogenous ATP: 1) autophosphorylation and activation of the insulin receptor; 2) tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1); 3) association of tyrosine-phosphorylated IRS-1 with phosphoinositide 3-kinase; 4) activation of the kinase Akt via its phosphorylation on Thr-308 and Ser-473; and 5) phosphorylation of glycogen synthase kinase-3 by activated Akt. The activation of Akt in vitro was abolished in the presence of the phosphoinositide 3-kinase inhibitor, wortmannin, thus recapitulating the most notable regulatory feature of Akt observed in vivo. Evidence is presented indicating that the critical spatial compartmentalization of signaling molecules necessary for efficient signal transduction is likely to be preserved in the cell-free system. Additionally, data are provided demonstrating that full Akt activation in this system is dependent on plasma membrane-associated IRS-1, cannot be mediated by robust cytosol-specific tyrosine phosphorylation of IRS-1, and occurs in the complete absence of detectable IRS-2 phosphorylation in the cytosol and plasma membrane.

Highlights

Insulin initiates multiple signaling pathways leading to numerous responses that regulate carbohydrate, fat, and protein metabolism [1]
Jarett and co-workers noted that the direct addition of insulin to a purified adipocyte plasma membrane fraction resulted in numerous effects, including alterations in the phosphorylation of several proteins [6] and increased calcium binding by the plasma membrane [7]
Characterization of the Cell-free System—We have attempted to reconstitute key aspects of the insulin-signaling pathway using subcellular fractions derived from 3T3-L1 adipocytes

Summary

Introduction

Insulin initiates multiple signaling pathways leading to numerous responses that regulate carbohydrate, fat, and protein metabolism [1]. Insulin-stimulated Akt Activation Does Not Require Cytosolic IRS Proteins in the Cell-free Assay—The preceding data demonstrate that early insulin signaling events dependent on PI 3-kinase up to and including Akt and GSK-3 phosphorylation appear to be faithfully reconstituted in the in vitro system.

Results

Conclusion