Characterizing the Interactions between the Two Subunits of the p101/p110γ Phosphoinositide 3-Kinase and Their Role in the Activation of This Enzyme by Gβγ Subunits

Sonja Krugmann,Phillip T Hawkins,Nancy Pryer,Sylvia Braselmann

doi:10.1074/jbc.274.24.17152

Sonja Krugmann, Phillip T Hawkins + Show 2 more

Open Access

https://doi.org/10.1074/jbc.274.24.17152

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Journal: Journal of Biological Chemistry	Publication Date: Jun 1, 1999
Citations: 91	License type: cc-by

Affiliation: Babraham Institute

Abstract

Recently, we have reported the purification and cloning of a novel G protein betagamma subunit-activated phosphoinositide 3-kinase from pig neutrophils. The enzyme comprises a p110gamma catalytic subunit and a p101 regulatory subunit. Now we have cloned the human ortholog of p101 and generated panels of p101 and p110gamma truncations and deletions and used these in in vitro and in vivo assays to determine the protein domains responsible for subunit interaction and activation by betagamma subunits. Our results suggest large areas of p101 including both N- and C-terminal portions interact with the N-terminal half of p110gamma. While modifications of the N terminus of p110gamma could modulate its intrinsic catalytic activity, binding to the N-terminal region of p101 was found to be indispensable for activation of heterodimers with Gbetagamma.

Highlights

Phosphoinositide 3-kinases (PI 3-kinases)1 are responsible for the phosphorylation of inositol phospholipids in the D-3 position of the inositol ring
Analysis of p101 Structure/Function in Vivo—To address issues such as (a) the possibility that purification and our handling of the p101 constructs had resulted in varying levels of denaturation and that this effect generated the differential binding we observed, and (b) that some binding only occurs in vitro in the absence of competing proteins found in the cell, we examined the ability of p101 derivatives to bind to p110␥ when the proteins are transiently co-expressed in COS-7 cells
Cytosolic fractions of harvested cells were subjected to anti-(EE) immunoprecipitations, half of which were used to estimate binding stoichiometries on gels or blots while the other half was assayed for PI 3-kinase activity in the presence or absence of G protein ␤␥ subunits

Summary

Introduction

Phosphoinositide 3-kinases (PI 3-kinases)1 are responsible for the phosphorylation of inositol phospholipids in the D-3 position of the inositol ring. All p101-p110␥ heterodimers were assayed for PI 3-kinase activity in the presence or absence of G␤␥ (Fig. 1D).

Results

Conclusion