Positive and Negative Roles of p85α and p85β Regulatory Subunits of Phosphoinositide 3-Kinase in Insulin Signaling

Kohjiro Ueki,David A Fruman,Claudine M Yballe,Mathias Fasshauer,Johannes Klein,Tomoichiro Asano,Lewis C Cantley,C Ronald Kahn,Carl Ronald Kahn

doi:10.1074/jbc.m305602200

Kohjiro Ueki, David A Fruman + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m305602200

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Abstract

Class IA phosphoinositide (PI) 3-kinase is composed of a p110 catalytic subunit and a p85 regulatory subunit and plays a pivotal role in insulin signaling. To explore the physiological roles of two major regulatory isoforms, p85 alpha and p85 beta, we have established brown adipose cell lines with disruption of the Pik3r1 or Pik3r2 gene. Pik3r1-/- (p85 alpha-/-) cells show a 70% reduction of p85 protein and a parallel reduction of p110. These cells have a 50% decrease in PI 3-kinase activity and a 30% decrease in Akt activity, leading to decreased insulin-induced glucose uptake and anti-apoptosis. Pik3r2-/- (p85 beta-/-) cells show a 25% reduction of p85 protein but normal levels of p85-p110 and PI 3-kinase activity, supporting the fact that p85 is more abundant than p110 in wild type. p85 beta-/- cells, however, exhibit significantly increased insulin-induced Akt activation, leading to increased anti-apoptosis. Reconstitution experiments suggest that the discrepancy between PI 3-kinase activity and Akt activity is at least in part due to the p85-dependent negative regulation of downstream signaling of PI 3-kinase. Indeed, both p85 alpha-/- cells and p85 beta-/- cells exhibit significantly increased insulin-induced glycogen synthase activation. p85 alpha-/- cells show decreased insulin-stimulated Jun N-terminal kinase activity, which is restored by expression of p85 alpha, p85 beta, or a p85 mutant that does not bind to p110, indicating the existence of p85-dependent, but PI 3-kinase-independent, signaling pathway. Furthermore, a reduction of p85 beta specifically increases insulin receptor substrate-2 phosphorylation. Thus, p85 alpha and p85 beta modulate PI 3-kinase-dependent signaling by multiple mechanisms and transmit signals independent of PI 3-kinase activation.

Highlights

Class IA phosphoinositide (PI) 3-kinase is composed of actions regulated by insulin [1, 2]
We have shown that in the Pik3r1 heterozygous knockout fibroblasts there is enhanced insulin-like growth factor (IGF)-1 action and a significant increase in PIP3 level despite no increase in PI 3-kinase activity [9], suggesting that the regulatory subunit can down-regulate IGF-1/insulin signaling by promoting clearance of PIP3
Through the use of pharmacological inhibitors and dominant negative mutants, PI 3-kinase has been shown to be required for a wide variety of the metabolic effects of insulin, including stimulation of glucose transport and glycogen synthesis [1, 28, 42,43,44]

Summary

Introduction

Class IA phosphoinositide (PI) 3-kinase is composed of actions regulated by insulin [1, 2]. Effects of Disruption of the Pik3r1 and Pik3r2 Genes on Differentiation of Adipocytes—To assess roles of the regulatory subunits of PI 3-kinase, we established brown preadipocyte cell lines from Pik3r1Ϫ/Ϫ (p85␣Ϫ/Ϫ), Pik3r1ϩ/Ϫ (p85␣ϩ/Ϫ), and Pik3r2Ϫ/Ϫ (p85␤Ϫ/Ϫ) neonatal mice as well as the wild-type control mice. In p85␤Ϫ/Ϫ cells, deletion of the p85␤ isoform did not appear to decrease in either p110 catalytic subunit proteins or the p851⁄7p110 complexes, maintaining PI 3-kinase activity associated with the p110␣ or p110␤ isoform (Fig. 2d).

Results

Conclusion