Different inhibition of G beta gamma-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a G beta gamma-dependent regulator of PI3K gamma enzymatic activity

摘要

Class IB phosphoinositide 3-kinases gamma (PI3K gamma) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3K gamma variants have one catalytic p110 gamma subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110 gamma and p101-p110 gamma allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110 gamma monoclonal antibody [mAb(A)(p110 gamma)] to block PI3K gamma enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110 gamma interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110 gamma, which was accompanied by conformational changes in the helical domain harbouring the G beta gamma-binding site. We then studied the modulation of phospholipid vesicles association of PI3K gamma by the antibody. p87-p110 gamma showed a significantly reduced G beta gamma-mediated phospholipid recruitment as compared with p101-p110 gamma. Concomitantly, in the presence of mAb(A)(p110 gamma), G beta gamma did not bind to p87-p110 gamma. These data correlated with the ability of the antibody to block G beta gamma-stimulated lipid kinase activity of p87-p110 gamma 30-fold more potently than p101-p110 gamma. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific G beta gamma-dependent regulation of p101 in PI3K gamma activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3K gamma variants downstream of GPCRs.