In B cells Phosphatidylinositol 5-phosphate 4-kinase $\alpha$ synthesizes PI(4,5)P$_2$ to impact on mTORC2 and Akt signalling

Abstract

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases whose physiological functions are incompletely understood, not least because genetic deletion and cell transfection have led to contradictory data. Here we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4K$\alpha$ was removed either stably by genetic deletion, or transiently (within one hour) by tagging the endogenous protein genomically with the auxin degron. In both cases removal impacted upon Akt phosphorylation, and by leaving one PI5P4K$\alpha$ allele present but mutating it to be kinase-dead or to have PI4P 5-kinase activity we show that all the effects on Akt phosphorylation were dependent upon the ability of PI5P4K$\alpha$ to synthesise PI(4,5)P$_2$ rather than to remove PI5P. Whilst stable removal of PI5P4K$\alpha$ resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, due in part to reduced plasma membrane PIP$_3$, its acute removal led to an increase in Akt phosphorylation only at Ser473. This invokes activation primarily of mTORC2, which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4K$\alpha$ as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P$_2$ and as a novel regulator of mTORC2. They also show a phenomenon similar to the ‘butterfly effect’ described for PI 3-kinase I$\alpha$ (Hart JR et al PNAS 112 1131-1136; 2015) whereby via apparently the same underlying mechanism the removal of a protein’s activity from a cell can have widely divergent effects, depending upon the time course of that removal.