Oncogenic Mutations of p110α Isoform of PI 3-Kinase Upregulate Its Protein Kinase Activity

Christina M Buchanan,Jackie D Kendall,James M J Dickson,Woo-Jeong Lee,Peter R Shepherd,Mark A Guthridge,Nils Cordes

doi:10.1371/journal.pone.0071337

Abstract

In addition to lipid kinase activity, the class-I PI 3-kinases also function as protein kinases targeting regulatory autophosphorylation sites and exogenous substrates. The latter include a recently identified regulatory phosphorylation of the GM-CSF/IL-3 βc receptor contributing to survival of acute myeloid leukaemia cells. Previous studies suggested differences in the protein kinase activity of the 4 isoforms of class-I PI 3-kinase so we compared the ability of all class-I PI 3-kinases and 2 common oncogenic mutants to autophosphorylate, and to phosphorylate an intracellular fragment of the GM-CSF/IL-3 βc receptor (βic). We find p110α, p110β and p110γ all phosphorylate βic but p110δ is much less effective. The two most common oncogenic mutants of p110α, H1047R and E545K have stronger protein kinase activity than wildtype p110α, both in terms of autophosphorylation and towards βic. Importantly, the lipid kinase activity of the oncogenic mutants is still inhibited by autophosphorylation to a similar extent as wildtype p110α. Previous evidence indicates the protein kinase activity of p110α is Mn2+ dependent, casting doubt over its role in vivo. However, we show that the oncogenic mutants of p110α plus p110β and p110γ all display significant activity in the presence of Mg2+. Furthermore we demonstrate that some small molecule inhibitors of p110α lipid kinase activity (PIK-75 and A66) are equally effective against the protein kinase activity, but other inhibitors (e.g. wortmannin and TGX221) show different patterns of inhibition against the lipid and protein kinases activities. These findings have implications for the function of PI 3-kinase, especially in tumours carrying p110α mutations.

Highlights

The class 1 phosphoinositide 3-kinases (PI 3-kinases) play a critical role in pathways regulating functions such as cell metabolism, cell growth and survival, cytoskeletal rearrangements and cell movement [1,2]
Previous studies using endogenous purified proteins have shown that phosphorylation of class-I PI 3-K reduces the lipid kinase activity of all isoforms except p110c [13,14,15,32,33], more recently a study comparing wildtype and mutant p110a reported that p85a phosphorylation had no impact on lipid kinase activity [34]
Previous studies suggested there would be no change in lipid kinase activity for p110c following autophosphorylation [36,37], and while p110c was less impacted than p110a and b, in our hands it was still reduced to less than 40% activity

Summary

Introduction

The class 1 phosphoinositide 3-kinases (PI 3-kinases) play a critical role in pathways regulating functions such as cell metabolism, cell growth and survival, cytoskeletal rearrangements and cell movement [1,2]. Evidence has been presented that the intrinsic phosphorylation of PI 3-kinase on Ser608 of the regulatory p85a subunit represents a form of negative feedback regulation [14]. This phosphorylation of Ser608 is stimulated by class 1a agonists [14,15] and yet was observed to result in a dosedependent decrease in PI 3-K lipid kinase activity [13,14], an effect which was reversible by treatment with protein phosphatase 2A [13,14] and alkaline phosphatase [13]. Some small molecules have been shown to differentially inhibit the lipid and protein kinase activities of PI 3-kinases [16,17] raising the possibility that some of the new drugs being developed to target PI 3-kinases may do the same

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Conclusion