New insights into the allosteric regulation of Arabidopsis PI4Kβ1 and effects of phosphorylation on protein function

Abstract

Phosphatidylinositol 4-kinase β1 (PI4Kβ1) is a key enzyme in plant phosphoinositide biosynthesis and mediates the ATP-dependent conversion of phosphatidylinositol (PI) to PI 4-phosphate (PI4P). The aim of this study was to describe the biochemical properties of PI4Kβ1, to clarify its orientation towards the membrane, and to characterize the effects of previously described phosphorylation events. In vitro activity tests with purified MBP-PI4Kβ1 protein showed a sigmoidal kinetic profile with increasing concentration of the substrate PI, indicating allosteric regulation of the enzyme. For the cosubstrate ATP, on the other hand, a hyperbolic kinetic behavior of PI4Kβ1 was determined. Using a 3D model, a C-terminal helix of the protein may interact with membrane lipids. The influence of this helix on the kinetic behavior of PI4Kβ1 as well as that of various MAP-kinase-mediated phosphorylation sites is also described.