Characterization of recombinant phosphatidylinositol 4-kinase beta reveals auto- and heterophosphorylation of the enzyme.

Phosphatidylinositol (PI) 4-kinases catalyze the synthesis of PI 4-phosphate, an important intermediate for the synthesis of membrane polyphosphoinositides, regulators of multiple cellular functions. Two mammalian PI 4-kinases have been cloned, a 230-kDa enzyme (alpha-form) and a 110-kDa (beta-form), both of which are inhibited by >0.1 microm concentrations of the PI 3-kinase inhibitor, wortmannin (WT). In the present study, we created a glutathione S-transferase-PI4Kbeta fusion protein for expression in Escherichia coli. The purified protein was biologically active and phosphorylated PI in its 4-position with WT sensitivity and kinetic parameters that were identical to those of purified bovine brain PI4Kbeta. In addition to its lipid kinase activity, the enzyme exhibited autophosphorylation that was enhanced by Mn(2+) ions and inhibited by WT and another PI 3-kinase inhibitor, LY 294002. The recombinant protein was unable to transphosphorylate, but its isolated C-terminal catalytic domain still displayed autophosphorylation, suggesting that the autophosphorylation site resides within the C-terminal catalytic domain of the protein and is held in position by intramolecular interactions. Autophosphorylation inhibited subsequent lipid kinase activity, which was reversed upon dephosphorylation, by protein phosphatases, PP1 and PP2A(1), suggesting that it may represent a regulatory mechanism for the enzyme. Phosphorylation of endogenous or overexpressed PI4Kbeta was also observed in COS-7 cells; however, the in vivo phosphorylation of the expressed protein was only partially inhibited by WT and also occurred in a catalytically inactive form of the enzyme, indicating the presence of additional phosphorylation site(s). Successful bacterial expression of PI4Kbeta should aid research on the structure-function relationships of this protein as well as of other, structurally related enzymes.