Characterization of recombinant phosphatidylinositol 4-kinase beta revealsauto- and heterophosphorylation of the enzyme

Phosphatidylinositol (PI) 4-kinases catalyze the synthesis of PI 4-phosphat e, an important intermediate for the synthesis of membrane polyphosphoinosi tides, regulators of multiple cellular functions. Two mammalian PI 4-kinase s have been cloned, a 230-kDa enzyme (alpha-form) and a 110-kDa (beta-form) , both of which are inhibited by >0.1 mu M concentrations of the PI 3-kinas e inhibitor, wortmannin (WT), In the present study, we created a glutathion e S-transferase-PI4K beta fusion protein for expression in Escherichia coil . 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 PI4K beta, In addition to its lipid kinas e activity, the enzyme exhibited autophosphorylation that was enhanced by M n2+ 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 interaction s. Autophosphorylation inhibited subsequent lipid kinase activity, which wa s reversed upon dephosphorylation, by protein phosphatases, PPI and PP2A(1) , suggesting that it may represent a regulatory mechanism for the enzyme. P hosphorylation of endogenous or overexpressed PI4K beta was also observed i n COS-7 cells; how ever, the in vivo phosphorylation of the expressed prote in was only partially inhibited by WT and also occurred in a catalytically inactive form of the enzyme, indicating the presence of additional phosphor ylation site(s). Successful bacterial expression of PI4K beta should aid re search on the structure-function relationships of this protein as well as o f other, structurally related enzymes.