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J Biol Chem, Vol. 275, Issue 19, 14642-14648, May 12, 2000

Characterization of Recombinant Phosphatidylinositol 4-Kinase  Reveals Auto- and Heterophosphorylation of the Enzyme^*

Xiao-Hang Zhao, Tzvetanka Bondeva, and Tamas Balla

From the Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892

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 (-form) and a 110-kDa (-form), both of which are inhibited by >0.1 µM concentrations of the PI 3-kinase inhibitor, wortmannin (WT). In the present study, we created a glutathione S-transferase-PI4K 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 PI4K. In addition to its lipid kinase activity, the enzyme exhibited autophosphorylation that was enhanced by Mn²⁺ 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 PP2A₁, suggesting that it may represent a regulatory mechanism for the enzyme. Phosphorylation of endogenous or overexpressed PI4K 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 PI4K should aid research on the structure-function relationships of this protein as well as of other, structurally related enzymes.

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