Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant enzyme and determination of multiple phosphorylation sites

Human phosphatidylinositol 4-kinase, isoform PI4K92, was expressed as His6 tagged protein in Sf9 cells reaching a level of approximately 5% of cellular protein. The Enzyme can be purified nearly to homogeneity in a single step by absorption/desorption on Ni/nitriloacetic acid agarose Magnetic Beads. High Km values in the millimolar range for ATP and PtdIns as well as only a moderate inhibition by adenosine and a sensitivity to Wortmannin (IC50 approximately 300 nM) characterize the Enzyme as a type 3 PI4K. The Enzyme produces PtdIns4P as product. The isolated Enzyme is a phosphoprotein, additionally phosphate is incorporated by incubation with ATP/Mg or ATP/Mn. Phosphorylation sites were mapped by MALDI-MS and LC-MS/MS at the following positions: S258, T263, S266, S277, S294, T423, S496, T504. Accordingly, a stretch of 81 Amino acids between the common and the C-terminal catalytic domain was designated phosphorylation domain.