Human phosphatidylinositol 4-kinase isoform P14K92 - Expression of the recombinant enzyme and determination of multiple phosphorylation sites

Human phosphatidylinositol 4-kinase, isoform PI4K92, was expressed as His(6 ) tagged protein in Sf9 cells reaching a level of approximately 5% of cellu lar protein. The enzyme can be purified nearly to homogeneity in a single s tep by absorption/desorption on Ni/nitriloacetic acid agarose magnetic bead s. High K-m values in the millimolar range for ATP and PtdIns as well as on ly a moderate inhibition by adenosine and a sensitivity to Wortmannin (IC50 approximate to 300 nM) characterize the enzyme as a type 3 PI4K. The enzym e produces PtdIns4P as product. The isolated enzyme is a phosphoprotein, ad ditionally phosphate is incorporated by incubation with ATP/Mg or ATP/Mn. P hosphorylation sites were mapped by MALDI-MS and LC-MS/MS at the following positions: S258, T263, S266, S277, S294, T423, S496, T504. Accordingly, a s tretch of 81 amino acids between the common and the C-terminal catalytic do main was designated phosphorylation domain.