Functional expression and characterisation of a new human phosphatidylinositol 4-kinase PI4K230

By constructing DNA probes we have identified and cloned a human PtdIns 4-k inase, PI4K230, corresponding to a mRNA of 7.0 kb. The cDNA encodes a prote in of 2044 amino acids. The C-terminal part of ca. 260 amino acids represen ts the catalytic domain which is highly conserved in all recently cloned Pt dIns 4-kinases. N-terminal motifs indicate multiple heterologous protein in teractions. Human PtdIns 4-kinase PI4K230 expressed in vitro exhibits a spe cific activity of 58 mu mol mg(-1) min(-1). The enzyme expressed in Sf9 cel ls is essentially not inhibited by adenosine, it shows a high K-m, for ATP of about 300 mu M and it is half-maximally inactivated by approximately 200 nM wormannin. These data classify this enzyme as type 3 PtdIns 4-kinase. A ntibodies raised against the N-terminal part moderately activate and those raised against the C-terminal catalytic domain inhibit the enzymatic activi ty. The coexistence of two different type 3 PtdIns 4-kinases, PI4K92 and PI 4K230, in several human tissues, including brain, suggests that these enzym es are involved in distinct basic cellular functions. (C) 1999 Elsevier Sci ence B.V. All rights reserved.