PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides - Effect of insulin

One or more free hydroxyls of the phosphatidylinositol (PtdIns) head group undergo enzymatic phosphorylation, yielding phosphoinositides (PIs) with ke y functions in eukaryotic cellular regulation. Two such species, PtdIns 5-P and PtdIns 3,5-P-2, have now been identified in mammalian cells, but their biosynthesis remains unclear. We have isolated a novel mammalian PI kinase , p235, whose exact substrate specificity remained to be determined (Shishe va, A., Sbrissa, D,, and Ikonomov, O. (1999) Mol. Cell. Biol. 19, 623-634). Here we report that recombinant p235 expressed in COS cells, like the auth entic p235 in adipocytes, displays striking specificity for PtdIns over PI substrates and generates two products identified as PtdIns 5-P and PtdIns 3 ,5-P, by HPLC analyses. Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P, but to a substantially lesser extent than PtdIns isolated from natural sources. Important properties of the p235 PI 5-kinase include high sensitivity to nonionic detergents and relative resistance to wortman nin and adenosine. By analyzing deletion mutants in a heterologous cell sys tem, we determined that in addition to the predicted catalytic domain other regions of the molecule are critical for the p235 enzymatic activity. HPLC resolution of monophosphoinositide products, generated by p235 immune comp lexes derived from lysates of 3T3-L1 adipocytes acutely stimulated with ins ulin, revealed essentially the same PtdIns 5-P levels as the corresponding p235 immune complexes of resting cells. However, the acute insulin action r esulted in an increase of a wortmannin-sensitive PtdIns 3-P peak, suggestiv e of a plausible recruitment of wortmannin-sensitive PI 3-kinase(s) to p235 . In conclusion, mouse p235 (renamed here PIKfyve) displays a strong in vit ro activity for PtdIns 5-P and PtdIns 3,5-P, generation, implying PIKfyve h as a key role in their biosynthesis.