PIKfyve lipid kinase is a protein kinase: Downregulation of 5 '-phosphoinositide product formation by autophosphorylation

A subset of phosphoinositide 3-kinase family members are dual specificity e nzymes; their protein kinase activity is thought to bring about an addition al level to their intracellular regulation. Here we have examined whether t he 5'-phosphoinositide kinase PIKfyve, reported previously to catalyze the formation of PtdIns 5-P and PtdIns 3,5-P-2 in vitro [Sbrissa et al. (1999) J. Biol. Chem. 274, 21589-21597], displays dual specificity. We now report that PIKfyve possesses an intrinsic protein kinase activity inseparable fro m its lipid kinase activity and, besides itself, can phosphorylate exogenou s proteins in a substrate-specific manner. Both the autophosphorylation and transphosphorylation were demonstrated with PIKfyve immunopurified or affi nity-purified from heterologously transfected COS cells, infected Sf9 cells , or native 3T3-L1 adipocytes. Conversely, no protein kinase activity was a ssociated with immunopurified lipid kinase dead point (K1831E) or truncated (Delta 1812-2052) PIKfyve mutants. PIKfyve autophosphorylation or transpho sphorylation engaged Ser but not Thr or Tyr residues. PIKfyve autophosphory lation was largely abrogated upon pretreatment with PIKfyve lipid substrate s or phosphatases. The impact of autophosphorylation on the PIKfyve lipid k inase activity was further examined with purified PIKfyve preparations. A d ecrease of 70% in the lipid product formation was associated with PIKfyve a utophosphorylation, which was reversed upon treatment with phosphatases. In the cellular context, PIKfyve, or a fraction of it, was found in a phospho rylated form. Collectively, these results indicate that PIKfyve is a dual s pecificity kinase, which can generate and relay protein phosphorylation sig nals to regulate the formation of its lipid products, and possibly other ev ents, in the context of living cells.