D. Sbrissa et al., PIKfyve lipid kinase is a protein kinase: Downregulation of 5 '-phosphoinositide product formation by autophosphorylation, BIOCHEM, 39(51), 2000, pp. 15980-15989
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.