The dual specificity mammalian enzyme PIKfyve phosphorylates in vitro posit
ion D-5 in phosphatidylinositol (PtdIns) and PtdIns 3-P, itself or exogenou
s protein substrates, Here we have addressed the crucial questions for the
identity of the lipid products and the role of PIKfyve enzymatic activity i
n mammalian cells. CHO, HEK293, and COS cells expressing PIKfyve(WT) at hig
h levels and > 90% efficiencies increased selectively the intracellular Ptd
Ins 3,5-P-2 production by 30-55%. In these cell types PtdIns 5-P was undete
ctable. A kinase-deficient point mutant, pIKfyve(K1831E), transiently trans
fected into these or other cells elicited a dramatic dominant phenotype, Su
bsequent to a dilation of the PIKfyve-containing vesicles, PIKfyve(K1831E)-
expressing cells progressively accumulated multiple swollen lucent vacuoles
of endosomal origin, first in the perinuclear cytoplasm and then toward th
e cell periphery. Despite their drastically altered morphology, the PIKfyve
(K1831E)-expressing cells were viable and functionally active, evidenced by
several criteria. This phenotype was completely reversed by introducing PI
KfyveW(T) into the PIKfyve(K1831E)-transfected cells. Disruptions of the lo
calization signal in the PIKfyve kinase-deficient mutant yielded a pIKfyve(
K1831E Delta fyve) protein, incompetent to vacuolate cells, implying that a
n active PIKfyve enzyme at distinct late endocytic membranes is crucial for
normal cell morphology, This was further substantiated by examining the va
cuolation-induced potency of several pharmacological stimuli in cells expre
ssing high PIKfyve(WT) levels. Together, the results indicate that PIKfyve
enzymatic activity, possibly through the generation of PtdIns 3,5-P-2, and/
or yet to be identified endogenous phosphoproteins, is critical for cell mo
rphology and endomembrane homeostasis.