Mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 6-kinase PIKfyve

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.