Mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 5-kinase PIKfyve

Abstract

The dual specificity mammalian enzyme PIKfyve phosphorylates in vitro position D-5 in phosphatidylinositol (PtdIns) and PtdIns 3-P, itself or exogenous protein substrates. Here we have addressed the crucial questions for the identity of the lipid products and the role of PIKfyve enzymatic activity in mammalian cells. CHO, HEK293, and COS cells expressing PIKfyveWT at high levels and >90% efficiencies increased selectively the intracellular PtdIns 3,5-P2 production by 30-55%. In these cell types PtdIns 5-P was undetectable. A kinase-deficient point mutant, PIKfyveK1831E, transiently transfected into these or other cells elicited a dramatic dominant phenotype. Subsequent to a dilation of the PIKfyve-containing vesicles, PIKfyveK1831E-expressing cells progressively accumulated multiple swollen lucent vacuoles of endosomal origin, first in the perinuclear cytoplasm and then toward the cell periphery. Despite their drastically altered morphology, the PIKfyveK1831E-expressing cells were viable and functionally active, evidenced by several criteria. This phenotype was completely reversed by introducing PIKfyveWT into the PIKfyveK1831E-transfected cells. Disruptions of the localization signal in the PIKfyve kinase-deficient mutant yielded a PIKfyveK1831EΔfyve protein, incompetent to vacuolate cells, implying that an active PIKfyve enzyme at distinct late endocytic membranes is crucial for normal cell morphology. This was further substantiated by examining the vacuolation-induced potency of several pharmacological stimuli in cells expressing high PDXfyveWT levels. Together, the results indicate that PIKfyve enzymatic activity, possibly through the generation of PtdIns 3,5-P2, and/or yet to be identified endogenous phosphoproteins, is critical for cell morphology and endomembrane homeostasis. © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.