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Molecular and Cellular Biology, December 2004, p. 10437-10447, Vol. 24, No. 23
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.23.10437-10447.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Mammalian Ortholog of Saccharomyces cerevisiae Vac14 That Associates with and Up-Regulates PIKfyve Phosphoinositide 5-Kinase Activity{dagger}

Diego Sbrissa, Ognian C. Ikonomov, Jana Strakova, Rajeswari Dondapati, Krzysztof Mlak, Robert Deeb, Robert Silver, and Assia Shisheva*

Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan

Received 1 July 2004/ Returned for modification 22 July 2004/ Accepted 16 September 2004

Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P2, produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P2 synthesis and endomembrane homeostasis in mammalian cells.

* Corresponding author. Mailing address: Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. Phone: (313) 577-5674; Fax: (313) 577-5494; E-mail: ashishev{at}med.wayne.edu.

{dagger} Supplemental material for this article may be found at http://mcb.asmusa.org.

Molecular and Cellular Biology, December 2004, p. 10437-10447, Vol. 24, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.23.10437-10447.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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