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Molecular and Cellular Biology, January 1999, p. 623-634, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cloning, Characterization, and Expression of a Novel Zn2+-Binding FYVE Finger-Containing Phosphoinositide Kinase in Insulin-Sensitive Cells

Assia Shisheva,* Diego Sbrissa, and Ognian Ikonomovdagger

Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201

Received 29 July 1998/Returned for modification 18 September 1998/Accepted 19 October 1998

Signaling by phosphorylated species of phosphatidylinositol (PI) appears to regulate diverse responses in eukaryotic cells. A differential display screen for fat- and muscle-specific transcripts led to identification and cloning of the full-length cDNA of a novel mammalian 2,052-amino-acid protein (p235) from a mouse adipocyte cDNA library. Analysis of the deduced amino acid sequence revealed that p235 contains an N-terminal zinc-binding FYVE finger, a chaperonin-like region in the middle of the molecule, and a consensus for phosphoinositide 5-kinases at the C terminus. p235 mRNA appears as a 9-kb transcript, enriched in insulin-sensitive cells and tissues, likely transcribed from a single-copy gene in at least two close-in-size splice variants. Specific antibodies against mouse p235 were raised, and both the endogenously and heterologously expressed proteins were biochemically detected in 3T3-L1 adipocytes and transfected COS cells, respectively. Immunofluorescence microscopy analysis of endogenous p235 localization in 3T3-L1 adipocytes with affinity-purified anti-p235 antibodies documented a punctate peripheral pattern. In COS cells, the expressed p235 N-terminal but not the C-terminal region displayed a vesicular pattern similar to that in 3T3-L1 adipocytes that became diffuse upon Zn2+ chelation or FYVE finger truncation. A recombinant protein comprising the N-terminal but not the C-terminal region of the molecule was found to bind 2.2 mole equivalents of Zn2+. Determination of the lipid kinase activity in the p235 immunoprecipitates derived from 3T3-L1 adipocytes or from COS cells transiently expressing p235 revealed that p235 displayed unique preferences for PI substrate over already phosphorylated PI. In conclusion, the mouse p235 protein determines an important novel class of phosphoinositide kinases that seems to be targeted to specific intracellular loci by a Zn-dependent mechanism.

* 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}moose.med.wayne.edu.

dagger Present address: Department of Psychiatry, Wayne State University School of Medicine, Detroit, MI 48201.

Molecular and Cellular Biology, January 1999, p. 623-634, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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