PIKfyve, a Mammalian Ortholog of Yeast Fab1p Lipid Kinase, Synthesizes 5-Phosphoinositides

doi:10.1074/jbc.274.31.21589

PIKfyve, a Mammalian Ortholog of Yeast Fab1p Lipid Kinase, Synthesizes 5-Phosphoinositides

EFFECT OF INSULIN*

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

Abstract

One or more free hydroxyls of the phosphatidylinositol (PtdIns) head group undergo enzymatic phosphorylation, yielding phosphoinositides (PIs) with key functions in eukaryotic cellular regulation. Two such species, PtdIns 5-P and PtdIns 3,5-P₂, have now been identified in mammalian cells, but their biosynthesis remains unclear. We have isolated a novel mammalian PI kinase, p235, whose exact substrate specificity remained to be determined (Shisheva, A., Sbrissa, D., and Ikonomov, O. (1999)Mol. Cell. Biol. 19, 623–634). Here we report that recombinant p235 expressed in COS cells, like the authentic p235 in adipocytes, displays striking specificity for PtdIns over PI substrates and generates two products identified as PtdIns 5-P and PtdIns 3,5-P₂ by HPLC analyses. Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P₂ but to a substantially lesser extent than PtdIns isolated from natural sources. Important properties of the p235 PI 5-kinase include high sensitivity to nonionic detergents and relative resistance to wortmannin and adenosine. By analyzing deletion mutants in a heterologous cell system, we determined that in addition to the predicted catalytic domain other regions of the molecule are critical for the p235 enzymatic activity. HPLC resolution of monophosphoinositide products, generated by p235 immune complexes derived from lysates of 3T3-L1 adipocytes acutely stimulated with insulin, revealed essentially the same PtdIns 5-P levels as the corresponding p235 immune complexes of resting cells. However, the acute insulin action resulted in an increase of a wortmannin-sensitive PtdIns 3-P peak, suggestive of a plausible recruitment of wortmannin-sensitive PI 3-kinase(s) to p235. In conclusion, mouse p235 (renamed here PIKfyve) displays a strong in vitro activity for PtdIns 5-P and PtdIns 3,5-P₂ generation, implying PIKfyve has a key role in their biosynthesis.

Footnotes

↵* This work was supported by the American Diabetes Association Career Development Award (to A. S.), Juvenile Diabetes Foundation International Research Grant 1-1999-40 (to A. S.), and Morris Hood Jr. Comprehensive Diabetes Center, Michigan (to A. S.). Parts of this study have been presented at the 1st International Motor City Diabetes Symposium, Detroit, MI, October 1998.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Present address: Dept. of Psychiatry, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201.
↵§ To whom correspondence and reprint requests should be addressed: Dept. of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. Tel.: 313-577-5674; Fax: 313-577-5494; E-mail: ashishev@moose.med.wayne.edu.
Abbreviations:

PtdIns

phosphatidylinositol

PI

phosphoinositide

P

phosphate

P₂

bisphosphate

P₃

trisphosphate

GroPIns

glycerophosphorylinositol

K

kinase

HPLC

high pressure liquid chromatography

GST

glutathione S- transferase

HA

hemagluttinin
- Received March 25, 1999.
- Revision received May 12, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.