Cloning, characterization, and expression of a novel Zn2+-binding FYVE finger-containing phospboinositide kinase in insulin-sensitive cells

Signaling by phosphorylated species of phosphatidylinositol (PI) appears to regulate diverse responses in eukaryotic cells. A differential display scr een for fat- and muscle-specific transcripts led to identification and clon ing 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 ac id sequence revealed that p235 contains an N-terminal zinc-binding FYVE fin ger, a chaperonin-like region in the middle of the molecule, and a consensu s for phosphoinositide 5-kinases at the C terminus. p235 mRNA appears as a 9-kb transcript, enriched in insulin-sensitive cells and tissues, likely tr anscribed from a single-copy gene in at least two close-in-size splice vari ants. Specific antibodies against mouse p235 were raised, and bath the endo genously and heterologously expressed proteins were biochemically detected in 3T3-L1 adipocytes and transfected COS cells, respectively. Immunofluores cence microscopy analysis of endogenous p235 localization in 3T3-L1 adipocy tes with affinity-purified anti-p235 antibodies documented a punctate perip heral pattern. In COS cells, the expressed p235 N-terminal but not the C-te rminal region displayed a vesicular pattern similar to that in 3T3-L1 adipo cytes 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+. Determinat ion of the lipid kinase activity in the p235 immunoprecipitates derived fro m 3T3-L1 adipocytes or from COS cells transiently expressing p235 revealed that p235 displayed unique preferences for PI substrate over already phosph orylated PI. In conclusion, the mouse p235 protein determines an important novel class of phosphoinositide kinases that seems to be targeted to specif ic intracellular loci by a Zn-dependent mechanism.