A. Shisheva et al., Cloning, characterization, and expression of a novel Zn2+-binding FYVE finger-containing phospboinositide kinase in insulin-sensitive cells, MOL CELL B, 19(1), 1999, pp. 623-634
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.