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Cloning, characterization, and expression of a novel Zn2+-binding FYVE finger-containing phosphoinositide kinase in
A Shisheva1, D Sbrissa, O Ikonomov
1Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Abstract:
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.
Insights
Researchers identified a novel mouse protein, p235, involved in phosphatidylinositol signaling. This protein functions as a phosphoinositide kinase and is targeted to specific cellular locations via a zinc-dependent mechanism.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Phosphatidylinositol (PI) signaling regulates crucial cellular functions in eukaryotes.
- Identifying novel proteins involved in PI metabolism is key to understanding cellular regulation.
Purpose of the Study:
- To identify and characterize novel mammalian proteins involved in phosphatidylinositol signaling.
- To elucidate the function and localization mechanism of the newly discovered p235 protein.
Main Methods:
- Differential display screening of mouse adipocyte cDNA libraries.
- Full-length cDNA cloning and amino acid sequence analysis of p235.
- Antibody generation for protein detection and immunofluorescence microscopy.
- Biochemical assays to determine lipid kinase activity and Zn2+ binding.
Main Results:
- A novel mouse protein, p235, was identified, featuring a FYVE finger, chaperonin-like region, and phosphoinositide 5-kinase consensus.
- p235 mRNA is enriched in insulin-sensitive tissues.
- Immunofluorescence revealed a punctate peripheral localization pattern for p235, dependent on the N-terminal FYVE finger and Zn2+.
- p235 exhibits unique phosphoinositide kinase activity, preferring PI substrates.
Conclusions:
- Mouse p235 represents a novel class of phosphoinositide kinases.
- The protein's intracellular targeting is mediated by a zinc-dependent mechanism involving its FYVE finger.