The cDNA cloning and characterization of inositol polyphosphate 4-phosphatase type II. Evidence for conserved alternative splicing in the 4-phosphatase family.
Inositol polyphosphate 4-phosphatase (4-phosphatase) is a Mg2+-independent enzyme that catalyzes the hydrolysis of the 4-position phosphate of phosphatidylinositol 3,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 3,4-bisphosphate. We have isolated cDNA encoding a 105,257-Da protein that is 37% identical to the previously cloned 4-phosphatase. Recombinant protein was expressed in Escherichia coli and shown to hydrolyze all three 4-phosphatase substrates with enzymatic properties similar to the original enzyme. We designate the original 4-phosphatase and the new isozyme as inositol polyphosphate 4-phosphatase types I and II, respectively. 4-Phosphatase II is highly conserved with the human and rat enzymes having 90% amino acid identity. A conserved motif between 4-phosphatase I and II is the sequence CKSAKDRT that contains the Cys-Xaa5-Arg active site consensus sequence identified for other Mg2+-independent phosphatases. Northern blot analysis indicated that 4-phosphatase II is widely expressed with the highest levels occurring in the skeletal muscle and heart. In addition, cDNA encoding alternatively spliced forms of human 4-phosphatase I (107, 309 Da) and rat 4-phosphatase II (106,497 Da) were also isolated that encode proteins with a putative transmembrane domain near their C termini. These alternatively spliced forms were expressed as recombinant proteins in E. coli and SF9 insect cells and found to possess no detectable enzymatic activity suggesting that additional factors and/or processing may be required for these alternatively spliced isozymes.
Norris
F A
FA
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Atkins
R C
RC
Majerus
P W
PW
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010744:Phosphoric Monoester Hydrolases; C053153:phosphatidylinositol-3,4-bisphosphate 4-phosphatase