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+-indepen dent 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-phosph atase, Recombinant protein was expressed in Escherichia coli and shown to hydrolyze all three 4-phosphatase substrates with enzymatic proper ties similar to the original enzyme. We designate the original 4-phosp hatase and the new isozyme as inositol polyphosphate 4-phosphatase typ es I and II, respectively. 4-Phosphatase II is highly conserved with t he human and rat enzymes having 90% amino acid identity, A conserved m otif between 4-phosphatase I and II is the sequence CKSAKDRT that cont ains the Cys-Xaa(5)-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 occu rring in the skeletal muscle and heart. In addition, cDNA encoding alt ernatively 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 alte rnatively spliced forms were expressed as recombinant proteins in E. c oli and SF9 insect cells and found to possess no detectable enzymatic activity suggesting that additional factors and/or processing may be r equired for these alternatively spliced isozymes.