Cloning, and characterization of a 72-kDa inositol-polyphosphate 5-phosphatase localized to the Golgi network

The inositol-polyphosphate B-phosphatase enzyme family removes the B-positi on phosphate from both inositol phosphate and phosphoinositide signaling mo lecules. We have cloned and characterized a novel B-phosphatase, which demo nstrates a restricted substrate specificity and tissue expression. The 3.9- kb cDNA predicts for a 72-kDa protein with an N-terminal proline rich domai n, a central B-phosphatase domain, and a C-terminal CAAX motif. The 3.9-kil obase mRNA showed a restricted expression but was abundant in testis and br ain. Antibodies against the sequence detected a 72-kDa protein in the testi s in the detergent-insoluble fraction. Indirect immunofluorescence of the T era-1 cell line using anti-peptide antibodies to the 72-kDa 5-phosphatase d emonstrated that the enzyme is predominantly located to the Golgi. Expressi on of green fluorescent protein-tagged 72-kDa 5-phosphatase in COS-7 cells revealed that the enzyme localized predominantly to the Golgi, mediated by the N-terminal proline-rich domain, but not the C-terminal CAAX motif. In v itro, the protein inserted into microsomal membranes on the cytoplasmic fac e of the membrane. Immunoprecipitated recombinant 72-kDa B-phosphatase hydr olyzed phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3, 5-bisphosphate, forming phosphatidylinositol 3,4-bisphosphate and phosphati dylinositol S-phosphate, respectively. We propose that the novel 5-phosphat ase hydrolyzes phosphatidylinositol 3,4,5-trisphosphate and phosphatidylino sitol 3,5-bisphosphate on the cytoplasmic Golgi membrane and thereby may re gulate Golgi-vesicular trafficking.