The regulation of inositol trisphosphate by inositol polyphosphate 5-phosphatases

Agonist-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) results in the generation of the calcium mobilising intracellular second messenger molecule, inositol 1,4,5 trisphosphate (Ins(l,4,5)P3). The inositol polyphosphate 5-phosphatases (5phosphatase) are an emerging family of signal terminating enzymes that hydrolyse the 5-position phosphate and thereby inactivate Ins(l,4,5)P3. At least two distinct classes of 5-phosphatase enzymes have recently been characterised. The Type I 5-phosphatases are of smaller molecular weight (approximately 43 kDa) and hydrolyse and inactivate only Ins(l,4,5)P3 and inositol 1,3,4,5 tetrakisphosphatc (lns(l,3,4,5)P4) with high affinity. The Type II 5-phosphatase have a larger molecular weight (75-160 kDa) and decreased affinity for both Ins(l,4,5)P3 and Ins(l,3,4,5)P4 compared to the Type I enzymes. In addition the Type II 5-phosphatases hydrolyse polyphosphoinositides including PtdIns(4,5)P2 and/or PtdIns(3,4,5)P3. The recent cloning of several novel 5-phosphatase enzymes has demonstrated that both Type I and II 5-phosphatases contain a conserved, approximately 150 amino acid domain, that may represent the catalytic or substrate binding site We have stably underexpressed the 43 kDa 5-phosphatase using an antiscnse strategy in normal rat kidney (NRK) cells. Cells wilh decreased 43 kDa 5-phosphatase demonstrated a 2-4 fold increase m Ins( 1,4,5)P, and lns( 1,3,4,5)P4 respectively, associated with a 1.9 fold increase in basal intracellular calcium levels. Cells underexpressing the enzyme demonstrated a transformed phenotype and formed tumours in nude mice These studies suggest the regulation of Ins(I,4,5)Pr induced calcium release by the 43 kDa 5-phosphatase is important for the control of cell growth. We have investigated the regulation of the Type II 75 kDa 5-phosphatase with reference to the enzyme's substrate specificity and its tissue and intracellular location. Isolation of several longer cDNA's encoding the enzyme and Northern blot analysis suggest the 75 kDa 5-phosphatase may be regulated by RNA splicing.