MOLECULAR-CLONING AND EXPRESSION OF A RAT HEPATIC MULTIPLE INOSITOL POLYPHOSPHATE PHOSPHATASE

The characterization of the multiple inositol polyphosphate phosphatas e (MIPP) is fundamental to our understanding of how cells control the signalling activities of 'higher' inositol polyphosphates. We now desc ribe our isolation of a 2.3 kb cDNA clone of a rat hepatic form of MIP P. The predicted amino acid sequence of MIPP includes an 18 amino acid region that aligned with approximately 60% identity with the catalyti c domain of a fungal inositol hexakisphosphate phosphatase (phytase A) ; the similarity encompassed conservation of the RHGXRXP signature of the histidine acid phosphatase family. A histidine-tagged, truncated f orm of MIPP was expressed in Escherichia coli and the enzymic specific ity of the recombinant protein was characterized: Ins(1,3,4,5,6)P-5 wa s hydrolysed, first to Ins(1,4,5,6)P-4 and then to Ins(1,4,5)P-3, by c onsecutive 3- and 6-phosphatase activities. Inositol hexakisphosphate was catabolized without specificity towards a particular phosphate gro up, but in contrast, MIPP only removed the beta-phosphate from the 5-d iphosphate group of diphosphoinositol pentakisphosphate. These data, w hich are consistent with the substrate specificities of native (but no t homogeneous) MIPP isolated from rat liver, provide the first demonst ration that a single enzyme is responsible for this diverse range of s pecific catalytic activities. A 2.5 kb transcript of MIPP mRNA was pre sent in all rat tissues that were examined, but was most highly expres sed in kidney and liver. The predicted C-terminus of MIPP is comprised of the tetrapeptide SDEL, which is considered a signal for retaining soluble proteins in the lumen of the endoplasmic reticulum; the presen ce of this sequence provides a molecular explanation for our earlier b iochemical demonstration that the endoplasmic reticulum contains subst antial MIPP activity [Ali, Craxton and Shears (1993) J. Biol. Chem. 26 8, 6161-6167].