Post-translational modification of human brain type I inositol-1,4,5-trisphosphate 5-phosphatase by farnesylation.

ABSTRACT

In brain, type I inositol-1,4,5-trisphosphate 5-phosphatase (InsP3 5-phosphatase) is the major isoenzyme hydrolyzing the calcium-mobilizing second messenger InsP3. Activity of this enzyme could be measured in both soluble and particulate fractions of tissue homogenates. The protein sequence showed a putative C-terminal isoprenylation site (CVVQ). In this study, two mutants have been generated. The first mutant (C409S) has a serine replacing a cysteine at position 409 of the wild-type enzyme. The second mutant (K407D1) is a deletion mutant that lacks the last five C-terminal amino acids. These constructs were individually expressed by transfection in COS-7 cells. Western blot analysis of wild-type transfected cells indicated that both soluble and particulate fractions had a 43-kDa immunoreactive band, with a higher proportion of the original homogenate associated with the particulate part. On the contrary, when the two mutated constructs were transfected in COS-7 cells, the phosphatase was predominantly soluble. Confocal immunofluorescence studies showed the wild-type enzyme to be present on the cell surface of transfected COS-7 cells and in subcellular compartments around the nucleus. This was not observed for the two mutants, where uniform immunofluorescence labeling was observed throughout the cytosol. Recombinant type I InsP3 5-phosphatase expressed in Escherichia coli was a substrate of purified farnesyltransferase. Altogether, the data therefore suggest a direct participation of Cys-409 in a C-terminally anchored InsP3 5-phosphatase by farnesylation.