Post-translational modification of human brain type I inositol-1,4,5-trisphosphate 5-phosphatase by farnesylation.
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.
De Smedt
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F
Interdisciplinary Research Institute, Université Libre de Bruxelles, Campus Erasme, 1070 Brussels, Belgium.
Boom
A
A
Pesesse
X
X
Schiffmann
S N
SN
Erneux
C
C
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D016466:CHO Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D017368:Protein Prenylation; D011689:Purkinje Cells / Q000201:enzymology; D051381:Rats; D011994:Recombinant Proteins
D017931:DNA Primers; D011994:Recombinant Proteins; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases