Post-translational Modification of Human Brain Type I Inositol-1,4,5-trisphosphate 5-Phosphatase by Farnesylation (*)
- Florence De Smedt(1)(§)(¶),
- Alain Boom(§)(2),
- Xavier Pesesse(1),
- Serge N. Schiffmann(2)(**) and
- Christophe Erneux(1)(§§)
- From the (1)Interdisciplinary Research Institute and the
- (2)Brain Research Unit, Laboratory of Neuropathology and Neuropeptides Research, Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, 1070 Brussels, Belgium
- §§ To whom correspondence should be addressed: IRIBHN, Campus Erasme, Bldg. C, 808 route de Lennik, 1070 Brussels, Belgium. Tel.: 32-2-555-41-62; Fax: 32-2-555-46-55.
Abstract
In brain, type I inositol-1,4,5-trisphosphate 5-phosphatase (InsP
5-phosphatase) is the major isoenzyme hydrolyzing the calcium-mobilizing second messenger InsP
. 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 InsP
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 InsP
5-phosphatase by farnesylation.
Footnotes
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↵§ Contributed equally to this work.
-
↵¶ Supported by the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture.
-
↵** Chercheur Qualifié of the Fonds National de la Recherche Scientifique.
-
↵* This work was supported in part by grants from the Ministère de la Politique Scientifique, the Communauté Francaise (Action Concertée), Fonds Recherche Scientifique Médicale, the Fondation Médicale Reine Elisabeth, and Boehringer Ingelheim. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- InsP
-
inositol 1,4,5-trisphosphate
- InsP
5-phosphatase -
inositol-1,4,5-trisphosphate 5-phosphatase
- InsP
-
inositol 1,3,4,5-tetrakisphosphate
- FPP
-
farnesyl pyrophosphate
- GGPP
-
geranylgeranyl pyrophosphate
- FTase
-
farnesyltransferase
- GGTase-1
-
geranylgeranyltransferase-1
- CHO
-
Chinese hamster ovary
- TBS
-
Tris-buffered saline.
- InsP
-
↵2 P. Casey, personal communication.
-
- Received October 19, 1995.
- Revision received December 13, 1996.
