Cloning and Characterization of a Mammalian Prenyl Protein-specific Protease*
- From the ‡Departments of Pharmacology and Cancer Biology and of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710-3686 and the ¶Gladstone Institute of Cardiovascular Disease, the Cardiovascular Research Institute, and the Department of Medicine, University of California, San Francisco, California 94141-9100
Abstract
Proteins containing C-terminal “CAAX” sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Gγ1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins.
Footnotes
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↵* This work was supported by American Cancer Society Grant BE-117 (to P. J. C.) and National Institutes of Health Grants GM46372 (to P. J. C.) and AG15451 (to S. G. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF121951.
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↵§ Supported by a fellowship from the Leukemia Society of America.
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↵‖ Supported by a Postdoctoral Fellowship Award for Physicians from the Howard Hughes Medical Institute.
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↵** To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710-3686. Tel.: 919-613-8613; Fax: 919-613-8642; E-mail:casey006{at}mc.duke.edu.
- Abbreviations:
- HUVEC
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human umbilical vein endothelial cell
- AdoMet
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S-adenosylmethionine
- EST
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expressed sequence tag
- PCR
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polymerase chain reaction
- bp
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base pair(s)
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- Received December 21, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
