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
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ABSTRACT |
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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 G A variety of proteins are modified with an isoprenoid lipid at a
cysteine that is initially four residues from the C terminus (1-3).
Such proteins contain the so-called CAAX motif, in which the
"C" is the modified cysteine, the "A" residues are
most commonly (but not always) aliphatic amino acids, and the
"X" residue can be one of several amino acids. The
X residue determines whether the protein is modified by the
15-carbon farnesyl lipid or the 20-carbon geranylgeranyl. If the
X residue is a leucine, the protein will be
geranylgeranylated; several other residues (e.g. Met, Ser,
Ala, and Gln) direct farnesylation. Following prenylation of the
protein, two additional processing steps occur (4, 5). First, a
specific protease cleaves the -AAX tripeptide from the protein, leaving the prenylated cysteine as the new C terminus. The
carboxyl group of the prenylcysteine is then methylated by a specific methyltransferase.
It is well established that protein prenylation plays a vital role in
the membrane localization and function of most prenylated proteins (6).
The role that the proteolysis and methylation of prenyl proteins play
in their function, however, is not as well understood. Studies on
peptides have demonstrated that each processing step significantly
increases the affinity of farnesylated peptides for membranes (7),
although the effect of the final two steps is not as great for
geranylgeranylated peptides. Proteolysis and methylation also increased
the hydrophobicity of Ras proteins processed in an in vitro
system (8). Prevention of the proteolysis of Ras in cells resulted in a
decrease in membrane localization (9, 10), and in yeast it resulted in
at least a partial loss of Ras function (10).
The enzymes responsible for prenylation of CAAX-containing
proteins, protein farnesyltransferase and protein
geranylgeranyltransferase I, have been cloned and studied in detail
(3). Additionally, a specific prenyl protein carboxymethyltransferase
has been identified in yeast as the product of the STE14
gene (11), and a human homologue of the STE14 gene product has recently
been described (12). These findings have left the prenyl protein
protease as the only member of the processing pathway yet to be
identified on a molecular level.
Recently, an elegant genetic screen in yeast resulted in the
identification of two candidate genes for prenyl protein proteases (10). The first gene, AFC1/STE24, appeared to be primarily involved in
the processing of the precursor to a-factor, a farnesylated yeast mating pheromone. AFC1/STE24 catalyzes two cleavage events on the
a-factor precursor, the first being the C-terminal proteolysis and the second being a cleavage occurring near the N
terminus of the peptide (10, 13, 14). Strong evidence was provided that
the second candidate gene, RCE1, was involved in the processing of the
yeast Ras proteins, in addition to the C-terminal processing of the
a-factor precursor (10, 15). Because RCE1 was linked to the
processing of a prenyl protein, it seemed likely that the RCE1 gene
product might have a broader range of substrates than AFC1/STE24. We
report here the identification, expression, and preliminary
characterization of a human homologue of RCE1, termed hRCE1. Evidence
is provided that hRCE1 is in fact a prenyl protein protease and that it
is involved in the processing of a variety of CAAX-type
prenylated proteins, including all known forms of Ras. These findings
open the door for molecular studies of this protease and will
facilitate studies aimed at determining the roles of the proteolysis
and methylation steps in the functions of CAAX-type prenyl proteins.
Materials--
The Cloning of hRCE1--
The deduced amino acid sequence of yeast
RCE1 was used to conduct a text-based search of the human expressed
sequence tag (EST) data base at the National Center for Biotechnology
Information. The clone W96411 was identified as a likely homologue.
Five additional clones that overlapped W96411 were present in the data
base, representing 725 nucleotides of the cDNA sequence. The primer
5'-GGGCTTCAGGCTGGAGGGCATTTT-3' was chosen from this sequence for use in
a GeneTrapper cDNA positive-selection cloning protocol. A partial
clone containing the hRCE1 open reading frame but lacking an initiation
codon was cloned from a pCMV.SPORT 2 human fetal brain cDNA plasmid
library. A probe was generated from the XcmI-PstI
fragment of that clone and used to screen a Northern Blot Analysis--
A 32P-labeled probe was
generated with random hexamer priming from the 1100-bp
BssHII-PstI hRCE1 cDNA fragment and
hybridized to a human multi-tissue poly(A)+ RNA blot
(obtained from CLONTECH); hybridization and washing were performed as described previously (21). The process was repeated
using a probe made to the cDNA of Production of Recombinant Proteins in Sf9
Cells--
Recombinant baculoviruses were produced with the pFASTBAC-1
vector and the protocol provided in the Bac-2-Bac Baculovirus Expression System kit. Recombinant baculoviruses were prepared for the
two human RCE1 constructs described above, for yeast RCE1, and for the yeast prenyl protein carboxymethyltransferase
STE14.
For production of recombinant proteins, Sf9 cells in
log phase growth were diluted to 1 × 106 cells/ml and
infected with recombinant baculoviruses at multiplicities of infection
ranging from 2 to 10. Cells producing yeast or human RCE1 were
harvested 72 h post-infection and resuspended in 50 mM
Tris-HCl, pH 7.7. Cells expressing yeast STE14 were harvested 60 h
post-infection, and resuspended in 5 mM NaHPO4,
pH 7.0, containing 5 mM EDTA (11) and a mixture of protease
inhibitors (22). In all cases, cells were disrupted by sonication,
nuclei and debris were removed by centrifugation at 500 × g for 5 min, and membranes were then pelleted by
centrifugation at 200,000 × g for 1.5 h. Membranes from RCE1 producing cells were resuspended in 50 mM Tris-HCl, whereas membranes from cells producing STE14
were resuspended in 5 mM NaHPO4 containing 5 mM EDTA and the protease inhibitor mixture (22). Final
protein concentrations of the membrane suspensions were 10-25 mg/ml.
The suspensions were flash-frozen in liquid nitrogen and stored at
Production of Prenylated Proteins--
Unprenylated Ki-Ras (18),
Ha-Ras (23), N-Ras (17), G
Farnesyl-Ki-Ras and geranylgeranyl-Ki-Ras were resolved from the
unprenylated precursor and modifying enzyme by applying the prenylation
reactions to a heptyl-Sepharose column, which was washed with buffer B
(50 mM Hepes, 3 mM MgCl2, 1 mM dithiothreitol, 5 µM GDP) containing 0.1%
lubrol (removing unprenylated Ki-Ras and the prenyltransferase), and
then eluting the purified prenyl-Ki-Ras with buffer B containing either
2% sodium cholate (farnesyl-Ki-Ras) or 4% sodium cholate
(geranylgeranyl-Ki-Ras). The eluted prenyl-Ki-Ras preparations were
diluted 20-fold in buffer B and applied to an S-Sepharose column. The
column was washed with buffer B, and the prenyl-Ki-Ras was eluted with
buffer B containing 0.1% octylglucoside and 750 mM NaCl.
Purified prenyl-Ki-Ras stocks were flash-frozen in liquid nitrogen and
stored at Prenyl Protein Protease Assays--
Protease reactions were
initiated by the addition of membranes containing the recombinant
prenyl protein protease to an assay mixture containing prenylated
proteins in 100 mM Hepes, pH 7.4, and 5 mM
MgCl2 in a total volume of 50 µl; reactions were
conducted at 37 °C. Following the proteolysis reaction, methylation
of proteolysed prenylated protein was initiated by addition of 20 µg
of membranes containing STE14 to the reaction in 17.5 µM
[3H]AdoMet (2000 Ci/mmol), 5 mM
NaHPO4, pH 7.0, 62.5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 10 mM 1,10-phenanthroline,
and 300 µM N-tosyl-L-phenylalanine
chlorophenyl ketone in a total volume of 20 µl. The addition of EDTA
and protease inhibitors served both to quench the proteolysis reaction
and to inhibit proteases present in the STE14 membranes. Stoichiometric
methylation was achieved within 20 min at 37 °C. Reactions were
terminated by adding 0.5 ml 4% SDS along with 50 µg of bovine brain
cytosol as carrier protein (27), and 3H-methylated protein
was quantitated using a filter assay (23, 25, 27).
Protease assays were also performed directly on prenylated proteins
following their prenylation. In these assays, a 45-µl prenylation
reaction was performed as described above, and the concentration of
prenylated protein generated in the reaction was determined. The
prenylated protein was diluted to the desired concentration in buffer
A, and a source of membranes was added to the mixture. The assay then
proceeded as described above.
The deduced amino acid sequence of yeast RCE1 was used to search a
human EST data base, and the entry W96411 was identified as a potential
homologue. Five additional overlapping ESTs were identified giving a
composite sequence of 725 amino acids. A full-length hRCE1 clone was
assembled from a clone isolated from a human fetal brain cDNA
library, which contained the 3' end of the cDNA, and a clone
isolated from a HUVEC cDNA library, which contained the 5' end of
the cDNA. The transcript was 1500 bp, not including polyadenylation, and had an open reading frame that encoded a 329-amino
acid protein (Fig. 1). The strongest
homology between the yeast and human proteins was found in a region
from Arg166 to Pro270 of hRCE1 (42% identity
and 68% similarity), suggesting that the active site of the enzyme
could reside in this region. This region was also highly conserved in a
potential homologue of RCE1 found in the Caenorhabditis
elegans genome data base (CEF48F5). As reported for yeast RCE1
(10), the deduced amino acid sequence of hRCE1 contained multiple
predicted transmembrane domains (not shown). Examination of hRCE1
expression by Northern blotting revealed a primary band of 1.8 kilobases, which was expressed in all tissues tested (Fig.
2A). A faint 2.4-kilobase band
was also evident in some tissues. Examination of RCE1 expression in
mouse tissues, using a probe corresponding to a sequence found in the
mouse data base, gave stronger evidence of the presence of larger
transcripts, which may represent alternatively spliced variants of RCE1
(Fig. 2B).
1 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
gt11 human umbilical vein endothelial cell
library (HUVEC)1 (16) was
provided by John York of this institution. The plasmid pRS315-RCE1
containing the cDNA for yeast RCE1 (10) was a gift from Jasper Rine
(University of California, San Francisco); the pATH-STE14 plasmid
containing the cDNA encoding the yeast methyltransferase STE14 (11)
was a gift from Susan Michaelis (Johns Hopkins Medical Center); the
QE31-N-Ras expression plasmid for N-Ras (17) was a gift from Robert
Bishop (Schering-Plow Research Institute); the pTrcHis-Rap1b expression
plasmid for hexahistidine-tagged Rap1b (18) was a gift from Guy James
(University of Texas Health Sciences Center, San Antonio). The reduced
farnesyl-peptide analogue RPI (19, 20) was a gift from Robert Rando
(Harvard Medical School). Oligonucleotide primers were synthesized at
the Duke University DNA Core Facility. The Bac-2-Bac Baculovirus
Expression System, the GeneTrapper cDNA Positive Selection System,
and the pCMV.SPORT 2 human fetal brain cDNA library were purchased
from Life Technologies, Inc. S-Adenosylmethionine (AdoMet)
was purchased from Research Biochemicals International.
[3H-methyl]-S-adenosylmethionine
([3H]AdoMet) was purchased from New England Nuclear.
Peptides were synhesized by Princeton Biomolecules.
gt11 HUVEC library (16).
That screen yielded a clone containing the likely initiation codon,
because it contained an upstream stop codon in-frame with the coding
sequence. The 5' end of the HUVEC cDNA containing the initiation
codon was removed as a BssHII-XcmI fragment and
ligated to the human fetal brain cDNA
XcmI-PstI fragment to generate a hRCE1 cDNA
containing the entire open reading frame. This
BssHII-PstI fragment was subcloned into the
vector pFASTBAC-1 to produce p-FASTBAC-1-hRCE1. In a second construct,
designated pFASTBAC-1-
hRCE1, the 5'-untranslated region of hRCE1 was
replaced with a portion of the 5'-untranslated region from the
baculovirus polyhedron gene (CCTATAAAT), and the codons specifying the
first 22 amino acids of hRCE1 were removed (see Fig. 1).
-actin as a control. Expression of RCE1 was also examined using a mouse multiple tissue Northern blot (CLONTECH). The blot was probed with
a 223-bp Rce1 cDNA fragment that was amplified from a
mouse liver cDNA library (CLONTECH) with
oligonucleotide primers 5'-TTGCCCTTGTGACCTGACAGATGG-3' and
5'-GAGTGGGCAGGTGAACACAGCAGG-3'.
80 °C in multiple aliquots.
1 (24), and Rap1b (18) were
expressed in Escherichia coli and purified essentially as
described previously. Ki-Ras, N-Ras, and Rap1b each had an N-terminal
hexahistidine tag, whereas Ha-Ras and G
1 were
unmodified. Ki-Ras, Ha-Ras, N-Ras, and G
1 were
farnesylated by incubation of 2 µM protein with 20 µg/ml purified farnesyltransferase (22), 6 µM farnesyl
diphosphate in buffer A (5 mM MgCl2, 5 µM ZnCl2, 2 mM dithiothreitol, 5 µM GDP, 50 mM Tris-HCl, pH 7.7) for 1 h
at 37 °C. Ki-Ras and Rap1b were geranylgeranylated by incubation of
2 µM protein with 20 µg/ml purified
geranylgeranyltransferase 1 (22), 6 µM geranylgeranyl diphosphate in buffer A for 1 h at 37 °C (23, 25).
80 °C in multiple aliquots. The peptides CVIM and
GSPCVLM were prenylated chemically as described previously (26) and
purified by high pressure liquid chromatography.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (78K):
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Fig. 1.
Sequence alignment of human and yeast
RCE1. The region of highest homology between yeast and human RCE1
originally identified in the Blast search is indicated by the
shaded region, and identical residues are boxed.
Human and yeast RCE1 are designated as hRCE1 and
yRCE1. The region of hRCE1 deleted in the pFASTBAC- hRCE1
construct is underlined.
View larger version (60K):
[in a new window]
Fig. 2.
Northern blot analysis of hRCE1 expression in
human tissues. Expression of RCE1 mRNA in various human
(A) and murine (B) tissues was assessed by
hybridization with 32P-labeled probes as described under
"Experimental Procedures." kb, kilobases.
High level expression of the yeast carboxymethyltransferase STE14 in
Sf9 cells has allowed development of a coupled assay for prenyl protein proteolysis that utilizes proteins prenylated in vitro as substrates and quantitates C-terminal
proteolysis by stoichiometric methylation. In preliminary experiments,
we found that expression of the cDNA encoding yeast RCE1 in
Sf9 cells by infection with a recombinant baculovirus
resulted in a dramatic increase (>1000-fold) in prenyl protein
protease activity in the cells (Fig. 3);
subfractionation of cell extracts revealed that all of the activity was
found in the membrane fraction (data not shown). It was this
demonstration that the yeast RCE1 gene encoded an authentic prenyl
protein protease that compelled us to search for and ultimately clone
hRCE1. Although attempts to express full-length cDNA encoding hRCE1
in insect cells were unsuccessful, when a construct in which the first
22 codons of the hRCE1 were deleted (designated hRCE1) was expressed
in Sf9 cells, a dramatic (>1000-fold) increase in
prenyl protein protease activity was observed in the membrane fraction
(Fig. 3).
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Recombinant hRCE1 produced by expression of the hRCE1 construct
utilized both farnesylated and geranylgeranylated Ki-Ras as substrates,
with both substrates exhibiting apparent Km values
of approximately 0.5 µM (Fig.
4A) and similar
kcat values, indicating that the enzyme has
similar catalytic efficiencies for farnesylated and geranylgeranylated
substrates. Farnesyl-Ha-Ras, farnesyl-N-Ras,
farnesyl-G
1, and geranylgeranyl-Rap1b were also found to
be substrates for hRCE1 (Fig. 4B), demonstrating that the
enzyme can utilize a broad range of CAAX-type prenyl protein
substrates. Competition studies were performed with peptides corresponding to the C termini of Ki-Ras (CVIM) and of mouse N-Ras (GSPCVLM) to determine whether the enzyme could recognize unprenylated as well as prenylated substrates. GSPCVLM and CVIM that contained farnesylated cysteine residues were able to compete for the processing of prenylated Ki-Ras, whereas the corresponding unprenylated peptides could not, demonstrating both that hRCE1 is specific for prenylated proteins and that it recognizes short prenylated peptides (Fig. 4C). Additionally, the activity of a previously identified
inhibitor of prenyl protease activity, a reduced farnesyl-peptide
analogue termed RPI (19, 20), was examined. The RPI compound was indeed an extremely effective inhibitor of hRCE1, exhibiting an
IC50 of approximately 5 nM (Fig.
4D).
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The first report of prenyl protein protease activity indicated that proteolysis (and methylation) occurs in a membrane compartment in cells (8). Subsequent in vitro studies have focused on membrane fractions in analysis of this processing step. Two distinct activities have been characterized. The first activity was tightly bound by membranes (28-31) and could only be released by detergents (20, 32, 33). The second activity was loosely associated with membranes and could be solubilized by a freeze-thaw process (34). For the purpose of comparison, these activities have been described as microsomal and soluble (35). The microsomal enzyme exhibits an apparently higher affinity for the prenyl peptide CVIM (Km of 0.65 µM) (20) than the soluble enzyme (Km of 32 µM) (34). Based on competition-type assays, the microsomal enzyme exhibited a 250-fold specificity for prenylated peptides over unprenylated peptides, whereas the soluble enzyme reportedly had only a 5-fold specificity (35). Additionally, only the microsomal enzyme was inhibited by the RPI compound (19, 20, 35). The activity described herein for hRCE1 is most similar to the above-mentioned microsomal activity, based on its apparent Km for farnesyl-Ki-Ras (~0.5 µM), its specificity for prenylated proteins, and the potent inhibition observed with the RPI compound.
The variety of substrates that hRCE1 processed in vitro
suggests that it plays a major role in the processing of prenylated proteins in cells. Indeed, in an independent study, fibroblasts prepared from mouse embryos in which the RCE1 gene was disrupted failed
to process the Ras proteins, as well as other prenylated proteins (36).
Identification of the hRCE1 cDNA and the ability to produce
substantial quantities of enzyme in Sf9 cells will provide several major advantages for its study. First, the high level
expression of hRCE1 will allow detailed examination of its activity,
including screening for specific inhibitors, with minimal concerns of
background. Additionally, the high level production system will provide
a platform for the purification of the enzyme and will allow initiation
of structural approaches to its study. Finally, in combination with the
overexpression of yeast carboxymethyltransferase, the tools are now in
place to generate large amounts of processing intermediates of prenyl
proteins, which should be quite useful in examining the properties that
each of the processing steps import to the functions of prenylated proteins.
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ACKNOWLEDGEMENTS |
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We thank John York and Michael Howell for discussions on cloning strategies. We are indebted to Robert Rando for providing the RPI compound, to Jasper Rine for providing the yeast RCE1 cDNA, and to Susan Michaelis for providing the yeast STE14 cDNA. We thank John Moomaw, Carolyn Weinbaum, and Ying Chen for assistance with protein purification and Ted Meigs for assistance with Northern blotting and comments on the manuscript.
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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 GenBankTM/EMBL Data Bank with accession number(s) AF121951.
§ Supported by a fellowship from the Leukemia Society of America.
Supported by a Postdoctoral Fellowship Award for Physicians
from the Howard Hughes Medical Institute.
** 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.
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ABBREVIATIONS |
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The abbreviations used are: HUVEC, human umbilical vein endothelial cell; AdoMet, S-adenosylmethionine; EST, expressed sequence tag; PCR, polymerase chain reaction; bp, base pair(s).
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