From the Research Center for Cardiovascular Diseases, Institute of Molecular Medicine for the Prevention of Human Diseases, and the Division of Molecular Medicine, Department of Internal Medicine, University of Texas-Houston Health Science Center, Houston, Texas 77030
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
NEDD8 is a ubiquitin-like molecule that can be
covalently conjugated to a limited number of cellular proteins, such as
Cdc53/cullin. We have previously reported that the C terminus of NEDD8
is efficiently processed to expose Gly-76, which is required for
conjugation to target proteins. A combination of data base searches and
polymerase chain reaction cloning was used to identify a cDNA
encoding human UBA3, which is 38% identical to the yeast homologue,
22% identical to human UBA2, and 19% identical to the C-terminal
region of human UBE1. The human UBA3 gene is located on
chromosome 3p13 and gave rise to a 2.2-kilobase pair transcript that
was detected in all tissues. Human UBA3 could be precipitated with
glutathione S-transferase (GST)-NEDD8, but not with
GST-ubiquitin or GST-sentrin-1. Moreover, human UBA3 could form a
NEDD8 is a novel 81-amino acid polypeptide that is 60% identical
and 80% homologous to ubiquitin (1). Antiserum specific for NEDD8
detected a 6-kDa NEDD8 monomer and a series of NEDD8 multimers or
NEDD8-conjugated proteins (2). However, in all cell lines tested, the
conjugation pattern of NEDD8 is entirely different from that of
ubiquitin (2). Immunocytochemical analysis showed that NEDD8-conjugated
proteins were highly enriched in the nucleus. In contrast,
ubiquitin-conjugated proteins were detected equally well in the nucleus
and cytosol. Mutational analysis showed that the C terminus of NEDD8
was efficiently cleaved and that Gly-76 was required for conjugation of
NEDD8 to other proteins. The yeast homologue of NEDD8, Rub1, can also
be conjugated to a limited number of cellular proteins, including
Cdc53/cullin, a component of the SCF ubiquitin ligase (a complex
composed of Cdc53, Skp1, and an F-box protein) that plays a critical
role in the regulation of cell cycle progression (3, 4). A prominent 90-kDa NEDD8-modified protein was detected in all mammalian cells, which was consistent with the molecular mass of the NEDD8-cullin-1 conjugate (2). The substrate specificity of NEDD8 conjugation appears
to be strictly regulated because NEDD8 cannot conjugate to PML and
RanGAP1, two substrates of the sentrin family of ubiquitin-like proteins (2, 5). The function of NEDD8 modification is not known at
present. Yeast mutants defective in Rub1 are viable, but sensitive to
alterations in the levels of Cdc4, Cdc34, and Cdc53 (3). It is not
known whether NEDD8 conjugation is also involved in targeting proteins
to the proteasome.
Ubiquitin-mediated proteolysis is an important pathway of non-lysosomal
protein degradation. This pathway involves a cascade of enzymatic
reactions (6). The first step is the ATP-dependent activation of ubiquitin by a ubiquitin-activating enzyme
(E1),1 leading to the
formation of a thiol ester linkage between Gly-76 of ubiquitin and a
cysteine residue of E1. In the second step, activated ubiquitin is
transferred to a cysteine residue in one of several
ubiquitin-conjugating enzymes (E2) to form another thiol ester
conjugate. In the last step, an isopeptide bond is formed between
Gly-76 of ubiquitin and a lysyl In this paper, we report the molecular cloning of a human cDNA
encoding a protein homologous to yeast Uba3, which has recently been
shown to be involved in the activation of Rub1 (3, 4). We show that
UBA3 is homologous to the C-terminal half of E1 and can form a thiol
ester conjugate with NEDD8 in the presence of APP-BP1, which is
homologous to the N-terminal half of E1. Furthermore, we have cloned
human UBC12 and demonstrate that it could form a thiol ester linkage
with NEDD8 in the presence of APP-BP1 and UBA3. The identification of
the specific E1 complex and E2 for NEDD8 should provide further stimuli
to study the role of NEDD8 modifications in health and disease.
Materials--
All chemical reagents were purchased from Sigma
unless otherwise noted. DNA restriction endonucleases and T4 ligase
were from New England Biolabs Inc. (Beverly, MA) or Roche Molecular
Biochemicals. Human and mouse cDNA libraries, multiple-tissue
Northern blots, and the PCR amplification kit were obtained from
CLONTECH (Palo Alto, CA). The TNT®
T7-coupled reticulocyte lysate system was purchased from Promega (Madison, WI). ATP, [ Plasmid Construction--
All plasmid constructs were made using
standard techniques. Each construct was sequenced to verify the correct
frame as well as the proper sequence of any linker introduced during
the cloning procedure. The plasmids used as templates for in
vitro translation were constructed based on the vector pcDNA3.
A 1.3-kb cDNA containing the entire coding sequence of UBA3 was
amplified from a human placenta cDNA library by PCR using an
oligonucleotide forward primer
(5'-GCGAAGCTTAAATGGCTGTTGATGGTGGG-3') containing a
HindIII site (underlined) and a reverse primer
(5'-GCGGGTACCAAGAAGTAAAATGAAGTTTG-3') containing a
KpnI site (underlined). The PCR product was digested with
HindIII and KpnI and ligated with the 5.4-kb
HindIII-KpnI fragment from a derivative of
pcDNA3 to construct pcDNA3-UBA3. pcDNA3-APP-BP1 and
pcDNA3-UBC12 were constructed in a similar way, except that the
1.6-kb HindIII-KpnI fragment of
APP-BP1 and the 0.55-kb HindIII-KpnI
fragment of UBC12 were used. For the generation of the GST
fusion plasmid, a DNA segment encoding the first 76 amino acids of
NEDD8 was amplified by PCR using a 5'-primer (5'-GGCGAATTCATGCTGATTAAAGTGAAGACGCTG-3') containing an
EcoRI site (underlined) and a 3'-primer
(5'-ATTGTCGACTCATCCTCCTCTCAGAGCCAAC-3') containing an
XhoI site (underlined). The PCR product was cut with
EcoRI and XhoI and ligated with pGEX-5X1
previously linearized with EcoRI and XhoI to
construct pGEX-NEDD8. pGEX-UB and pGEX-sentrin-1 were constructed in a
similar way, except that the 0.23-kb EcoRI-XhoI fragment of ubiquitin and the 0.30-kb EcoRI-XhoI
fragment of sentrin-1 covering the first 97 residues were used.
Identification of Genes Homologous to UBA3 and UBC12--
To
clone the activating enzyme of NEDD8, we first performed a TBLASTN
search of the Tentative Human Consensus Sequence data base generated by
the Institute for Genomic Research using the partial amino acid
sequence of human UBE1 (amino acids 450-500) that contains the
ATP-binding region. This identified 17 positive THC fragments. After
further analysis, two THC fragments (THC197270 and THC206278) were
found to have highly conserved ATP-binding regions, but different from
each other. THC206278 was found to be more closely related to yeast
Uba2, which resulted in the discovery of an activating enzyme for
sentrin. An extensive search of the TIGR Tentative Human Consensus
Sequence data base using the nucleotide sequence of THC197270 revealed
another THC fragment (THC142086) that partially overlaps with
THC197270. We designed and synthesized two oligonucleotides (olig1 and
olig2) according to the information from both clones. Using the
oligonucleotides as primers, we obtained a 1.3-kb PCR fragment from
both human placenta and liver cDNA libraries. Sequence data of the
PCR products from two different cDNA libraries showed that they are
identical. Rapid amplification of cDNA ends was performed to extend
the cDNA sequence toward the 5'- and 3'-directions. Olig3 from the
internal sequence of the 1.3-kb PCR product (nucleotides 88-113) was
used as the antisense primer in combination with a sense primer of
olig4. The PCR product obtained spanned a region from the vector
flanking the 5'-end of the 1.3-kb fragment. The PCR product of 0.18 kb
was sequenced. To identify the 3'-end of the cDNA, a second PCR was
performed using olig5 as the sense primer together with olig6. The
product (0.8 kb) was sequenced. Using a pair of redesigned 5'- and
3'-primers compatible with the sequence revealed in the 5'- and
3'-anchored PCRs, we then amplified a 2.1-kb cDNA fragment from the
human placenta cDNA library by PCR.
For identification of mouse UBA3, the amino acid sequence of human UBA3
was used to search the mouse data base of expressed sequence tags
(ESTs). For identification of UBC12, the partial amino acid sequence
from human UBC9, YPssPPkckfepplfHPNvypsGtvCL, was used to search the
TIGR Tentative Human Consensus Sequence data base. One positive THC
fragment (THC210627) was identified and used for the PCR-based cloning.
PCR and Rapid Amplification of cDNA Ends of the cDNA
Fragment--
Unless otherwise specified, PCR was carried out in a
volume of 50 µl containing 50 mM KCl, 2 mM
MgCl2, 0.2 mM dNTP, 10 mM Tris-HCl,
pH 8.3, 100 ng of each primer, and 25 units of Pfu
polymerase (CLONTECH), which possesses proof
reading activity on a PTC-200 programmable thermal cycler (MJ Research
Inc.). The reaction mixture was denatured at 94 °C for 4 min, and
the amplification reaction consisted of 30 cycles of denaturation
(94 °C, 30 s), annealing (55 °C, 50 s), and extension
(72 °C, 1 min) with a final extension at 72 °C for 5 min. For
rapid amplification of cDNA ends, nested primers specific to human
UBA3 cDNA and nested adaptor primers were used in the primary and
secondary PCRs. A thermal cycling profile of 94 °C for 30 s,
65 °C for 30 s, and 72 °C for 90 s (25 cycles) and
human placenta or liver cDNA as a template were used in the primary
PCR. A cycling profile of 94 °C for 50 s and 70 °C for 3 min
(30 cycles) and 1 µl of the primary PCR product as a template were
used in the secondary PCR.
DNA Sequencing and Analysis--
The DNA sequences of the
identified clones were determined on both strands using Applied
Biosystems Prism dye termination DNA sequencing reagents and an Applied
Biosystems automated ABI 377 sequencer.
Southern and Northern Blot Analyses of Human UBA3--
To
determine the copy of the human UBA3 gene, human genomic DNA
was completely digested with either BamHI or
BglII and hybridized with a probe for UBA3. To
study the expression pattern of the UBA3 gene, a commercial
Northern blot (CLONTECH) containing RNA from eight
different tissues was used. The full-length coding human placenta
cDNA for UBA3 was amplified by PCR and gel-purified and
concentrated using a Geneclean kit (Bio 101, Inc., La Jolla, CA)
according to the manufacturer's instructions. A human Expression and Purification of GST Fusion Proteins--
The
plasmids pGEX-UB, pGEX-NEDD8, and pGEX-sentrin-1 were used for
expression of GST fusion proteins. The pGEX expression vectors were
introduced into competent BL21 bacteria, and expression of GST fusion
proteins was induced by
isopropyl- In Vitro Translation and Binding Assays--
For in
vitro translation of UBA3, APP-BP1, and UBC12 proteins,
pcDNA3-APP-BP1, pcDNA3-UBA3, and pcDNA3-UBC12 were
used as templates for in vitro transcription and translation
using T7 RNA polymerase (Promega) under conditions recommended by the
supplier. Translation reactions were performed in TNT T7-coupled rabbit reticulocyte lysates in a final volume of 50 µl. Reaction mixtures contained 25 µl of lysate, 40 µCi of [35S]methionine
(1000 Ci/mmol), 15 units of RNasin (Promega), 50 µM amino
acid mixture (minus methionine), and 1 µg of DNA. The reactions were
incubated for 90 min at 30 °C and stopped by the addition of 3 volumes of Laemmli buffer. Translation products were analyzed by 12%
SDS-polyacrylamide gel electrophoresis. Gels were fixed, treated in
Amplify solution (Amersham Pharmacia Biotech), dried, and processed for
autoradiography. For in vitro binding between NEDD8 and
UBA3, 10 µl of in vitro translated UBA3 with or without 20 µl of translated APP-BP1 were incubated with 50 µl of
glutathione-Sepharose beads containing ~1 µg of GST-ubiquitin, GST-NEDD8, or GST-sentrin-1 for 30 min at 25 °C. For thiol ester bond formation between NEDD8 and UBC12, 5 µl of in vitro
translated unlabeled UBA3 and APP-BP1 and 10 µl of translated
35S-labeled UBC12 were incubated with 50 µl of
glutathione-Sepharose beads containing GST fusion protein for 12 h
at 4 °C.
Molecular Cloning of the Human UBA3 cDNA--
A full-length
cDNA sequence encoding a protein with significant homology to the
human E1 protein was identified by a combination of data base searches
and PCR amplification described under "Experimental Procedures."
The nucleotide and deduced amino acid sequences of one of the cloned
human cDNAs are shown in Fig. 1. The
predicted amino acid sequence of the cloned human cDNA is most
similar to yeast Uba3 (38.3% identity) and less similar to yeast Uba2
(20.8% identity) and the C-terminal half of the E1 enzyme (19.2%
identity). We named this protein UBA3 because of its close similarity
to yeast Uba3 (Fig. 2) and its ability to
form a thiol ester bond with NEDD8 (see below). The UBA3
cDNA contains an open reading frame initiating with an ATG codon
(nucleotides 85-87) and terminating with a TAA codon (nucleotides
1411-1413). The ATG triplet is preceded by an in-frame stop codon at
nucleotides 13;15, and therefore, it is the most likely candidate for
the initiation codon. Another ATG codon is located at nucleotides
322-325, but the functional UBA3 was found to initiate from the first
ATG codon (see below). The 84 -base pair 5'-noncoding region has a high
AG content (72%). At the 3'-end of the cDNA, two potential
polyadenylation signals are observed: AAAATAAA is present at nucleotide
1779, and AATAAAA is located at nucleotide 2101. This open reading
frame encodes a protein (UBA3) of 442 amino acids residues. The
calculated molecular mass and pI, based on the cDNA-derived amino
acid sequence of human UBA3, are 49.3 kDa and 5.13, respectively.
Compared with the amino acid sequence for yeast Uba3, human UBA3 has an
extra 46 amino acids at its N terminus and an additional 90 amino acids at its C terminus (Fig. 2).
Two important motifs are present in the known E1 enzymes: one is the
consensus sequence for a nucleotide-binding site,
GXGXXGCE (amino acids 475-482 in human UBE1);
and the other is the consensus sequence PZCTXXXXP
surrounding the essential cysteine in E1 enzymes (amino acids 630-639
in human UBE1), which becomes linked to ubiquitin in an E1-ubiquitin
thiol ester linkage. As shown in Fig. 3,
both UBA2 2 and UBA3 have an almost identical ATP-binding
region and a significantly conserved active-site motif. The last amino
acid residue in the active-site motif of yeast and human E1 is
asparagine. In contrast, the last amino acid residue in the
active-site motif of yeast and human UBA2 is serine, whereas it is
arginine for yeast and human UBA3. Site-directed mutagenesis
experiments will be required to determine whether this substitution
contributes to the substrate preference for different activating
enzymes.
Southern and Northern Blot Analyses--
Southern blot analysis of
human genomic DNA completely digested with either BamHI or
BglII using a probe for UBA3 revealed the
presence of a single hybridization signal (data not shown). This
observation indicates the existence of one copy of the UBA3 gene per chromosome. Northern blot analysis revealed a single 2.2-kb
band in all tissue samples, but the message level of UBA3 was highest in the heart and skeletal muscle and lowest in the liver
(Fig. 4).
Chromosomal Localization of the Human UBA3 Gene by EST
Mapping--
When the human UBA3 cDNA sequence was used
to search the GenBankTM EST data base using the BLASTN
program, 63 positive EST clones were identified. All of these EST
clones were used to check the Human Gene Map data base. Three of them,
W73812 (nucleotides 1601-2120), N71576 (nucleotides 1810-2120), and
T17227 (nucleotides 1815-2120), have been mapped to chromosome 3 between D3S125 and D3S131 microsatellite markers at 91-97 centimorgans
(National Center for Biotechnology Information). Thus, the
UBA3 gene is located on chromosome 3p13. The E1-like gene,
UBE1L, is also located on chromosome 3 (3p21), whereas
UBE1 is located on chromosome X, and UBA2 is
located on chromosome 19.2
Isolation and Characterization of Mouse UBA3--
We also searched
the mouse EST data base to identify mouse UBA3. A number of positive
ESTs, such as W62229 (amino acids 1-118), AA231430 (amino acids
17-170), AA204189 (amino acids 161-283), AA670660 (amino acids
213-382), and AA073106 (amino acids 323-441) have been identified.
Next, an attempt was made to isolate the mouse Uba3 gene by
using PCR. Two primers were designed from W62229 and AA073106. This led
to the isolation of a 1.8-kb cDNA from a mouse liver cDNA
library. The open reading frame encodes a protein of 441 amino acids
with a predicted molecular mass of 49,314 Da and a pI of 5.24. When
compared with the human UBA3 sequence, the predicted mouse UBA3
sequence is one amino acid shorter at the C terminus. Nucleotide
sequence comparison between mouse and human UBA3, using BestFit with
default parameters, showed 94% identity between the two sequences. The
level of conservation between the two proteins is even greater (98.7%
identical) (Fig. 2). Interestingly, both human and mouse UBA3 contain
almost identical N- and C-terminal amino acids, which are absent from
yeast Uba3. The structure/function relationship of these N- and
C-terminal extensions awaits further investigation.
Human UBA3 and APP-BP1 Are Required for NEDD8
Activation--
Since UBA3 has a conserved ATP-binding site and an
active site of E1, we tested whether UBA3 would be able to form a thiol ester linkage with either ubiquitin or ubiquitin-like proteins. As
shown in Fig. 5A (lane
1), in vitro translated UBA3 resulted in two major
bands (40 and 49 kDa). This is most likely due to differential usage of
the two ATG codons (nucleotides 13-14 and 322-325) in the
UBA3 sequence. The in vitro translated UBA3 was incubated with GST-ubiquitin (lane 2), GST-NEDD8 (lane
4), or GST-sentrin-1 (lane 6) at room temperature for
30 min in the presence of ATP. The GST beads were extensively washed,
and the precipitated proteins were analyzed by SDS-polyacrylamide gel
electrophoresis. As shown, two bands (40 and 49 kDa) could be
precipitated by GST-NEDD8, but not by GST-ubiquitin or GST-sentrin-1.
Thus, UBA3 appears to bind preferentially to NEDD8. However, a higher
molecular mass UBA3-NEDD8 conjugate could not be detected (lane
4). Since UBA3 is homologous to the C-terminal half of E1, it is
likely that another protein homologous to the N-terminal half of E1 is
required for the formation of a UBA3-NEDD8 conjugate. Initially, we
tested whether human AOS1 would be able to complement UBA3 in the
formation of the UBA3-NEDD8 conjugate. However, human AOS1 could not
complement UBA3 and appeared to be specific for the sentrinization
pathway.2 Thus, we tested an AOS1 homologue, APP-BP1, for
its ability to complement UBA3 in the formation of the UBA3-NEDD8
conjugate. APP-BP1 was initially identified in a yeast two-hybrid
cloning experiment as a binding protein for the amyloid precursor
protein (13). In vitro translated APP-BP1 resulted in a
predicted single band of 51 kDa (lane 7). In
vitro translated UBA3 and APP-BP1 were co-incubated with
GST-ubiquitin (lane 3), GST-NEDD8 (lane 5), and
GST-sentrin-1 (lane 8); washed; and analyzed by
SDS-polyacrylamide gel electrophoresis. A high molecular mass band of
82 kDa, consistent with the molecular mass of GST-NEDD8-UBA3
conjugates, was observed in the GST-NEDD8 precipitate (lane
5). This 82-kDa band cannot be a APP-BP1-NEDD8 conjugate because
APP-BP1 does not contain the active-site cysteine. Furthermore, the
UBA3 C216S mutant, which contains a substitution in the active site,
could not form a 82-kDa conjugate (Fig. 5B). The thiol ester
bond formation between UBA3 and NEDD8 was dependent on the presence of
ATP (Fig. 5C). Next, we showed that the 82-kDa band was
sensitive to 5% Identification and Cloning of Human UBC12--
We have previously
identified UBC9 as a specific conjugating enzyme in the sentrinization
pathway (12). While testing the specificity of the conjugating enzymes
in the sentrinization pathway, we used NEDD8 as a control and failed to
observe a thiol ester bond formation between NEDD8 and eight known
human E2 proteins, including UBCH5B, UBCH6, HIP2, HHR6A, HHR6B, E2-EPF,
UBCH-ben, and UBC9 (data not shown). Thus, we sought to identify
additional human E2 proteins that could conjugate to NEDD8. We used the
amino acid sequence from the conjugation site of human UBC9
(YPssPPkckfepplfHPNvypsGtvCL) as a query to search the TIGR
Tentative Human Consensus Sequence data base. 18 positive THC fragments
were identified. After further analysis, one THC fragment (THC210627)
was found to have a conserved sequence different from known E2
proteins. We designed two primers, oligo7 and oligo8, according to the
information from THC210627. PCR amplification from a human placenta
cDNA library resulted in the isolation of a 750-base pair cDNA.
The nucleotide sequences and predicted amino acids are shown in Fig.
7A. The cloned cDNA contains an open reading frame initiating with an ATG codon
(nucleotides 4-6) and terminating with a TAG codon (nucleotides
553-555). The cloned cDNA (human UBC12) encodes a protein of 183 amino acid residues that is 40.4% identical and 61.2% similar to the
yeast Ubc12 protein (Fig. 7B). Both human and yeast Ubc12
proteins have an almost identical motif,
HPNIDLXGNVCLNILREDW, around the cysteine residue
(a site needed for the formation of a thiol ester bond), which strongly
suggests that the cloned human cDNA is UBC12, not another E2. This
motif was also found in mouse (AA881308) and Drosophila
melanogaster (AA952265) by analysis of EST clones from EST data
bases.
Conjugation of NEDD8 to UBC12 Requires the Involvement of APP-BP1
and UBA3--
Next, the ability of human UBC12 to form thiol ester
conjugates with ubiquitin, NEDD8, or sentrin-1 was determined. As shown in Fig. 8 (lanes 2-4), only
GST-NEDD8, but not GST-ubiquitin or GST-sentrin-1, could specifically
precipitate in vitro translated UBC12. However, we could not
observe an NEDD8 and UBC12 conjugate in the absence of E1 (lane
3). When APP-BP1 and UBA3 were added to the reaction mixture, a
55-kDa conjugate could be detected (lane 5). This band most
likely represents GST-NEDD8 conjugated to UBC12 via a thiol ester
linkage because it disappeared when the sample was reduced with 5%
Distinct Pathways for Activation and Conjugation of Ubiquitin-like
Proteins--
Ubiquitination has proven to be one of the major protein
modification pathways and plays a critical role in many cellular processes (14-16). The complexity of the ubiquitination system is
further compounded by the identification of other ubiquitin-like molecules, such as UCRP (ubiquitin
cross-reactive protein), sentrin, and NEDD8. UCRP is a type 1 interferon-inducible protein that contains
two ubiquitin domains (17). UCRP has been shown to conjugate to a large
number of intracellular proteins (18). Proteins modified by UCRP have
not yet been identified. Recent results from the Haas laboratory (19)
suggest that UCRP conjugation proceeds through an enzyme pathway
distinct from that of ubiquitin, at least with respect to the
activation step. However, the true identity of the activating enzyme
for UCRP is not known. The sentrin family of ubiquitin-like proteins
consists of three family members that have similar substrate
specificity. We have shown that sentrin-1, sentrin-2, and sentrin-3 are
able to conjugate to RanGAP1 and PML (5). The yeast homologue of
sentrin is Smt-3, which utilizes an activating enzyme complex
consisting of Uba2 and Aos1 (10). A similar complex is also required
for the activation of the sentrin family members.2
Interestingly, both sentrin and Smt-3 utilize UBC9 as the conjugating enzyme (10, 12). We have shown previously that NEDD8 can be covalently
conjugated to a limited number of cellular proteins (2). In this
report, we cloned and characterized both the activating and conjugating
enzyme complexes for NEDD8. Our results are consistent with reports
from the yeast system (3, 4). Taken together, the enzymatic machinery
responsible for the activation and conjugation of ubiquitin-like
proteins diverged from the ubiquitination system early in evolution
(Fig. 9).
-mercaptoethanol-sensitive conjugate with NEDD8 in the presence of
APP-BP1, a protein with sequence homology to the N-terminal half of
ubiquitin-activating enzyme. We have also cloned human UBC12 and
demonstrated that it could form a thiol ester linkage with NEDD8 in the
presence of the activating enzyme complex. Identification of the
activating and conjugating enzymes of the NEDD8 conjugation pathway
should allow for a more detailed study of the role of NEDD8
modification in health and disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-amino group within a substrate
protein, a reaction catalyzed either directly by the E2 enzyme or via a
third enzyme designated ubiquitin-protein ligase. A single E1 enzyme
has been characterized from human, mouse, wheat, and
Saccharomyces cerevisiae (7). In yeast, Uba1 is essential for viability and encodes a 114-kDa activating enzyme required for
ubiquitin conjugation (8). Another E1-like protein, Uba2, which encodes
a 71-kDa protein that is similar to the C terminus of E1 proteins and
bears a cysteine residue at a position similar to the active-site
cysteine of Uba1, has been identified (9). Johnson et al.
(10) demonstrated that Uba2 cooperates with another 40-kDa protein,
termed Aos1 (activation of Smt3),
the yeast homologue of the sentrin family of ubiquitin-like proteins
(11). Interestingly, both human and yeast Aos1 and Uba2 share extensive
homology with the N- and C-terminal halves of yeast
ubiquitin-activating enzyme, Uba1.2
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]dCTP,
L-[35S]methionine, Hyperfilm MP, and the GST
protein purification kit were from Amersham Pharmacia Biotech.
Prestained molecular mass markers and SDS-polyacrylamide gel
electrophoresis reagents were obtained from Bio-Rad. Oligonucleotides
were synthesized with a Beckman DNA synthesizer (Model olig1000M).
The sequences of the primers used in this study are as follows:
olig1, CTGGATCCAAATGGCTGTTGATGGTGGG; olig2,
GGAATTCTTATGCATATTGGCCTCTATGG; olig3, CTGGAGAGATTCAGTGCTCGGTTCGA; olig4, TATCTATTCGATGATGAAGATACC; olig5, CAGACTGTACTATTCAAACTTCATTTT; olig6, TGCACAGTTAAGTGAACTTGCGG; olig7, AGGATGATCAAGCTGTTCTCGCTGAAG; and
olig8, GCAGTAACATTCCCCTTTAGTTAGCC. Escherichia coli
strains XL1-Blue and BL21 were used for cDNA cloning and expression
of GST fusion proteins, respectively.
-actin probe
was used as a control to determine equal RNA loading. The cDNA
probe was labeled with [
-32P]dCTP (3000 Ci/mmol) using
the Random Primers DNA labeling system (Life Technologies, Inc.), and
unincorporated nucleotide was removed by chromatography on a NucTrap
column (Stratagene, La Jolla, CA). The filter was prewashed for 1 h at 42 °C in 0.5× SSPE (1× SSPE = 0.16 M NaCl,
20 mM NaPO4, and 1 mM
Na4EDTA, pH 7.2), 0.5% SDS, and 100 µg/ml herring sperm
DNA and prehybridized for 16 h at 42 °C in 30% formamide.
Hybridization was performed with the 32P-labeled probe
overnight. The nylon sheet was washed twice with 2× SSC and 0.1% SDS
at room temperature for 20 min and with 0.2× SSC and 0.1% SDS at
55 °C for 10 min. Autoradiograms were obtained by exposing the blot
to x-ray film with an intensifying screen at
80 °C for 2 days.
-D-thiogalactopyranoside and purified as
described previously (12).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (58K):
[in a new window]
Fig. 1.
Nucleotide sequence of the coding and
flanking regions of human UBA3. The deduced amino acid
sequence is given below the nucleotide sequence in single-letter code.
The asterisk indicates the stop codon, and the potential
polyadenylation signal is underlined. The nucleotide
sequence was determined on both strands by automated sequencing.
View larger version (37K):
[in a new window]
Fig. 2.
Sequence alignment of human, yeast, and mouse
UBA3. Amino acids that are identical to the corresponding position
in yeast (Y), human (H), and mouse (M)
are shaded. The numbers correspond to the
sequence of UBA3.
View larger version (13K):
[in a new window]
Fig. 3.
Amino acid alteration in the ATP-binding
region and active-site motif. Identical amino acids are in
boldface. GenBankTM accession numbers M58028,
X55386, Z48725, and Y16891 are for human UBE1 and yeast Uba1, Uba2, and
Uba3, respectively.
View larger version (47K):
[in a new window]
Fig. 4.
Northern blot analysis of human
UBA3 mRNA from various human tissues. Human
tissue blot (CLONTECH) was probed with
32P-labeled cDNA of UBA3. Hybridization and washing
were carried out according to the manufacturer's manual. RNAs were
from the tissues indicated.
-mercaptoethanol treatment, suggesting that it
behaved like a thiol ester conjugate (Fig.
6). These results suggest that formation
of a UBA3-NEDD8 conjugate is dependent on the presence of APP-BP1. Our
results are also consistent with recent reports that Rub1, the yeast
homologue of NEDD8, can form a thiol ester bond with yeast Uba3 (3, 4). Moreover, Ula1/Enr2, the yeast homologue of APP-BP1, is also required for the activation of Rub1(3, 4). Taken together, there is a remarkable conservation in the enzymology in the activation of NEDD8 and Rub1.
View larger version (26K):
[in a new window]
Fig. 5.
In vitro interaction of ubiquitin,
NEDD8, and sentrin-1 with UBA3 and a UBA3 mutant. A,
in vitro translated UBA3 (lane 1) was
precipitated by GST-NEDD8 (lane 5), but not by GST-ubiquitin
(UB; lane 3) or GST-sentrin-1 (lane
8), in the presence of APP-BP1. The position of the GST-NEDD8-UBA3
conjugate (82 kDa) is indicated. Lanes 2 and 3,
UBA3 precipitated by GST-ubiquitin in the absence and presence of
APP-BP1, respectively; lanes 4 and 5, UBA3
precipitated by GST-NEDD8 in the presence and absence of APP-BP1,
respectively; lanes 6 and 8, UBA3 precipitated by
GST-ubiquitin in the absence and presence of APP-BP1, respectively;
lanes 1 and 7, in vitro translated
UBA3 and APP-BP1, respectively. B, in vitro
translated UBA3 (lane 1) and UBA3 C216S (lane 3)
were precipitated by GST-NEDD8 in the presence of APP-BP1. The position
of the GST-NEDD8-UBA3 conjugate (82 kDa) is indicated. C,
thiol ester conjugate formation between UBA3 and NEDD8 could be
observed only in the presence of both APP-BP1 and ATP (lane
1), but not in the absence of ATP (lane 3).
View larger version (24K):
[in a new window]
Fig. 6.
The UBA3-NEDD8 conjugate is sensitive to
-mercaptoethanol treatment. The position of
the GST-NEDD8-UBA3 conjugate (82 kDa) formed in the presence of APP-BP1
is indicated. Lane 1 was run under nonreducing conditions;
lane 2 was treated with 5%
-mercaptoethanol
(2-ME).
View larger version (58K):
[in a new window]
Fig. 7.
cDNA and amino acid sequence of human
UBC12 and alignment of yeast and human UBC12. A, the
sequence of human UBC12 cDNA is shown above the putative
peptide sequence. The asterisk indicates the stop codon. The
nucleotide sequence was determined on both strands by automated
sequencing. B, the peptide sequence for human (H)
UBC12 is shown above the sequence of the yeast (Y) protein
(GenBankTM accession number X99442). The numbers
correspond to the sequence of UBA3. Dashes were inserted to
allow for optimal alignment. Identical amino acids are indicated by
vertical bars. Amino acid similarity is indicated by a
plus sign.
-mercaptoethanol (lane 6). Thus, UBC12 is the only E2
tested that was able to form a conjugate with NEDD8. This observation
is also consistent with the recent report of the critical role of yeast
Ubc12 in the conjugation of Rub1 (4).
View larger version (22K):
[in a new window]
Fig. 8.
In vitro interaction of ubiquitin,
NEDD8, and sentrin-1 with UBC12. In vitro translated
UBC12 was precipitated by GST-NEDD8 in the presence of UBA3 and APP-BP1
(lane 5). The position of the GST-NEDD8-UBC12 conjugate (55 kDa) is indicated. UBC12 could not be precipitated by GST-ubiquitin
(UB) and GST-NEDD8, respectively, in the presence of UBA3
and APP-BP1 UBC12 (lanes 2 and 4); lane
5, precipitated by GST-NEDD8 in the presence of both UBA3 and
APP-BP1; lane 6, the sample from lane 5 treated
with 5% -mercaptoethanol (2-ME).
View larger version (14K):
[in a new window]
Fig. 9.
Summary of activating enzymes for
the ubiquitin and ubiquitin-like proteins, sentrin, and NEDD8. The
sizes for UBE1, AOS1, APP-BP1, UBA2, and UBA3 are 1011, 346, 532, 670, and 442 base pairs, respectively. APP-BP1 is 22% identical and 46%
similar to the N-terminal region of UBE1 (37-426 amino acids) and 21%
identical and 43% similar to AOS1 (L. Gong and E. T. H. Yeh,
manuscript in preparation). UBA3 is 19% identical and 42% similar to
the C-terminal region of UBE1 (427-1011 amino acids) and 22%
identical and 44% similar to UBA2 and AOS1 (L. Gong and E. T. H. Yeh, manuscript in preparation). The positions for the
ATP-binding region and the active-site motif are shown in Fig. 3.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. T. Kamitani for scientific discussions and B. Li and L. S. Caskey for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant HL-45851, the DREAM Project, and an Established Investigator award from the American Heart Association (to E. T. H. Y.). This is publication number 145-IMM from the Institute of Molecular Medicine for the Prevention of Human Disease, University of Texas-Houston Health Science Center.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) AF046024.
To whom correspondence should be addressed: Div. of Molecular
Medicine, Dept. of Internal Medicine, University of Texas-Houston Health Science Center, 6431 Fannin, Suite 4.200, Houston, TX 77030. Tel.: 713-500-6660; Fax: 713-500-6647; E-mail: eyeh{at}heart.med.uth.tmc.edu.
2 L. Gong and E. T. H. Yeh, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; PCR, polymerase chain reaction; kb, kilobase pair(s); EST, expressed sequence tag; THC, tentative human consensus.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|