BMB
reports
Direct characterization of E2-dependent target specificity and
processivity using an artificial p27-linker-E2 ubiquitination
system
1,2,#
1,2,#
3
3
1
1
Kyoung-Seok Ryu , Yun-Seok Choi , Junsang Ko , Seong-Ock Kim , Hyun Jung Kim , Hae-Kap Cheong , Young Ho
Jeon1,2, Byong-Seok Choi3,* & Chaejoon Cheong1,2,*
1
2
Magnetic Resonance Team, Korea Basic Science Institute, Department of Bio-Analytical Science, University of Science and Technology,
3
Daejeon 305-333, Department of Chemistry and National Creative Research Initiative Center, KAIST, Daejeon 305-701, Korea
Little attention has been paid to the specificity between E2 and
the target protein during ubiquitination, although RING-E3 induces a potential intra-molecular reaction by mediating the direct transfer of ubiquitin from E2 to the target protein. We have
constructed artificial E2 fusion proteins in which a target protein
(p27) is tethered to one of six E2s via a flexible linker.
Interestingly, only three E2s (UbcH5b, hHR6b, and Cdc34) are
able to ubiquitinate p27 via an intra-molecular reaction in this
system. Although the first ubiquitination of p27 (p27-Ub) by
Cdc34 is less efficient than that of UbcH5b and hHR6b, the additional ubiquitin attachment to p27-Ub by Cdc34 is highly
efficient. The E2 core of Cdc34 provides specificity to p27, and
the residues 184-196 are required for possessive ubiquitination
by Cdc34. We demonstrate direct E2 specificity for p27 and also
show that differential ubiquitin linkages can be dependent on E2
alone. [BMB reports 2008; 41(12): 852-857]
INTRODUCTION
Members of the E3 family of enzymes are mainly classified into two groups (HECT and RING) according to the nature of
their E2 binding domains (1). The RING-E3 group has no internal ubiquitin-accepting Cys residue and thus mediates the
direct transfer of ubiquitin from E2 to the target protein (1). It is
possible that the RING-E3 enzyme provides a scaffold whose
primary function is to bring together E2~Ub (ubiquitin thiolester of E2) and the target protein and thus induces an intramolecular interaction. Ubiquitination by the RING-E3 enzyme
is comprised of the following four interactions: E3-to-substrate,
*Corresponding author. Byong-Seok Choi; Tel: 82-42-869-2828; Fax:
82-42-869-2810; E-mail: [email protected]; Chaejoon Cheong;
Tel: 82-42-865-3431; Fax: 82-42-865-3405; E-mail: [email protected]
#
Contributed equally to this work
E3-to-E2, E2-to-ubiquitin, and E2-to-substrate. To interpret the
mechanism of ubiquitination the first three interactions (E3-tosubstrate, E3-to-E2, and E2-to-ubiquitin) have been taken into
account in most studies (2-4). However, little research has
been directed at the identification of a possible E2-to-substrate
interaction. A recent yeast two-hybrid analysis using a fused
BRCA1-BARD1 bait identified an additional six E2s (UbcH6,
Ube2e2, UbcM2, Ube2k, Ubc13, and Ube2w) in addition to
UbcH5 and UbcH7 that bind directly to the RING domain of
BRCA1 (5). Interestingly, UbcH7, Ubc13, and Ube2k cannot
perform the monoubiquitination of BRCA1 even in the presence of E3 (BRCA1-BARD1). It is thus possible that a direct interaction between E2 and the target protein provides additional target specificity during ubiquitination mediated by
RING-E3.
This hypothesis leads us to attempt to simplify the ubiquitination reaction by constructing an artificial system that does
not require the E3 enzyme. We constructed E2 fusion proteins
Kip1
by tethering a target substrate protein, p27 (p27), for one of
the various E2s via a flexible linker (-G4SG4SG4-) to increasing
the probability of contact between E2 and p27. The in vivo
ubiquitin-attachment site of any target protein preferentially
contains lysine residues located in an unstructured region (6)
and maps to a distinct spatial area with positional diversity (7).
p27 is an unstructured protein and has been reported to be
ubiquitinated at multiple lysine residues (134, 153, and 165)
by UbcH5 in vitro and by Cdc34 in vivo in the presence of
Skp2
SCF -E3 (7,8).
Here, we studied the direct specificity of E2 for p27 using
p27-linker-E2 proteins with a focus on the following discrete
steps (Fig. 1A and B): (i) first ubiquitination (the transfer of
ubiquitin from E2~Ub to a unubiquitinated target protein) and
(ii) consecutive ubiquitination (the transfer of ubiquitin from
E2~Ub to a ubiquitin molecule that is already attached to the
target protein).
Received 11 July 2008, Accepted 12 September 2008
Keywords: Direct target specificity of E2, p27-E2 fusion protein,
Processive ubiquitination, Ubiquitin, Ubiquitin-linkage
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Fig. 1. Ubiquitination of p27 by the tethered E2 enzymes. (A) Monoubiquitination is achieved after the first ubiquitin attachment to the
target protein (1a). First ubiquitination also includes additional multiple monoubiquitinations at several ubiquitination sites within the target
protein (1b). Consecutive ubiquitination refers to the additional ubiquitin attachments that occur after the first ubiquitination, and it can also be classified as nonprocessive (2a) and processive (2b) ubiquitination. (B) An artificial E3-independent ubiquitination system consists of
a target protein, a flexible linker, and one of the various E2 enzymes, and thus E2~Ub formation by E1 and subsequent in vitro ubiquitination are a coupled reaction. The flexible linker mimics the primary function of E3, which is to bring E2~Ub and the target protein into close proximity to each other and enhance the probability of contact between the target protein and E2~Ub. (C) The reaction time is
indicated at the top of each panel. The E2 fusion protein bands are indicated with open arrows and the ubiquitin self-adducts are indicated using closed arrows (di and tri). His-tagged ubiquitin was used for the ubiquitination of p27-Ubc7, and PK-ubiquitin was used for
all other p27-E2 proteins. The N-terminal tag of PK-ubiquitin was partially digested by trace protease activity that remained in the purified
p27-Cdc34, which resulted in diubiquitin with a reduced molecular size (di-f). A diubiquitin with a nonclassical ubiquitin linkage is observed for p27-hHR6b (di* in p27-hHR6b section). The p27 fragments that remained after the purification of the E2 fusion proteins are
marked with a pound sign (#). The protein bands that were generated by the autoubiquitination of Ubc13 are marked with stars (*). The
ubiquitinated forms of the E2 fusion proteins are labeled as in section A.
RESULTS
Only certain E2 enzymes efficiently ubiquitinate p27 in the
context of an E2 fusion protein
p27 was tethered to a single E2 protein, either UbcH5b,
Cdc34, hHR6b (human homolog of yeast Rad6), UbcH7,
Ubc13, or Ubc7 (Ube2g1). This E2 fusion system had positive
effects on both KM and VMAX values for first and consecutive
ubiquitination reactions. The linker mimicked the E3 activity
for only Cdc34, UbcH5b, and hHR6b E2 enzymes, but not for
UbcH7, Ubc13, or Ubc7 (Fig. 1C). The attachment of ubiquitin to the p27-E2 proteins was confirmed using anti-ubiquitin
Western blot analysis (data not shown). Mutation of the active
Cys of UbcH5b to Ser (C85S) in the p27-UbcH5b protein completely abolished the ubiquitination of p27; thus p27 ubiquitination is directly mediated by the tethered E2 (labeled as
“C85S” in the p27-UbcH5b section). We also investigated the
influence of reduced linker length on the ubiquitination activity of p27-UbcH7, as a short linker (-GGSGG-) might lead to
an increased relative local concentration of p27 and UbcH7.
However, the ubiquitination activity of this smaller version
was also negligible (data not shown). The different versions of
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the hybrid proteins, in which the positions of p27 and E2 were
exchanged (UbcH5b-p27 and Cdc34- p27), were also capable
of p27-ubiquitination to a similar degree as that of the original
p27-E2 hybrids (Supplemental Material 1).
To confirm that E2 alone has specificity for p27, we performed in vitro ubiquitination of p27 in the presence of E2
alone using high concentrations of the reaction components.
The same E2s (Cdc34, UbcH5b, and hHR6b), which were able
to ubiquitinate p27 in the E2 fusion context, also generated
smeared bands in the upper region of the original p27 band
(Fig. 2). The Western blot analysis using antibodies to p27 and
ubiquitin (data not shown) revealed that these bands represented ubiquitinated p27. In addition we identified weak autoubiquitination of hHR6b and Ubc13. Ubc13 was previously
shown to be auto-ubiquitinated at Lys92 and Lys94 (9).
We also tested the E2 fusion system using the human PCNA
(hPCNA) target protein, because PCNA is known to be monoubiquitinated by Rad6 and Rad18 (10). hPCNA was also ubiquitinated by hHR6b tethered via a flexible linker peptide containing a thrombin-cleavage site (Supplemental Material 2).
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Fig. 2. Ubiquitination of p27 by separate E2 enzymes. The in vitro ubiquitination reaction was performed for 2 h with increasing concentrations of reaction components to observe any weak p27 ubiquitination activity of the various E2s. (A) Both Cdc34 and p27 have similar
electrophoretic mobility, and thus these protein bands were not separated in this SDS-PAGE analysis. The p27 protein was partially digested by trace protease activity that remained in the protein preparations (p27-f). hHR6b appeared to give rise to two different diubiquitin bands (the hHR6b section); the upper band is diubiquitin with a nonclassical ubiquitin linkage (di*) and the lower band is diubiquitin
with a K48 linkage (di). The p27 fragments that remained after the purification of the E2 fusion proteins are marked with a pound sign
(#). Autoubiquitination was observed for hHR6b and Ubc13 (E2*). (B) The boxed region of the gel shown in A is the same shown in B
following Western blotting using a polyclonal antibody to p27 protein.
Ubiquitination of p27-linker-E2 proteins is an intramolecular
reaction
We performed in vitro ubiquitination of the E2 fusion proteins using varying protein concentrations so as to discriminate between
intra- and intermolecular reactions, because the activation of
ubiquitin by E1 and the transfer of ubiquitin from E2 to p27 are
coupled in the E2 fusion system. We found that the ubiquitination activity did not decrease with decreasing concentrations (40
to 2 μM) of p27-UbcH5b, p27-hHR6b, or p27-Cdc34 (Fig. 3). The
p27-E2 protein likely mimics E3-mediated ubiquitination, because the ubiquitination that occurs in the E2 fusion system is an
intramolecular reaction.
Cdc34 is more processive during the polyubiquitination of
p27, but less potent during the monoubiquitination of p27
than are UbcH5b or hHR6b
The in vitro ubiquitination reactions with various p27-E2 proteins displayed different activities during the two discrete ubiquitination steps (first and consecutive ubiquitination). The accumulation of first-ubiquitinated forms was observed for both
p27-UbcH5b and p27-hHR6b, and their efficiencies for consecutive ubiquitination were lower than that of p27-Cdc34
(Fig. 1). No clear accumulation of the first-ubiquitinated forms
was observed for p27-Cdc34; instead, discrete consecutiveubiquitination products were efficiently produced (as represented by the ladders shown in Fig. 1C and Supplemental
Material 1). Moreover, a large amount of unubiquitinated p27Cdc34 remained after the ubiquitination reaction was
terminated. The low level of p27-Cdc34 first-ubiquitination
products observed did not result from a low level of Cdc34 activation by E1, because nonreducing SDS-PAGE analysis con854 BMB reports
Fig. 3. Concentration-dependent ubiquitination of p27-E2 proteins.
The in vitro ubiquitination reaction was performed with p27-UbcH5b
(A), p27-hHR6b (B), and p27-Cdc34 (C) for 0.5 h, while varying their
concentrations from 40 to 2 μM. The concentrations of all other components were constant. The loading-sample volumes for SDS-PAGE
analysis were adjusted to equal amounts of E2 fusion proteins.
Therefore, the E1 and E1 contaminant protein bands (E1#) are increased as the concentration of the p27-E2 protein is decreased. The
remaining E2 fusion protein bands are indicated using open arrows.
The p27 fragments that remained during the purification are marked
with a pound sign (#). The ubiquitinated forms of the E2 fusion proteins are classified as 1a (single monoubiquitination), 1b (multiple
monoubiquitination), 2a (nonprocessive ubiquitination), and 2b
(processive ubiquitination).
firmed that more than half of p27-Cdc34 exists as a ubiquitin
thiolester form (data not shown). Therefore, these derivatives
likely result from differences in the tendency toward consecutive ubiquitination by Cdc34 (which gives rise to more discretely sized products) vs. first ubiquitination of p27 by hHR6b
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Ubiquitination of p27-linker-E2 fusion proteins
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and UbcH5b.
Although UbcH5b and hHR6B are Class-I E2 enzymes,
which only possess the E2 core domain (150-170 residues),
Cdc34 belongs to Class II and has a long C-terminal extension
in addition to an E2 core (11). Interestingly, highly processive
consecutive ubiquitination by the tethered Cdc34 was largely
dependent on the presence of residues 184-196, because (i)
p27-Cdc34 (1-183) was able to do first ubiquitination, however its tendency for consecutive ubiquitination was greatly reduced, and (ii) the consecutive ubiquitination activity of
p27-Cdc34 (1-196) was comparable to that of the wild-type
p27-Cdc34 (Fig. 4A). The ubiquitination activity of p27-Cdc34
(1-183) indicates that the specificity of Cdc34 for p27 is located in its E2 core domain, which forms E2~Ub via the active Cys residue. It was previously reported that the presence
of residues 170-194 of Cdc34 is important for the multiubiquitin assembly by Cdc34 in the presence of the
ROC1-CUL1 core ubiquitin ligase (12). The presence of residues 184-196 of Cdc34 was also critical for effective multiubiquitin synthesis by Cdc34 alone (Fig. 4B).
Ubiquitin linkages are readily observed in the E2 fusion system
Yeast proteomics has revealed that all 7 lysine residues in
ubiquitin serve as sites for polyubiquitin chain assembly
(ubiquitin linkage) in vivo (13, 14). The ubiquitin self-assembly
Fig. 4. Effect of the Cdc34 C-terminal extension on in vitro
ubiquitination. All E2 and p27-E2 proteins tested in this experiment
are indicated using open arrows. (A) The N-terminal region of
PK-ubiquitin was occasionally digested by trace protease activity,
which resulted in a diubiquitin species with a smaller molecular size
(di-f). The p27-tethered Cdc34 (1-196) is able to perform consecutive
ubiquitination of p27, and the efficiency of ubiquitination is similar
to that of Cdc34. However, the p27-tethered Cdc34 (1-183) cannot
perform consecutive ubiquitination, although it still retains its ability
to carry out first ubiquitination. The ubiquitinated forms of the E2 fusion proteins are classified as 1a (single monoubiquitination) and 2b
(processive ubiquitination). (B) His-tagged ubiquitin was used for ubiquitination by nontethered E2s, instead of PK-ubiquitin, since it is more
resistant to trace protease activity. Both Cdc34 and Cdc34 (1-196) efficiently produced ubiquitin self-adducts. Interestingly, the efficiency of
diubiquitin synthesis by Cdc34 (1-183) is significantly lower than that
of wild-type Cdc34.
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by E2 alone is generally inefficient, and thus the identification
of E2-dependent ubiquitin linkage is difficult. The E2 fusion
system would be useful for identifying E2-dependent ubiquitin
linkages, because the consecutive ubiquitination of the E2 fusion protein was relatively efficient. We observed that both
p27-Cdc34 and p27-UbcH5b retained the classical K48 ubiquitin linkage during consecutive ubiquitination (Supplemental
Material 3). However, we found that polyubiquitination of
p27-hHR6b occurred by all Lys-to-Arg mutant ubiquitins.
These findings imply the presence of at least dual ubiquitin
linkages in hHR6b.
DISCUSSION
Although the E3 enzyme primarily provides target specificity to
E2 by recruiting a specific E2~Ub during the ubiquitination of
the target protein, the presence of binding specificity between
E3 and E2 can not completely explain the E2-dependent target
specificity that has previously been characterized (5). The in vitro ubiquitination of p27-E2 fusion proteins demonstrates that
E2 alone has direct specificity for the target protein, although it
is less stringent than that of E3. The p27-E2 fusion is a highly
simplified system, and the absence of the E3 enzyme makes it
possible to evaluate the direct target specificity that is solely dependent on E2 itself. Cdc34 and UbcH5b were able to ubiquitinate p27 in the fusion context, and these E2 enzymes are
Skp2
known to ubiquitinate p27 in the presence of SCF -E3.
However, the in vivo function of hHR6b during the ubiquitination of p27 is still uncertain, because hHR6b has been reported
to be a functional E2 during the monoubiquitination of the
Skp2
PCNA protein. It is possible that SCF -E3 may not recruit
hHR6b during the in vivo ubiquitination of p27.
The first ubiquitination of a target protein shares common
characteristic with the sumoylation reaction, except that (i) first
ubiquitination is critically dependent on the presence of E3
and (ii) many E2 species participate in ubiquitination, while
only one Ubc9 can carries out sumoylation. SUMO-conjugated Ubc9 directly recognizes the sumoylation motif
(ΨKxE/D, Ψ is a bulky aliphatic residue) within target proteins
and transfers the SUMO moiety to a Lys side chain in the target protein (15-17). Ubiquitination by a RING-E3 enzyme in
vivo would be more complicate than during in vitro ubiquitination in the E2 fusion protein system. It has been reported
that the yeast Cdc34ΔC-Sic1 fusion protein is not autoubiquitinated in the absence of SCF-E3 (Skip/Cullin/F-box) (18). It is
likely that the presence of a RING-E3 can influence the specificity of yeast Cdc34 (yCdc34) for the Sic1 protein by supplying
an additional interaction to yCdc34 or Sic1. Moreover, an unstructured protein, like p27, generally interacts with many other cellular proteins, and thus its in vivo ubiquitination of unstructured proteins is likely to be more complicate. Nevertheless, E2 specificity for a target protein could provide an additional point of regulation during the RING-E3 mediated ubiquitination and could explain (i) the reason why some E2s bind
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RING-E3 but are not able to ubiquitinate a target protein (19,
20) and (ii) the presence of many E2 and UEV (ubiquitin E2
variant) proteins.
The ubiquitination of p27-Cdc34 has characteristics that are
similar to SCF-mediated ubiquitination of the Sic1 protein, in
which the first ubiquitination by Cdc34 is also the rate-limiting
step (21). We have characterized E2s as target selective
(UbcH5b and hHR6b) or processive (Cdc34) based on the results regarding the first and consecutive ubiquitination of E2
fusion proteins. Recently, it was reported that Ubc4 and Ubc1
are sequentially involved in the efficient ubiquitination of target proteins by the anaphase-promoting complex, E3 (22). We
speculate that two different E2 enzymes (target selective and
processive) are generally involved in the ubiquitination of a
target protein by RING-E3. Cdc34 might function as a processive E2 for the ubiquitination of many target proteins. This
could be one reason why the yeast cdc34 deletion strain can
not be obtained (23).
A detailed structural mechanism has been determined for
the K63 ubiquitin linkage, in which the Ubc13/Mms2
(E2/UEV) complex guides K63 of the acceptor ubiquitin to the
active Cys of Ubc13 (24). However, almost nothing is known
about the other ubiquitin linkages, including the classical K48
linkage. Moreover, the noncovalent binding surfaces of ubiquitin for hHR6b, UbcH5b, and Cdc34, which were determined
using a chemical shift perturbation NMR technique, are almost
identical (unpublished data). Although our results indicated
the presence of at least dual ubiquitin linkages during the in vitro ubiquitination of p27-hHR6b, its in vivo function is still
unclear. Rad6 is known to monoubiquitinate the yeast PCNA
in the presence of Rad18 (E3) (10). The nonclassical ubiquitin
linkage observed for hHR6b may be related to its low ability to
perform consecutive ubiquitination. UbcH5 has also been reported to have a nonclassical linkage (25), and its activity for
consecutive ubiquitination is also significantly lower relative
to that of Cdc34.
The acidic loop that is present in Cdc34, but not in hHR6b
and UbcH5b, has been reported to be critical for efficient
ubiquitination via the K48 linkage, and the loss of this loop results in a non-K48 ubiquitin linkage (19). However, our data
indicate that the presence of residues 184-196 is also critical
for the highly processive ubiquitination by Cdc34. It is likely
that the determination of the molecular mechanism for the efficient synthesis of K48-linked ubiquitin self-adducts by processive E2 could also provide an insight on the mechanism of
the other ubiquitin linkages. The E2 fusion system makes ubiquitination an intramolecular reaction, and thus the ubiquitin-linker-E2 proteins could be used for studying the characteristics of E2-dependent consecutive ubiquitination. This system can also be adapted to obtain a large amount of ubiquitin-modified proteins for the purpose of structural or clinical
studies, as shown for the PCNA-hHR6B fusion protein. In summary, the presence of direct p27 specificity in E2 alone was
clearly identified using p27-E2 fusion proteins; however, it is
856 BMB reports
still remains to be elucidated the exact determinants that provide E2 with a p27 specificity at the molecular level.
MATERIALS AND METHODS
Protein preparation
All the human E2 genes (cdc34, ubch5b, hhr6b, ubch7,
ubc13, and ubc7) were cloned into the pGEX-4T-1 vector. All
ubiquitin genes and E2 fusion genes were cloned into either
the pET15b or pET28a expression vector. PK-ubiquitin was
generously provided by prof. pan (26). The p27 protein was
obtained by inserting a stop codon into the p27-linkerUbcH5b construct. Point mutations were obtained using the
Quick- change method (Stratagene).
All proteins were expressed in E. coli (Rosetta DE3). GSTfused E2 proteins were purified using a Hitrap-GST column
(GE Healthcare), and the GST moiety was removed by thrombin digestion. His-tagged proteins were first purified with a
Histrap column (GE Healthcare), and then additional Hitrap-SP
or Hitrap-Q column (GE Healthcare) chromatography was performed to obtain high-purity protein. All proteins were dialyzed in buffer (pH 7.5) containing 20 mM Tris, 100 mM
NaCl, and 1 mM DTT.
In vitro ubiquitination assay
The reaction buffer (pH 8.0) for in vitro ubiquitination contained 50 mM Tris ․ HCl, 1 mM DTT, 2 mM ATP, 5 mM
MgCl2, 10 mM creatine phosphate, and 1 unit each of pyrophosphatase and creatine phosphokinase. Trace protease activity remaining in the protein preparations was inhibited by
adding 1-2 μg/ml of protease inhibitors (antipain, aprotinin,
bestatin, and leupeptine). Ubiquitination of the p27-linker-E2
protein was performed for 0.5 or 1 h at 25ºC using 0.02 mM
E2 fusion protein, 0.2 mM ubiquitin, and ~0.5 μM E1. The
concentrations of E2, p27, and E1 (0.03 mM, 0.05 mM, and
~1.0 μM) and reaction time (2 h) were increased for ubiquitination by E2 alone.
Western blot analysis
The ubiquitin and p27 bands were detected with a mouse anti-ubiquitin monoclonal IgG1 and a rabbit anti-p27 (M-197)
polyclonal antibody (Santa Cruz Biotech. Inc.), respectively,
and visualized using an ECL-detection system (GE Healthcare).
Acknowledgements
This work was supported by the National Creative Research
Initiatives from the Korean Ministry of Science and Technology
(grant to B. -S. Choi) and by the NMR Research Program from
the KBSI (grant to Y. H. Jeon). We thank Y. S. Seo (KAIST;
ubcH5b and mouse uba1 genes), C. H. Chung (Seoul National
University; ubc13 gene), and J. H. J. Hoeijmakers (Erasmus
Medical Center; hHR6b gene) for kindly providing various
cloned genes. We also thank J. H. Kim (Department of
Biological Science, KAIST) for useful discussions and J. W. Lee
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(KBSI) for kindly providing a ubiquitin antibody.
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