Accumulation of polyubiquitinated proteins by
overexpression of RBCC protein interacting with protein
kinase C2, a splice variant of ubiquitin ligase RBCC protein
interacting with protein kinase C1
Nobuo Yoshimoto
1,2
, Kenji Tatematsu
1
, Toshihide Okajima
3
, Katsuyuki Tanizawa
1
and Shun’ichi Kuroda
1,2
1 Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Japan
2 Laboratory of Industrial Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan
3 Department of Nanobiology, Institute of Scientific and Industrial Research, Osaka University, Japan
Keywords
26S proteasome; RING-IBR; S5a; splice
variant; ubiquitin
Correspondence
K. Tatematsu and S. Kuroda, Laboratory of
Industrial Biosciences, Graduate School of
Bioagricultural Sciences, Nagoya University,
Furo-cho, Chikusa, Nagoya 464-8601, Japan
Fax: +81 52 789 5227
Tel: +81 52 789 5227
E-mail: and

(Received 10 June 2009, revised 26 August
2009, accepted 3 September 2009)

MINT-7261462: Rbck1 (uniprotkb:Q62921) physically interacts (MI:0915) with Ubiquitin
(uniprotkb:
P62988)byanti tag coimmunoprecipitation (MI:0007)
l
MINT-7261503: S5a (uniprotkb:P55036) binds (MI:0407)toUbiquitin (uniprotkb:P62988)by
pull down (
MI:0096)
l
MINT-7261477: Rbck1 (uniprotkb:Q62921) binds (MI:0407)toUbiquitin (uniprotkb:P62988)
by pull down (
MI:0096)
Abbreviations
CBP, CREB-binding protein; E3, ubiquitin ligase; GST, glutathione S-transferase; HA, hemagglutinin; HEK293, human embryonic kidney 293;
HHR23, human homolog of Rad23; HRP, horseradish peroxidase; IBR, in-between-RING fingers; NES, nuclear export signal; NLS, nuclear
localization signal; NZF, novel zinc finger; PML, promyelocytic leukemia protein; RBCK1, RBCC protein interacting with protein kinase C1;
RBCK2, RBCC protein interacting with protein kinase C2; UBA, ubiquitin-associated; UBL, ubiquitin-like; UIM, ubiquitin-interacting motif.
FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS 6375
Introduction
RBCC protein interacting with protein kinase C1
(RBCK1), consisting of 498 amino acids, has been iso-
lated from a rat brain cDNA library by using a yeast
two-hybrid system with the regulatory region of
protein kinase Cb as bait [1] (Fig. 1A). The protein
contains a ubiquitin-like (UBL) domain, a novel zinc
finger (NZF) motif between two coiled-coil regions, a
first RING finger (RING1), an ‘in-between-RING
fingers (IBR) motif, and a second RING finger
(RING2) from the N-terminus to the C-terminus [1–3].
A RING finger coordinating two zinc ions is found in
many transcription factors and ubiquitin ligases (E3s).

Fig. 1. Effect of overexpression of RBCK2
on accumulation of polyubiquitinated pro-
teins. (A) Schematic drawing of RBCK1 and
RBCK2. Amino acid residue numbers of the
N-termini and C-termini are indicated for the
whole fragments and their domains: UBL,
ubiquitin-like; C-C, coiled-coil; RING1, first
RING finger; IBR, in-between-RING fingers;
RING2, second RING finger; and NZF, novel
zinc finger. The consensus amino acids of
the NZF motif are indicated in the gray box.
The cysteine residues essential for the
formation of zinc clusters are indicated in
the black box. (B) Effects of overexpression
of RBCK2 on the amount of polyubiquitinat-
ed protein in cells. An expression plasmid
for the N-terminally HA-tagged RBCK2 was
cotransfected into HEK293 cells with an
expression plasmid for FLAG-tagged
ubiquitin. The upper and lower panels show
the detection of polyubiquitinated proteins
and overexpressed RBCK2 in the cell lysate,
respectively. MG132, a specific inhibitor of
the proteasome, was used.
Potential polyubiquitinated protein carrier RBCK2 N. Yoshimoto et al.
6376 FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS
localizes preferentially in the cytoplasm [11]. When
RBCK2 is coexpressed with RBCK1 in human embry-
onic kidney 293 (HEK293) cells, RBCK1 localizes
preferentially in the cytoplasm, indicating that RBCK2

proteins was also observed in the HEK293 cells treated
with MG132, a proteasomal inhibitor (Fig. 1B, lane 2).
As RBCK2 lacks a RING domain, which plays a piv-
otal role in the E3 activity (Parkin [13], MDM2 [14],
and RNF8 [15]), the overexpressed RBCK2 was postu-
lated to participate in either the enhancement of the
activity of endogenous E3s or the inhibition of prote-
asomal degradation of polyubiquitinated proteins.
Colocalization of RBCK2 with the 26S
proteasome
The subcellular localization of endogenous RBCK1 ⁄ 2
(RBCK1 and RBCK2) was investigated in HEK293
cells. As described previously [12], RBCK1 ⁄ 2 resides in
the cytoplasm and nuclear bodies (Fig. 2A). Immuno-
chemical observations showed that endogenous
RBCK1 ⁄ 2 colocalizes with the 20S catalytic core sub-
complex of the 26S proteasome mainly in the cyto-
plasm (Fig. 2A, upper panels). After the MG132
treatment, the 20S subcomplex was translocated from
the cytoplasm to the nucleus, as reported previously
(Fig. 2B,E) [16]. The RBCK1 ⁄ 2 proteins were also
translocated from the cytoplasm to the nucleus
(Fig. 2A,D), suggesting that RBCK1 ⁄ 2 is involved in
the 20S subcomplex.
Direct interaction of RBCK2 with S5a
The UBL domains from Parkin, an autosomal-recessive
juvenile parkinsonism-related protein [13], and a human
homolog of Rad23 (HHR23) directly interact with 19S
proteasomal subunit S5a (a subunit of the 19S regula-
tory subcomplex of the 26S proteasome) [17–19]. We

chains
The NZF motif of Npl4, an endoplasmic reticulum-
associated degradation-related protein [3], directly
N. Yoshimoto et al. Potential polyubiquitinated protein carrier RBCK2
FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS 6377
interacts with polyubiquitinated proteins [3]. The NZF
motif of RBCK2 was also assumed to interact with
polyubiquitinated proteins. The lysate of HEK293 cells
overexpressing RBCK2 was subjected to immuno-
precipitation analysis. Whereas a small amount of
polyubiquitinated protein was observed in the immu-
noprecipitates of RBCK2 (Fig. 4A, lane 2), the
amount was significantly increased by the MG132
treatment (Fig. 4A, lane 4), indicating that RBCK2
interacts with polyubiquitinated proteins. Polyubiquitin
chains linked with Lys48 of ubiquitin are efficiently
and specifically recognized by the 26S proteasome [23].
Therefore, the direct interaction of the Lys48-linked
polyubiquitin chain with RBCK2 was examined by an
in vitro pull-down assay with bacterially expressed
purified GST-fused proteins (Fig. 4B,C). As reported
previously [22], polyubiquitin chains larger than tetra-
ubiquitins directly interact with purified GST–S5a
(positive control; Fig. 4B, lane 2). RBCK2 was shown
to interact directly with polyubiquitin chains larger
than triubiquitin chains (Fig. 4B, lane 4). The ubiqu-
itin–RBCK2 interaction was abolished by quadruple
mutation of the NZF motif of RBCK2 (Fig. 4B, lane 5),
which is the replacement of four cysteines essential for
the NZF motif with serines (Fig. 1A). Taken together,

D
E
F
Fig. 2. Localization of endogenous RBCK1 ⁄ 2 and the 20S proteasome. (A–F) The intracellular localization of endogenous RBCK1 ⁄ 2 and the
20S proteasome was detected by indirect immunofluorescence. HEK293 cells were cultured with or without MG132 and stained with an
antibody against RBCK (A, D) or an antibody against the 20S proteasome (B, E). Merge, merged images (C, F).
Potential polyubiquitinated protein carrier RBCK2 N. Yoshimoto et al.
6378 FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS
Overexpression of either HHR23, hPlic-2 or RBCK2
was considered to disturb the delivery of polyubiquiti-
nated proteins to the 26S proteasome by depriving the
polyubiquitin chain of the ternary complex containing
the ‘ubiquitin carrier protein’, S5a, and the polyubiqu-
itin chain. On the basis of these findings, RBCK2 was
postulated to be a ‘polyubiquitinated protein carrier’
facilitating the delivery of polyubiquitinated proteins to
the 26S proteasome by forming a ternary complex con-
taining RBCK2, S5a, and the polyubiquitin chain
(Fig. 5A, upper model).
RBCK1, harboring both an NES and an NLS, shut-
tles between the cytoplasm and the nucleus and inter-
acts with the transcription factor promyelocytic
leukemia protein (PML) and the coactivator, CREB-
binding protein (CBP), in nuclear bodies [12]. RBCK2
is constitutively excluded from the nucleus by its NES
activity, and is a cytoplasmic tethering protein for
RBCK1 [11]. In this study, we found that RBCK2
induces the accumulation of polyubiquitinated proteins
in cooperation with the 26S proteasome. These results
strongly suggested that RBCK2 is a potential cytoplas-

proteins coimmunoprecipitated with an antibody against HA. The
middle panel shows the FLAG-tagged RBCK2 and RBCK2DUBL in
the cell lysate. The bottom panel shows HA-tagged S5a in the
immunoprecipitates obtained with an antibody against HA.
N. Yoshimoto et al. Potential polyubiquitinated protein carrier RBCK2
FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS 6379
RBCK2-interacting proteins
The 19S proteasomal subunit S5a and a yeast homo-
log, Rpn10, have two UIMs (UIM1 and UIM2) and
one UIM, respectively. Each UIM of S5a interacts
with the polyubiquitin chain [22]. In particular, the
C-terminal UIM (UIM2) of S5a interacts with the
UBL domains of HHR23 and hPlic-2 [20,21,26]. On
the basis of the NMR analysis for the complex of the
S5a UIM2 with the HHR23 UBL domain, Leu38,
Ile44, Gly47 and Val70 in the UBL domain are essen-
tial for the association with S5a (Fig. 5B) [20,27].
Three hydrophobic residues and glycine are well con-
served in HHR23, RBCK1, and RBCK2 (Fig. 5B). It
is plausible that these residues of RBCK2 play crucial
roles in the interaction with the UIM2 of S5a. This, in
turn, corroborates the formation of the ternary
RBCK2–polyubiquitinated protein–S5a complex in the
vicinity of the 26S proteasome.
In this study, the NZF motif of RBCK2 was found
to interact directly with the Lys48-linked polyubiquitin
chain (Fig. 4B), which is exclusively involved in prote-
asomal degradation [22,23]. Recently, various lysine
residues of ubiquitin (e.g. Lys6, Lys11, Lys27, Lys29,
Lys33, and Lys63) were revealed to be involved in the

6380 FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS
tumor suppressor 1 (BRCA1) [33] and Parkin [34], and
the BRCA1-associated RING domain 1 protein [35].
However, the biochemical analyses of these splice vari-
ants have not been fully pursued yet. This study dem-
onstrated for the first time that the splice variant of
the RING–IBR protein RBCK1, RBCK2, is expressed
endogenously in various tissues, and participates not
only in the transcriptional system [11], but also in the
ubiquitin–proteasome system. On the other hand,
Parkin, a RING–IBR protein, was shown to accom-
pany many splice variants in the transcripts of rat and
fetal human brain [34]. Interestingly, two of these
splice variants (TV4 and TV5 in [34]) retain the UBL
domain and lack the RING–IBR domain, as does
RBCK2.
Although these variants possess neither the NZF
domain nor the UIM ⁄ UBA domain, they interact with
polyubiquitinated proteins through the N-terminal
region [13]. It is postulated that these splice variants of
Parkin serve, like RBCK2, as polyubiquitinated pro-
tein carriers proximal to the 26S proteasome. Another
Parkin splice variant, which also retains the UBL
domain and lacks the RING–IBR domain, was origi-
nally expressed in the peripheral leukocytes of normal
humans. In patients with sporadic Parkinson’s disease,
the same splice variant was frequently found in brain
[36]. Moreover, the Parkin mutant generated by the
guanine 535 microdeletion, which harbors the UBL
domain only, was found to be closely associated with

ubiquitin–proteasome system.
Experimental procedures
Plasmids
Deletion and substitution mutants of the rat RBCK2 gene
were obtained with a QuickChange site-directed mutagene-
sis kit (Stratagene, La Jolla, CA, USA). For the expression
of N-terminally HA-tagged rat RBCK2 (HA–RBCK2),
N-terminally HA-tagged human S5a (HA–S5a), N-terminally
FLAG-tagged RBCK2 (FLAG–RBCK2) and the N-termi-
nally FLAG-tagged UBL domain (1–130 amino acids)-
truncated RBCK2 mutant (FLAG–RBCK2DUBL) in
mammalian cells, the plasmids pTB701–HA–RBCK2 [11],
pTB701–HA–S5a, pTB701–FLAG–RBCK2 and pTB701–
FLAG–RBCK2DUBL were constructed using pTB701–
FLAG or pTB701–HA [38]. The expression plasmid
pcDNA3.1–FLAG–ubiquitin [9] was used for the expres-
sion of N-terminally FLAG-tagged human ubiquitin
(FLAG–ubiquitin). The RBCK2DNZF gene was generated
by replacing the sequences encoding four cysteines (Cys187,
Cys190, Cys201, and Cys204) in the NZF motif with
sequences encoding serines. For bacterial expression of
GST-fused RBCK2 (GST–RBCK2) and RBCK2DNZF
(GST–RBCK2DNZF), plasmids pGEX-6P-1–RBCK2 and
pGEX-6P-1–RBCK2DNZF were constructed by inserting
cDNA fragments encoding full-length RBCK2 and
RBCK2DNZF into pGEX-6P-1 (GE Healthcare, Chalfont
St Giles, UK).
Western blot analysis
HEK293 cells were cultured in DMEM supplemented with
10% (v ⁄ v) fetal bovine serum at 37 °C under humidified air

Immunofluorescence analysis
Approximately 5 · 10
4
HEK293 cells were grown on a
2.7 cm cover glass, washed twice with NaCl ⁄ P
i
(pH 7.2),
and fixed with 100% (v ⁄ v) methanol at )20 °C for 10 min.
These cells were permeabilized with 0.15% (v ⁄ v) Triton
X-100 in NaCl ⁄ P
i
(pH 7.2) for 15 min at room tempera-
ture, washed twice with NaCl ⁄ P
i
(pH 7.2), incubated with
blocking buffer [NaCl ⁄ P
i
(pH 7.2) containing 0.03% (v ⁄ v)
Triton X-100, 2% (w ⁄ v) BSA, and 3% (v ⁄ v) normal goat
serum] for 30 min at room temperature, and incubated with
the blocking buffer containing a primary antibody (a rabbit
polyclonal antibody against RBCK1 ⁄ 2 (dilution, 1 : 200)
[12] and a mouse monoclonal antibody against the 20S
proteasome (clone HP810; BIOMOL, Plymouth Meeting,
PA, USA) (dilution, 1 : 50)] for 30 min at room tempera-
ture. The cells were washed four times with NaCl ⁄ P
i
(pH
7.2) containing 0.03% (v ⁄ v) Triton X-100 for 10 min at
room temperature, incubated with the blocking buffer con-

primary antibody, and an HRP-conjugated anti-rabbit IgG
(GE Healthcare) (dilution, 1 : 10 000) as a secondary
antibody.
Immunoprecipitation assay
HEK293 cells (approximately 1 · 10
7
cells) were transfected
with pTB701–FLAG–RBCK2 (5 lg), pTB701–FLAG–
RBCK2DUBL (5 lg) or pTB701–HA–S5a (2 lg) by electro-
poration, and cultured for 48 h. When MG132 was applied,
the cells were pretreated with 50 lm MG132 at 37 °C for
4 h. The cells were washed twice with NaCl ⁄ P
i
(pH 7.2),
and lysed in 1 mL of lysis buffer. The cleared lysate was
mixed with a mouse monoclonal antibody against S5a
(clone S5a-18; BIOMOL) (2 lg), a mouse monoclonal anti-
body against HA (clone 12CA5; Roche) (10 lg), or a
mouse monoclonal antibody against FLAG (clone M2;
Sigma) (10 lg), incubated on ice for 60 min, mixed with
30 lL of protein G Sepharose 4 Fast Flow [50% (v ⁄ v)
slurry] (GE Healthcare), and incubated with rotation at
4 °C for 30 min. The beads were washed four times with
lysis buffer, resuspended in 35 lL of Laemmli’s sample
buffer, and subjected to SDS ⁄ PAGE. The samples were
blotted onto a poly(vinylidene difluoride) membrane, and
western blot analysis was carried out using an HRP-conju-
gated antibody against FLAG, an antibody against S5a
(dilution, 1 : 500), an HRP-conjugated antibody against
HA, or a mouse monoclonal antibody against ubiquitin

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