KCTD5, a putative substrate adaptor for cullin3

ubiquitin ligases
Yolanda Bayon1, Antonio G. Trinidad1, Mara L. de la Puerta1, Mara del Carmen Rodrguez1,
Jori Bogetz2, Ana Rojas3, Jose M. De Pereda4, Souad Rahmouni5, Scott Williams2,
Shu-ichi Matsuzawa6, John C. Reed6, Mariano Sanchez Crespo1, Tomas Mustelin2 and
Andres Alonso1
1 Instituto de Biologa y Genetica Molecular, CSIC-Universidad de Valladolid, Spain
2 Program of Inflammation, Inflammatory and Infectious Disease Center, and Program of Signal Transduction, Burnham Institute for Medical
Research, La Jolla, CA, USA
3 Structural Bioinformatics Group, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain
4 Centro de Investigacion del Cancer, CSIC-Universidad de Salamanca, Spain
5 Department of Pathology B-35, University of Lie`ge, CHU of Lie`ge, Belgium
6 Program of Apoptosis and Cell Death, Burnham Institute for Medical Research, La Jolla, CA, USA

Keywords
BTB; cullin; E3 ligases; KCTD; ubiquitin
Correspondence
A. Alonso, Instituto de Biologa y Genetica
Molecular, CSIC-Universidad de Valladolid,
c Sanz y Fores s n, 47003 Valladolid, Spain
Fax: +34 983 184800
Tel: +34 983 184839
E-mail: andres@ibgm.uva.es
(Received 7 April 2008, revised 30 May
2008, accepted 3 June 2008)
doi:10.1111/j.1742-4658.2008.06537.x

Potassium channel tetramerization domain (KCTD) proteins contain a

bric-a-brac, tramtrak and broad complex (BTB) domain that is most similar to the tetramerization domain (T1) of voltage-gated potassium channels. Some BTB-domain-containing proteins have been shown recently to
participate as substrate-specic adaptors in multimeric cullin E3 ligase reactions by recruiting proteins for ubiquitination and subsequent degradation
by the proteasome. Twenty-two KCTD proteins have been found in the
human genome, but their functions are largely unknown. In this study, we
have characterized KCTD5, a new KCTD protein found in the cytosol of
cultured cell lines. The expression of KCTD5 was upregulated post-transcriptionally in peripheral blood lymphocytes stimulated through the T-cell
receptor. KCTD5 interacted specically with cullin3, bound ubiquitinated
proteins, and formed oligomers through its BTB domain. Analysis of the
interaction with cullin3 showed that, in addition to the BTB domain, some
amino acids in the N-terminus of KCTD5 are required for binding to
cullin3. These ndings suggest that KCTD5 is a substrate-specic adaptor
for cullin3-based E3 ligases.

The BTB (bric-a-brac, tramtrak and broad complex) POZ (poxvirus zinc nger) domain is a protein
protein interaction domain rst described in several
proteins of Drosophila melanogaster and poxvirus [1,2].
BTB POZ domain-containing proteins constitute a
diverse group of proteins involved in transcriptional
repression, cytoskeletal regulation, and ion channel
function [3]. More recently, some BTB proteins have
been characterized as substrate-specic adaptors for

cullin(CUL)3-based E3 ligases [47]. The BTB domain

of these substrate-specic adaptors binds to CUL3,
whereas additional domains in these polypeptides, such
as zinc ngers, meprin and traf homology (MATH)
domain, and Kelch repeats, work as substrate recognition domains. The rst protein shown to be regulated
by a CUL3 ligase was MEI-1 in Caenorhaditis elegans.
This protein is part of the katanin-like microtubule
severing complex [5,6] and is recruited to CUL3 by the

Abbreviations
AU, arbitrary unit; BTB, bric-a-brac, tramtrak and broad complex; CT, cycle threshold; CUL, cullin; GFP, green fluorescent protein; GST,
glutathione S-transferase; HA, hemagglutinin; IL-2, interleukin-2; KCTD, potassium channel tetramerization domain; MATH, meprin and traf
homology; PBL, peripheral blood lymphocyte; PHA, phytohemagglutinin; PMA, 4b-phorbol 12-myristate 13-acetate; POZ, poxvirus zinc finger;
Ub, ubiquitin.

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Y. Bayon et al.

BTB protein MEL-26. In mammalian cells, a few other

BTB proteins, e.g. SPOP, a BTB-MATH protein, and
KEAP1, a BTB-KELCH protein, have been described
as adaptors of CUL3-based E3 ligases [8]. CUL3 is
one of the seven cullins found in the human genome
(CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5 and
CUL7), and most of them bind to adaptors through
their BTB domains, which, in turn, bind to additional
proteins that work as substrate-specic adaptors. Thus,
in SKP1CUL1F-box, the archetypical cullin E3
ligase, CUL1 binds on the N-terminus to the adaptor
Skp1 that associates with an F-box protein working as
substrate-specic adaptor, and on the C-terminus to
the RING domain-containing protein Roc1 Rbx Hrt
[9]. Cullin E3 ligases are multimeric RING E3 ligases
that participate in protein ubiquitination, a process
mediated by a three-step enzymatic cascade. Ubiquitin
(Ub) is initially activated by the Ub-activating enzyme
(E1) and then transferred to a Ub-conjugating enzyme
(E2), which associates with a third protein, the Ub
ligase (E3), involved in recruiting the substrates for
ubiquitination and, therefore, providing specicity to
this process [10]. Ubiquitination is involved in a wide
range of cellular functions, such as cell proliferation,
differentiation, and apoptosis, mainly by targeting proteins for degradation by the 26S proteasome, but it is
also involved in protein transport and signaling
through additional mechanisms [10,11].
Although the human genome might include about
400 BTB proteins [8], only a few have been shown to
work as substrate-binding proteins for CUL3 E3 ligases.
In this connection, potassium channel tetramerization
domain (KCTD) proteins form a group of proteins
containing a BTB domain, the function of which is largely unknown. Herein, we report the characterization
of KCTD5, a new POZ BTB protein that is a putative
new substrate-specic adaptor for CUL3-based E3
ligases.

Results and Discussion

KCTD5 was identied in a yeast two-hybrid screening
for the dual-specicity phosphatase VHR while looking for adaptors that help us to understand how this
phosphatase targets its substrates, Erk and Jnk. The
clone obtained in this assay contained a cDNA
sequence present in public databases with the Genbank
accession number NM_018992. Next, we tested VHR
interaction with KCTD5 in mammalian cells, and
could not nd evidence for this. Nevertheless, we continued the study of this new protein. First, we studied
the expression of this gene, nding that its mRNA was
expressed in all the tissues and cell lines tested

KCTD5, a new substrate-specific adaptor for Cul3

(Fig. 1A). On the contrary, protein expression was

only observed in transformed cells and was absent
from primary cells, such as peripheral blood leukocytes
(PBLs), mouse brain cells or human brain cells
(Fig. 1B, lanes 6, 9 and 10), thus suggesting that its
expression was upregulated post-transcriptionally.
Prompted by these results, especially by the differences
observed between the expression of mRNA and protein in PBLs, we hypothesized that KCTD5 might be
induced by mitogens such as as phytohemagglutinin
(PHA) and interleukin-2 (IL-2) in these cells. Using
these stimuli, we observed a 2.5-fold increase (Fig. 1D,
lane 5) in mRNA expression and an 84.7-fold increase
in protein expression at 48 h (Fig. 1C, lane 7) in PBLs
stimulated with PHA. To investigate whether other
stimuli known to induce T-cell proliferation increase
KCTD5 protein, 4b-phorbol 12-myristate 13-acetate
(PMA) plus ionomycin and a combination of antibodies for the T-cell and CD28 receptors, which mimic
antigen stimulation, were used. As shown in Fig. 1E,
these stimuli increased KCTD5 protein to an extent
similar to that observed for PHA. As RT-PCR assays
lack enough sensitivity to detect changes in the amount
of mRNA, quantitative PCR assays were conducted in
order to detect subtle changes in KCTD5 mRNA. As
shown in Fig. 1F, there was only a slight decrease of
KCTD5 mRNA after PHA stimulation of PBLs. These
data suggested that these stimuli regulated KCTD5 at
a post-transcriptional level, by increasing either the
translation or the stability of KCTD5 protein. In the
latter case, this would imply that KCTD5 is an unstable protein in the absence of stimuli. In this regard,
treatment of resting PBLs with MG132, a proteasome
inhibitor, has no effect on KCTD5 protein (data not
shown), meaning that stimulus-dependent translation is
involved in increasing the quantity of KCTD5 protein
in PBLs. Altogether, these data suggest that KCTD5
expression is mainly regulated by a post-transcriptional
mechanism in PBLs, possibly at the translational level.
Several databases were searched to nd homologs of
KCTD5, using as query its BTB domain. Although the
BTB domain is present in proteins from all eukaryotic
groups, when the query included the KCTD5 C-terminal region in addition to the BTB domain, homologs
were only found among the metazoans. However, no
protein was found with a BTB domain followed by the
C-terminus of KCTD5 in plants and fungi. An alignment of KCTD5 orthologs in several species is shown
in Fig. 2A. Among BTB proteins, KCTD5 is grouped
with potassium channels. The similarity with potassium channels is restricted to the T1 domain, which is
a BTB domain. Whereas cullins are present in all
eukaryotes, KCTD5-like proteins appeared later in

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KCTD5, a new substrate-specific adaptor for Cul3

Fig. 1. KCTD5 expression. (A) RNA from different tissues and cell lines was analyzed by RT-PCR using specific primers for KCTD5. A plasmid encoding KCTD5 was used as a positive control (lane 1) for the RT-PCR. (B) Expression of KCTD5 in different cell types detected by
immunoblot with antibody to KCTD5 (upper panel). b-Actin was detected by immunoblot on the same membrane as an internal control of
protein loading (lower panel). (C) Time course of the expression of KCTD5 protein in PBLs stimulated with PHA. (D) Time course of the
expression of KCTD5 mRNA in PBLs stimulated with PHA, where numbers indicate hours of stimulation. (E) Expression of KCTD5 protein in
PBLs subjected to various stimuli. (F) Levels of KCTD5 mRNA assayed by quantitative PCR in PBLs stimulated with PHA. TCR+CD28 indicates antibodies specific for T-cell receptor plus CD28.

evolution in multicellular organisms, most likely to full a new function, which is at the present time
unknown. Searches for human paralogs, using as query
the BTB domain of KCTD5 to generate a phylogenetic
tree (Fig. 2B), gave 22 sequences. Some of these
human paralogs are found in highly similar groups
with conserved sequences out of the BTB domain used
for this analysis, e.g. the group constituted by KCTD5,
KCTD2, and KCTD17. Elements recently cloned have
been included in two groups: (a) the group formed by
polymerase d and proliferating cell nuclear antigeninteracting proteins, tumor necrosis factor-a-induced
protein 1 [12], KCTD13 product polymerase deltainteracting protein 1 [12], and KCTD10 [13]; and (b)
the group formed by the leftover-related proteins
KCTD8, Pfetin (predominantly fetal expressed T1
domain) (KCTD12), and KCTD16, which are involved
in development [14]. For the remaining sequences there
are clear paralogy relationships, which indicate close
relationships within the sequences, as in the case of
KCTD3 and Q8TBC3, a human homolog of mouse
seta-binding protein-1 [15], KCTD1 and KCTD15,
and KCTD21 and KCTD6. Most of these sequences
remain uncharacterized. This analysis of KCTD
sequences shows that they form a group clearly differentiated from the voltage-gated potassium channels,
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not only by the absence of transmembrane domains,

but also on the basis of the differences in BTB
sequences.
To determine the subcellular localization of KCTD5,
green uorescent protein (GFP)KCTD5 was transfected and detected by confocal microscopy (Fig. 3A).
Whereas GFP alone is found in the nucleus as well as
in the cytosol, fusion of KCTD5 to GFP restricts the
expression of the fusion protein, GFPKCTD5, to the
cytosol. Furthermore, HEK293 cells were transfected
with a plasmid expressing mycKCTD5, and this
protein was detected by immunocytochemistry in the
cytosol (Fig. 3B). As it has been recently reported that
deletion of the C-terminus of KCTD5 [16] changes its
location to the nucleus, cells were transfected with
different deletion mutants of KCTD5. Immunocytochemistry of these cells showed that these constructs
were again detected in the cytosol (Fig. 3B). Therefore,
in our hands, KCTD5 is detected only in the cytosol.
As we had a specic antibody for KCTD5, we tried
several times to reveal the endogenous protein with
this antibody, but we could not see any specic binding, so we consider that this antibody is not suitable
for immunocytochemistry.
Although it has been proposed that all the proteins
containing a BTB domain are substrate-specic

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Y. Bayon et al.

KCTD5, a new substrate-specific adaptor for Cul3

Fig. 2. Analysis of KCTD5 homologs. (A) Multiple protein sequence alignment of various KCTD5 orthologous sequences from different species. (B) Phylogenetic tree built from human paralogs of KCTD5 using the BTB domain of 23 peptides. The BTB domain (T1 domain) of the
voltage-gated potassium channel KCNC1 protein is included in the analysis to root the tree.

adaptors for cullin ubiquitin ligases [5,6], in the case of

CUL3, most of the adaptors described so far belong to
the Kelch group. Thus, we investigated whether

KCTD5 could interact with CUL3. To test this interaction, HEK293 cells were transfected with plasmids
encoding CUL1, CUL2, CUL3, CUL4A and CUL4B

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KCTD5, a new substrate-specific adaptor for Cul3

Fig. 3. Subcellular localization of KCTD5. (A) Left panels: fluorescence images of HEK293 cells transfected with either GFP or GFP
KCTD5. Right panels: phase contrast images of the same cells. (B)
Immunofluorescence staining of HEK293 cells transfected with
plasmids encoding KCTD5 and several deletion mutants with mAb
to myc followed by a secondary antibody labeled with Alexa
Fluor 594.

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along with KCTD5. Total lysates were prepared from

these cells and used for immunoprecipitation assays. A
specic interaction of KCTD5 with CUL3 was
observed (Fig. 4A, lane 6), but not with the other cullins (Fig. 4A, lane 5 for CUL1 and data not shown).
This interaction was conrmed in primary cells by carrying out immunoprecipitation assays in lysates from
PBLs stimulated with PHA for 2 days. Under these
conditions, CUL3 was detected by immunoblot in
KCTD5 precipitates (Fig. 4B, lane 2), but not when
the immunoprecipitation was carried out with an irrelevant antibody (Fig. 4B, lane 1). Then, the ability to
form a functional E3 ligase complex with CUL3 and
Rbx1 was assayed. Expression vectors for these proteins were transfected into HEK293 cells, and cell
lysates were subjected to immunoprecipitation with
antibody to myc. As KCTD5 was precipitated when
CUL3 was present in the lysate (Fig. 4C, lane 5), this
result indicates that KCTD5 is part of a canonical cullin-based E3 ligase complex. A faint band is also seen
in Fig. 4C (lane 2) that is probably due to the interaction of Rbx1 with endogenous CUL3.
We also addressed whether KCTD5 could be ubiquitinated, based on the fact that other BTB adaptor proteins have been shown to be substrates of E3 ligases.
To do this, we transfected cells with expression vectors
for mycUb and hemagglutinin (HA)KCTD5, and
cell lysates were immunoprecipitated with an antibody
specic for HA. The precipitates showed the presence
of ubiquitinated proteins (Fig. 4D) by immunoblotting.
To distinguish between covalent and noncovalent Ub
binding to KCTD5, we repeated this experiment, lysing the cells with a highly denaturing buffer containing
8 m urea. Under these conditions, no smear was
detected in the KCTD5 immunoprecipitation and nor
was a KCTD5 ladder observed in Ub precipitates,
which is typical of ubiquitinated proteins (data not
shown). In addition, we could not detect KCTD5
ubiquitination in in vitro assays (data not shown).
Thus, unlike to what has been described for other
BTB proteins that work as substrate-specic adaptors,
KCTD5 is not ubiquitinated.
The interaction between BTB proteins and CUL3 is
considered to be mediated by the BTB domain and
the N-terminal region of CUL3 [8], mainly on the
basis of assays in which deletion of the BTB domain
and the N-terminal region of CUL3 is accompanied
by loss of binding. To analyze in detail KCTD5 binding to CUL3, pull-down and immunoprecipitation
assays with a series of deletion mutants of KCTD5
and CUL3 were carried out (Fig. 5A,C). These experiments showed that the C-terminal region of KCTD5
was dispensable for CUL3 interaction, whereas the

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KCTD5, a new substrate-specific adaptor for Cul3

Fig. 4. KCTD5 interacts with CUL3 and ubiquitinated proteins. (A) HEK293 cells were transfected with plasmids encoding mycKCTD5, HA
CUL1, and HACUL3, as indicated. Cell lysates were subjected to immunoprecipitation (IP) with antibody to myc followed by immunoblotting
with antibodies to HA and myc. Expression of the tagged proteins is shown in the lower panels as WCL (whole cell lysate). (B) Lysates from
PBLs treated with PHA for 2 days (upper panel) were immunoprecipitated with either KCTD5 or an irrelevant IgG antibody and then blotted
with antibodies to CUL3 (upper panel) and KCTD5. The panels marked WCL show the expression levels of KCTD5 and CUL3 in the PBL
whole cell lysates. (C) HEK293 cells were transfected with plasmids encoding mycRbx1, HACUL3 and GSTKCTD5, lysates from these
cells were processed for pull-down with GST beads, and the presence of KCTD5 in the precipitates was checked by western blotting with
antibody to GST, followed by anti-HA and anti-myc blots to detect HACUL3 and mycRbx1. WCLs were immunoblotted with antibodies to
GST, HA and myc to assess the expression of the tagged proteins. (D) HEK293 cells were transfected with plasmids encoding for
HAKCTD5 and mycubiquitin. Cell lysates were immunoprecipitated with antibody to HA, and ubiquitinated proteins that interact with
KCTD5 were detected with antibody to myc. WCLs were immunoblotted with the antibodies to HA and myc to assess the expression of
the tagged proteins.

BTB domain alone (45145 amino acids), although

essential for this interaction, was not sufcient
(Fig. 5B). In fact, it required additional amino acids
(4045) on the N-terminus, outside of the BTB fold,
as the 40145 amino acid peptide is the smallest moiety able to interact with CUL3. Studies with other
BTB proteins, e.g. SPOP [17]or the BTB protein
At1g21780 from Arabidopsis thaliana [18], have also
shown that other parts of their sequence, in addition

to the BTB domain, are involved in the association

with CUL3. On the other hand, the CUL3 region
involved in this interaction was the N-terminus, as
described for other BTB proteins, because a deletion
of 75 amino acids in the N-terminus of CUL3 completely abrogated the binding of KCTD5 to CUL3
(Fig. 5D, lane 8). Therefore, this detailed study on
the interaction of KCTD5 with CUL3 shows that the
sole BTB domain of KCTD5 does not support this

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KCTD5, a new substrate-specific adaptor for Cul3

Fig. 5. Mapping the interaction of KCTD5 with CUL3. (A) Schematic diagram of the several KCTD5 deletion mutants used in this study. (B)
Plasmids for KCTD5 and different deletion mutants expressed as GST fusion proteins were transfected, along with HACUL3, in HEK293
cells. Lysates were subjected to pull-down assays with glutathioneSepharose beads, and the presence of CUL-3 in the precipitates was
detected by immunoblot with antibody to HA, followed by antibody to GST. The expression of the proteins was checked in the whole cell
lysate (WCL) by western blot with antibodies to HA and CUL3. (C) Schematic diagram of the CUL3 deletion mutants used in this study. (D)
mycKCTD5 was expressed in HEK293 cells along with different deletion mutants of HACUL3. The presence of the different CUL3 peptides was checked in the myc immunoprecipitates by western blot with an antibody to HA. mycKCTD5 was detected in the immunoprecipitates by immunoblot with antibody to myc. The same antibodies were used to show the expression in the WCL (lower panels).

association and requires additional amino acids in the

N-terminus of this domain.
As the BTB domain is responsible for homo-oligomerization in BTB proteins [3], we addressed whether
KCTD5 might form homo-oligomers. For this purpose,
HEK293 cells were transfected with different constructs
of KCTD5 to show this association by either immunoprecipitation or pull-down assays (Fig. 6A,B). We found
the BTB domain to be essential for KCTD5 oligomerization, as peptides expressing the KCTD5 N-terminal
region (N55) or the C-terminal sequence (POZCO,
amino acids 145234) could not interact with themselves
(Fig. 6B). As the POZ BTB domain of KCTD5 is distantly related to the T1 domain of voltage-gated potassium channels, this fact was taken as a hint that KCTD5
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could also tetramerize. To address this issue, gel exclusion chromatography was run with recombinant
KCTD5 protein and KCTD5 was collected in fractions
consistent with the estimated molecular mass of an octamer (Fig. 6C), which in turn can be explained by the
formation of two tetramers.
Taken together, our results show that the BTB
domain of KCTD5 is not able to bind alone to CUL3,
indicating that although it is critical for this association, other sequences contribute to the binding of substrate-specic adaptors to CUL3, namely, ve amino
acids in the N-terminus of the BTB domain. In addition to the BTB fold, KCTD5 presents two other
regions: 40 amino acids in the N-terminal sequence,
which include a low-complexity region (1233 amino

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Y. Bayon et al.

Fig. 6. KCTD5 oligomerization. (A) HEK293 cells were transfected

with plasmids encoding for GSTKCTD5 and different deletion
mutants of KCTD5, and cell lysates were subjected to pull-down
with GlutathioneSepharose beads and immunoblotted with a specific antibody to FLAG followed by antibody to GST. (B) HEK293
cells were transfected with HAKCTD5 and several plasmids that
expressed different deletion mutants of KCTD5 as GST-fusion proteins. Anti-HA immunoprecipitates of the cell lysates were analyzed
by immunoblot with antibody to GST followed by antibody to HA.
(C) Gel filtration chromatography of KCTD5 recombinant protein
produced in bacteria. The presence of KCTD5 in the fractions was
analyzed by immunoblot with antibody to KCTD5. Numbers under
the arrows indicate the chromatography fractions in which molecular mass markers are eluted.

acids), and 88 amino acids in the C-terminus (POZCO). Taking into account that KCTD5 could be an
adaptor of CUL3 E3 ligases, we favor the hypothesis
that the POZCO region could participate in substrate

KCTD5, a new substrate-specific adaptor for Cul3

recognition, and that this could be a new protein interaction domain conserved through evolution, as seen in
orthologs. The fact that KCTD5 can form octamers
and the recent description of heterodimerization of
CUL3 [19] would indicate that complexes of higher
order could be formed among CUL3 and BTB
substrate adaptors, implying the recruitment of a great
number of substrates by these E3 ligases.
Although scarce, the information available about
KCTD proteins suggests that these proteins might be
involved in development and cellular differentiation.
For example, in zebra sh, three members of this group
lov (leftover), ron (righton), and dex (dexter) are
expressed asymmetrically in the left and right zebrash
diencephalons [14]. Pfetin, a human ortholog of lov and
ron genes, encoded by human gene KCTD12, is detected
as mRNA preferentially expressed in fetal organs [20],
with the highest expression levels in the cochlea.
Another KCTD protein, KCTD11 REN, is also regulated developmentally in the nervous system [21], and it
has been implicated in the regulation of the Hedgehog
pathway [22]. The information presented in this article
would indicate that KCTD proteins might function by
recruiting specic substrates involved in development
and cellular differentiation for ubiquitination by CUL3
Ub ligases and degradation by the proteasome. As
regards KCTD5, there is another report that shows its
ability to interact with two viral regulatory proteins,
Rep68 and Rep78, of the adeno-associated virus type 2,
which are essential for viral DNA replication and gene
expression [16], although no relationship was established
with CUL3.
In summary, in this study we present evidence that
KCTD5 is a new substrate-specic adaptor for CUL3based Ub ligases. Our data indicate that a relevant
mechanism underlying the physiological role of KCTD
proteins includes recruitment of proteins to CUL3based E3 Ub ligases for degradation in the proteasome. As identication of substrates recruited to the
proteasome would be very valuable for understanding
the function of these proteins, we are pursuing
the identication of KCTD5-interacting proteins,
especially those that are ubiquitinated.

Experimental procedures
Antibodies and reagents
Tissue culture reagents were from Cambrex (Verviers,
Belgium). The 12CA5 mAb against HA was from Roche
(Indianapolis, IN, USA), anti-HA clone HA.11 was from
Covance (Berkely, CA, USA), anti-glutathione S-transferase
(GST) and mAb against myc (9E10) were from Santa Cruz

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KCTD5, a new substrate-specific adaptor for Cul3

Biotechnology Inc. (Santa Cruz, CA, USA), anti-cullin 3 was

from Abcam (Cambridge, UK), and mAbs against b-actin,
PHA, FLAG M2 mAb and PMA were from Sigma Chemical
Co. (St Louis, MO, USA). Antibodies against CD3
(UCHT1) and CD28 (clone CD28.2) were from BD Pharmingen (Franklin Lakes, NJ, USA). MG-132 was from Calbiochem (Darmstadt, Germany). IL-2 was from PreprotechEC
(Rocky Hill, NJ, USA). Goat anti-(mouse IgG) conjugated
with Alexa Fluor 594 was from Molecular Probes (Eugene,
OR, USA). A mouse mAb was raised against recombinant
full-length KCTD5. Human MTC panel II was from
Clontech (Mountain View, CA, USA).

Plasmids and mutagenesis

Standard molecular biology techniques were used to generate the different constructs used in this study. All constructs
were veried by nucleotide sequencing. KCTD5 from a
Jurkat cDNA library obtained from Origene (Rockville,
MD, USA) was cloned in the pEF plasmid and served as a
template for the different KCTD5 plasmids used in this
study. HAcullin1 and HAcullin3 expression plasmids
were a kind gift of C. Geisen (Department of Medical
Oncology, Dana-Farber Cancer Institute, Boston, MA,
USA). Cullin4A and cullin4B were generously provided by
K. Tanaka (Department of Molecular Oncology, Tokyo
Metropolitan Institute of Medical Science, Japan) [23].

Cell culture and transfections

PBLs were isolated from buffy coats of healthy donors by
centrifugation at 700 g for 30 min on FicollHypaque (GE
Healthcare) cushions. Monocytes macrophages were eliminated by adherence to plastic for 1 h at 37 C. Proliferation
was induced by PHA and IL-2, which was added after 48 h
with PHA, antibodies to CD3 plus antibodies to CD28, or
PMA plus ionomycin. Jurkat T-leukemia cells were kept at
logarithmic growth in RPMI-1640 medium supplemented
with 10% fetal bovine serum, 2 mm l-glutamine, 1 mm
sodium pyruvate, nonessential amino acids, 100 UmL)1
penicillin G, and 100 lgmL)1 streptomycin. Transfection
of Jurkat T cells was performed as described previously
[24]. HEK293 cells were maintained at 37 C in DMEM
supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 UmL)1 penicillin G, and 100 lgmL)1 streptomycin. For transient transfection, HEK293 cells were
transfected using the calcium phosphate precipitation
method [25].

Immunoprecipitation, GST pull-down,

SDS PAGE, and immunoblotting
These procedures were performed done as reported previously [24]. Briey, cells were lysed in 20 mm Tris HCl,

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pH 7.5, 150 mm NaCl, 5 mm EDTA containing 1% NP-40,

1 mm Na3VO4, 10 lgmL)1 aprotinin and leupeptin, and
1 mm phenylmethanesulfonyl uoride, and claried by
centrifugation at 16 000 g for 10 min. The claried lysates
were preabsorbed on protein G-Sepharose and then incubated with antibody for 2 h; this was followed by overnight
incubation with protein G-Sepharose beads. Immune complexes were washed three times in lysis buffer and resuspended in SDS sample buffer. Proteins resolved by
SDS PAGE were transferred to a nitrocellulose membrane,
and immunoblotted with optimal dilutions of specic antibodies followed by the appropriate anti-IgGperoxidase
conjugate. Blots were developed by the enhanced chemiluminescence technique (ECL kit; GE Healthcare) according
to the manufacturers instructions. Pull-down of GST
fusion proteins was performed with glutathioneSepharose
beads (GE Healthcare) incubated with the claried lysates
for 2 h. The complexes were then washed and processed as
explained above for the immunoprecipitation. Some blots,
after being developed by chemiluminescence, were visualized with a Bio-Rad VersaDoc chemiluminescence imager.
In this case, quantitation was carried out using quantity
one software from Bio-Rad.

RT-PCR
Total cellular RNA was extracted by the TRIzol method
(Life Technologies, Grand Island, NY, USA). The conditions for cDNA rst-strand synthesis and PCR reactions
were as described previously [26]. To address more exactly
the expression of KCTD5 mRNA, real-time RT-PCR was
carried out in RNA samples treated with DNase (TurboDNA freeTM; Ambion, Austin, TX, USA). The resulting
cDNA was amplied in a PTC-200 apparatus equipped
with a Chromo4 detector (BioRad Laboratories), using
SYBR Green I mix containing HotStart polymerase
(ABgene, Epsom, UK). b-Actin was used as a housekeeping
gene to assess the relative abundance of KCTD5 mRNA,
using the comparative cycle threshold (CT) method for
relative expression. This method allows the relative
expression for a given cDNA using the formula: 2)DCT,
where DCT DCTKCTD5  DCTbactin [27]. Therefore, one arbitrary unit (AU) corresponds to the expression of b-actin.

Indirect immunofluorescence and confocal

microscopy
HEK293 cells were cultured on coverslips and transiently
transfected with the indicated plasmids. Cells transfected
with GFP plasmids were xed with 3.7% paraformaldehyde
and mounted on microscope slides, and GFP was then visualized on an MRC-1024 confocal laser scanning microscope
(Bio-Rad). Phase contrast images were also taken. Immunouorescence staining of transfected KCTD5 was performed

FEBS Journal 275 (2008) 39003910 2008 The Authors Journal compilation 2008 FEBS

Y. Bayon et al.

as described previously [24]. HEK293 cells were washed in

NaCl Pi, xed in 3.7% formaldehyde, permeabilized with
0.1% saponin in NaCl Pi, and blocked in the same medium
supplemented with 2.5% normal goat serum for 30 min at
room temperature. Primary and secondary antibodies were
diluted in the same buffer and incubated with the cells for 1 h
each at room temperature. After three washes with NaCl Pi,
the cells were mounted onto glass slides and viewed under a
confocal laser scanning microscope.

Gel filtration chromatography

For gel ltration chromatography, we used recombinant
KCTD5 produced in bacteria as His6-KCTD5 after
removal of the His-tag with thrombin. The protein solution
was fractionated through a Superdex 200 fast protein liquid
chromatography column (GE Healthcare), and collected in
fractions of 500 lL. Protein was precipitated with 10% trichloroacetic acid and washed with acetone before addition
of SDS sample buffer and analysis by 10% SDS PAGE.

Sequence analysis and alignments

For sequence retrieval, the BTB domain of human KCTD5
was used as query to retrieve the orthologs from the
UniPROT (http://www.ebi.uniprot.org/index.shtml) database using the blast algorithm [28]. psi-blast [29] searches
retrieved 22 human paralogs. Multiple sequence alignments
of the BTB domain were conducted using muscle [30] and
probcons [31] in both the orthologs and the paralogs. To
generate reliable phylogenetic trees, Bayesian inference
using mrbayes v3.1.2 software was applied [32]. Multiple
alignments were done in two independent runs, with four
independent Markov chains in each run. One thousand ve
hundred samples were used to estimate the posterior probability distribution. The amino acid model is a xed rate
model using a mixture of xed models. To compute a consensus tree, we sampled 2502 from a total of 3002 trees in
two independent les (thus discarding 16% of the initial
samples prior to convergence). To root the tree, the
sequence of the BTB domain (T1) of the voltage potassium
channel KCNC1_HUM is included in the analysis.

Acknowledgements
We are grateful to Dr Keiji Tanaka for the CUL4A
and CULB cDNAs, to Dr Cristoff Geisen for the
CUL1 and CUL3 plasmids, and to Dr Joan Conaway
for the mycRbx1 plasmid. We thank the staff of
Centro de Hemoterapia y Hemodonacion de Castilla y
Leon for its help with the separation of leukocytes.
This work was supported by a grant from Programa
Nacional de Biolog a Fundamental (Grant BFU200601203 BMC), Red Cardiovascular from Instituto de

KCTD5, a new substrate-specific adaptor for Cul3

Salud Carlos III. Y. Bayon is under contract within

the Ramon y Cajal Program of the Ministerio de Educacion y Ciencia of Spain, co-funded by the European
Social Fund through FEDER-FSE.

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