Role of Plant-Specific N-Terminal Domain of Maize CK2b1
Subunit in CK2b Functions and Holoenzyme Regulation
Marta Riera1., Sami Irar1., Isabel C. Ve ´lez-Bermu ´dez1, Lorenzo Carretero-Paulet1,2¤, Victoria
Lumbreras1, Montserrat Page `s1*
1Department of Molecular Genetics, Centre for Research on Agricultural Genomics CRAG (CSIC-IRTA-UAB), Barcelona, Spain, 2Department of Applied Biology (Area of
Genetics). University of Almerı ´a, Spain
Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a
heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2a) and the regulatory (CK2b). The CK2b
subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions
independently of the CK2 tetramer. CK2b regulatory subunits in plants differ from their animal or yeast counterparts, since
they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any
previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2b subunit
sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites.
By using maize CK2b1 and a deleted version (DNCK2b1) lacking the N-terminal domain, we have demonstrated that CK2b1
is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2a1/DNCK2b1 is able to
phosphorylate different substrates more efficiently than CK2a1/CK2b1 or CK2a alone. Transient overexpression of CK2b1
and DNCK2b1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation
in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence
complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to
the individual CK2a/b subunits mainly observed in the nucleus. All together, our results support the hypothesis that the
plant-specific N-terminal domain of CK2b subunits is involved in the down-regulation of the CK2 holoenzyme activity and in
the stabilization of CK2b1 protein. In summary, the whole amount of data shown in this work suggests that this domain was
acquired by plants for regulatory purposes.
Citation: Riera M, Irar S, Ve ´lez-Bermu ´dez IC, Carretero-Paulet L, Lumbreras V, et al. (2011) Role of Plant-Specific N-Terminal Domain of Maize CK2b1 Subunit in
CK2b Functions and Holoenzyme Regulation. PLoS ONE 6(7): e21909. doi:10.1371/journal.pone.0021909
Editor: Arthur J. Lustig, Tulane University Health Sciences Center, United States of America
Received December 23, 2010; Accepted June 14, 2011; Published July 15, 2011
Copyright: ? 2011 Riera et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: MR was financed by I3P-CSIC2006, ICV-B by predoctoral fellowship FPI2007 from MICINN (Spain) and LC-P by Juan de la Cierva Programme, MICINN
(Spain). This work was also supported by grant BIO2009-13044-CO2-01 from MICINN (Spain) and CONSOLIDER-INGENIO 2010 MEC (CSD2007-00057). The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
¤ Current address: Integrative Systems Biology Group, Institute for Plant Molecular and Cell Biology - IBMCP (CSIC-UPV), Valencia, Spain
Protein kinase CK2 is a constitutively active, highly conserved
serine/threonine protein kinase that is ubiquitously distributed in
eukaryotes. CK2 is one of the most pleiotropic kinases known, able
to phosphorylate and interact with multiple cellular proteins [1,2].
In mammals the typical CK2 holoenzyme is a heterotetrameric
complex composed of two catalytic (CK2a and CK2a9) and two
regulatory (CK2b) subunits. The CK2b regulatory subunits are
inactive and present no homology to regulatory subunits or
domains of other protein kinases. In the classical model of CK2
tetrameric holoenzyme, CK2b regulatory subunits are involved in
the assembly of CK2 tetrameric complexes, in enhancing catalytic
activity and stability of CK2a and in modulation of the substrate
specificity of CK2 . However, CK2b subunits also have
additional functions in addition to regulation of the holoenzyme,
since they can interact with and regulate other proteins in the
absence of CK2a subunits [4,5]. Structural analysis by X-ray
crystallographic assays shows that CK2 tetramers are subject to
disassembly and re-assembly . In addition, localization studies
of individual CK2 subunits indicate that both types of subunits
have been found in different compartments [7,8]. These findings
indicate that individual CK2 subunits may have an independent
role. All these evidences support the idea of the independent role
of the individual CK2 subunits versus the classical holoenzyme.
In plants CK2 is involved in relevant processes such as plant
growth and light-regulated gene expression , circadian rhythm
[10,11], cell-cycle regulation and development [12,13], salicylic
acid mediated defense  and abiotic stress responses .
CK2a/b subunits family is expanded in plant genomes relative to
animal genomes, since they belong to multigenic families
composed by up to 4 genes. As reported in animals, differential
subcellular localization of plant CK2 subunits suggests specific
functions for each CK2 subunit or CK2 isoform [15,16]. This
hypothesis is also supported by new findings showing that specific
CK2 holoenzyme isoforms can regulate the initiation of translation
in Arabidopsis . In maize, three genes for each CK2a/b have
been described to date [18–20]. A fourth CK2b gene (CK2b4) has
PLoS ONE | www.plosone.org1 July 2011 | Volume 6 | Issue 7 | e21909
been found in the Maize Genomic Database (MaizeGD) and is
included in this paper. Since it was crystallized , maize CK2a1
subunit has been widely studied as a model of CK2 structure and it
has been used successfully to design inhibitors of the holoenzyme
. This is due to the biochemical characteristics of maize
CK2a, which is highly stable and has more specific activity than
the human holoenzyme. Comparative studies demonstrate that the
maize holoenzyme is less stable than the human counterpart .
However, despite copious data on CK2a, little is known about
CK2b regulatory subunits and CK2 holoenzyme in maize. Plants
have a greater diversity of CK2b subunits than animals or yeasts
. Although plant CK2bs preserve in their central core the
characteristic CK2b features, they lacked 20 aminoacids from the
C-terminal domain and contain a specific N-terminal extension of
about 90 aminoacids. This N-terminal region shares no homology
with any previously characterized functional domain. The absence
of functional data about this domain prompted us to investigate its
putative role in: (i) CK2b functions and (ii) CK2 holoenzyme
regulation. Using maize CK2b1 and a deleted version lacking N-
terminal domain (DNCK2b1) we demonstrate that this plant-
specific N-terminal extension affects both CK2b and CK2
holoenzyme properties. In addition, we postulate a new role for
CK2b subunits in plants, since CK2b1 releases CK2a1 subunits
from the nucleolus and the CK2 holoenzyme can be found all over
the cell. These findings show that in vivo localization of the plant
CK2 holoenzyme is different from that of the independent CK2a/
b subunits alone. Even though the N-terminal domain of CK2b is
not involved in this export mechanism, the data reported here
indicates a role of this domain in regulation of both CK2b subunits
and CK2 holoenzyme in plants.
Sequence and evolutionary analysis of the N-terminal
domain of plant CK2b subunits
All plant CK2b subunits display an extra domain located N-
terminal to the highly conserved CK2b central region. These N-
terminal CK2b domains are poorly conserved both in length and
in primary sequence. At the amino acid composition level, they are
significantly enriched in phosphorylable residues such as Ser
(averaging ca. 10%), Thr and Tyr. Using the N-terminal domain
of maize CK2b1 as a query, BLAST searches were performed in
different protein databases, including the whole proteome of
selected plant species (Table S1). As a result, 34 sequences
corresponding to CK2b from 13 species representative of the main
land plant evolutionary lineages were identified (Table S2).
Additional searches of the protein databases were performed
through HMMer using as a query a hidden Markov models
(HMM) profile constructed on the basis of the alignment of 33 N-
terminal domains. Despite HMM profiles perform better in
detecting remote homologies , only land-plant species CK2b
sequences were detected. The architecture of conserved motifs
throughout the N-terminal domains was examined and represent-
ed over the corresponding alignment (Figure 1). Despite the high
degree of divergence within the N-terminal domain, 15 short
conserved motifs were found, some of them matching the
consensus phosphorylation sites for specific protein kinases (Table
S4), including putative CK2 autophosphorylation sites (motifs 1
and 5). Some motifs were highly conserved across almost every
sequence examined (e.g. motif 1, particularly rich in acidic amino
acids and including at least six Ser and/or Thr residues consensus
of CK2 phosphorylation) while many others were apparently
specific to certain evolutionary lineages (e.g. motif 5).
Genomic structure provides an independent criterion to assess
the evolutionary relatedness among genes and functional domains.
Exon/intron organization of plant CK2b for which the genomic
sequences were available was determined. In all land-plant CK2b
genes, location of the first intron was conserved at the same
relative position of the N-terminal domain, just before motif 1.
The first intron always showed phase 0 at the junction with exon 1
and phase 1 at the junction with exon 2 (Figure 1), supporting the
acquisition of the N-terminal domain by land plants as encoded by
a single exon.
To gain further insights into the evolutionary history of the land
plant CK2b N-terminal domain, we performed a phylogenetic
analysis of CK2b proteins from different eukaryotic kingdoms. For
this purpose, we constructed a sequence dataset of 69 CK2b
protein sequences, including sequences from animals and from
several non-land plant species (algae, fungi, and protists) also
displaying N-terminal extensions (Tables S2 and S3). Phylogenetic
Figure 1. Multiple Sequence Alignment of N-terminal domains of land plant CK2b regulatory subunits. Conserved not-overlapping
motifs identified in the MEME analysis are background-coloured. Positions in bold correspond to Serine and Threonine (S, T) residues predicted as
CK2 phosphorylation sites. Location of the first intron is underlined.
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analyses were conducted using two independent methods:
Neighbor Joining (NJ) and Maximum Likelihood (ML) [26–29].
A clade clustering all land-plant CK2b subunits could be
unambiguously retrieved in both NJ and ML trees (Figure S1
and S2) and is clearly separated from other clades grouping CK2b
from other organisms and also containing N-terminal extensions.
The N-terminal domain of maize CK2b1 affects the CK2
To ascertain whether the plant specific N-terminal domain of
maize CK2b1 affects CK2 holoenzyme regulation, we first
analyzed if the domain is needed for CK2 holoenzyme assembly,
CK2b/CK2b dimerization or interaction with CK2 substrates.
We prepared constructs harbouring different deletions of the
CK2b1 protein (Figure 2A) to perform two-hybrid assays. No
significant interaction was detected between empty AD/BD-clone
combinations (data not shown). Deletion del1 DNCK2b1 (80–276)
strongly interacts with other CK2a catalytic subunits (CK2a2) as
well as with full-length CK2b1. However, deletions del2 (180–
276), corresponding to CK2b without N-terminal domain and
acidic region and del3 (1–80), which corresponds to the N-
terminal domain alone, do not interact neither with CK2a2 nor
with CK2b1 subunits (Figure 2A). Therefore, these results
demonstrate that CK2b N-terminal domain is not essential for
We have previously demonstrated that recombinant maize
CK2a and CK2b subunits can assemble in a functional tetrameric
complex, and autophosphorylation of CK2b subunits demon-
strates the functionality of the holoenzyme . As previously
observed in animals, dimerization of CK2b subunits seems to be a
pre-requisite for holoenzyme formation . Here we have
reconstituted the active holoenzyme using the CK2a1 catalytic
subunit and the deleted version of CK2b1 subunit (del1
DNCK2b1 (80–276)) and we found that in absence of the N-
terminal domain of CK2b1, the CK2a1/DNCK2b1 holoenzyme
is also functional and autophosphorylable (Figure 2B). Compar-
CK2a1/CK2b1 and CK2a1/DNCK2b1 have been done and
quantification of the autophosphorylation of both holoenzymes
shows that CK2a1/CK2b1 was about 25% more phosphorylated
than CK2a1/DNCK2b1 (Figure 2B, right). It is noteworthy that
when GST-CK2b1 is overexpressed in E coli, a lower band (L) of
about 30 kDa appears in addition to a higher band (H)
corresponding to the fusion protein (56 kDa). Both bands are
highly phosphorylated by CK2a in vitro. Purification and
subsequent protein sequencing of this lower band demonstrate
that it corresponds to intermediate products containing the N-
terminal region of CK2b1. Moreover, the region corresponding to
the CK2b1 N-terminal alone (1–80) fused to GST (fusion protein
of 31 kDa) and overexpressed in E coli was also highly
phosphorylated by CK2a1 in vitro (Figure 2B). Taken together,
all these results suggest that autophosphorylation of CK2b1 occurs
in high proportion at the residues located in the N-terminal
To test whether CK2 activity was affected by the N-terminal
domain of CK2b subunits, we compared the ability of both CK2
holoenzymes (CK2a1/CK2b1 and CK2a1/DNCK2b1) to phos-
phorylate in vitro substrates as b-casein, in vivo substrates as Rab17
or interacting partners as maize transcription factor ZIM-like.
Interestingly, the holoenzyme composed by CK2a1/DNCK2b1 is
able to phosphorylate b-casein, Rab17 and ZIM-like in greater
amount than CK2a1 alone or CK2a1/CK2b1 (Figure 2C). These
results points towards a possible role of the N-terminal domain of
CK2b subunits as a negative regulator of CK2 activity. To
confirm this hypothesis, we added increasing amounts of CK2b1
N-terminal domain (1–80) to the in vitro phosphorylation assays
with the holoenzyme composed by CK2a1/DNCK2b1. The
addition of exogenous CK2b1 N-terminal domain to the CK2a1/
DNCK2b1 holoenzyme decreases its phosphorylation efficiency
towards the substrates tested, ZIM-like (Figure 2D), b-casein and
Rab17 (Figure S3). In conclusion, these results suggest that the N-
terminal domain of CK2b subunits competes with the substrate for
phosphorylation and down-regulate CK2a activity.
The N-terminal domain of CK2b1 enhances stability of
CK2b1 against proteasome degradation
To determine whether the N-terminal domain of CK2b
subunits is involved in regulation of their subcellular localization,
the deleted version of CK2b1 (del1 DNCK2b1 (80–276)) was fused
to GFP and examined by confocal microscopy in different plant
systems: immature maize embryos (10 DAP) transformed by
particle bombardment, agroinfiltrated tobacco leaves and onion
epidermal cells (Figure 3 and Figure S4).
In all plant systems examined the results obtained show that
both CK2b1 and DNCK2b1 are mainly located in the nucleus,
but whereas in the transformation with CK2b1 most of cells
presented nuclear speckles, in cells transformed with DNCK2b1
we found two different patterns: cells presenting a diffuse nuclear
pattern as well as cells showing nuclear speckles. Any nuclear
speckle structures were observed in cells transformed with GFP
alone. Since the total number of transformed cells after maize
bombardment is much lower than in tobacco cells, we counted the
percentage of DNCK2b1 cells presenting speckles vs. a diffuse
pattern in agroinfiltrated tobacco leaves (Figure 3B). Only 29% of
cells transformed with DNCK2b1 presented speckles vs. 71% with
diffuse pattern, whereas for cells transformed with full-lenght
CK2b1 96% of the cells presented speckles. Therefore, the
absence of nuclear aggregates in the DNCK2b1 cells could be
linked to the deletion of the N-terminal domain.
To test if the better ability of CK2b1 vs. DNCK2b1 to form
nuclear aggregates affects the protein stability, we performed cell-
free degradation assays. Total protein extracts from tobacco leaves
transformed with CK2b1 and DNCK2b1 fused to GFP were
maintained for 10, 30, 60 min at 30uC without protein inhibitors,
and aliquots were analyzed by Western blot using anti-GFP
antibody (Figure 4A, left). Results obtained suggest that the
amount of both CK2b1 and DNCK2b1 decreased over time.
Subsequently, we added proteasome inhibitor MG132 to test
whether the degradation observed was due to the proteasome
pathway. In samples treated with MG132 the fusion protein
remained stable, indicating that MG132 protects CK2b1 and
DNCK2b1 against proteasome degradation. The relative amount
of remaining proteins was estimated from these data and plotted,
and the rates of protein degradation for DNCK2b1 was
considerably higher than the rates for CK2b1 (Figure 4A, right).
These results suggest that the protein lacking the N-terminal
domain is more susceptible to degradation by proteasome than the
full-length CK2b1. To examine the effect of the N-terminal
domain on CK2b1 degradation by the proteasome pathway, we
treated transformed tobacco leaves with cycloheximide (CHX) to
inhibit de novo protein synthesis and we observed samples by
confocal microscopy for up to 24 h (Figure 4B). After 4 h of
treatment with CHX, the immunofluorescent signal was visible in
both CK2b1 and DNCK2b1 samples. In parallel, we have taken
samples of treated cells at different times and analyzed them by
Western blot. In agreement with the results obtained by confocal
analysis, the in vivo stability at short times is similar for both
proteins (Figure S5). However, after 24 h, we detected the
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immunofluorescent signal only in cells transformed with CK2b1,
indicating the requirement of ongoing protein synthesis to
maintain steady-state levels of DNCK2b1 protein. When samples
were treated with MG132 and CHX+MG132, we are able to
detect cells transformed with DNCK2b1 after 24 h of treatment,
indicating that proteasome inhibition protects DNCK2b1 from
Localization of CK2a1/CK2b1 holoenzyme is different
from its CK2 individual subunits
Different localization of plant CK2 subunits have been
previously demonstrated . In maize all CK2a subunits
described to date (CK2a1 to CK2a3) present nuclear localization
with high accumulation in nucleolus; whereas CK2b1 and CK2b2
are mainly located in nuclear speckles and CK2b3 can be found in
both nucleus and cytoplasm . However, nothing is known
about plant CK2 holoenzyme localization. To investigate that, we
conducted Bimolecular Fluorescence Complementation (BiFC)
assays in agroinfiltrated tobacco leaves [31,32]. In that system,
CK2a1 and CK2b1 split YFP tagged proteins must interact in vivo
to reconstitute YFP fluorescence. In CK2 heterotetramer the two
CK2b subunits associate as a stable dimer in the core of the
holoenzyme whereas the two CK2a are located in the external
part without interacting among themselves . Using BiFC we
show that CK2b1 subunits dimerize and present the same nuclear
speckled localization described for CK2b1 fused to GFP
(Figure 5A). Interestingly, the CK2 holoenzyme CK2a1/CK2b1
is located not only in nucleus but also in cytoplasmic aggregates
(Figure 5B). Next, we performed BiFC reconstituting the
DNCK2b1 interacted with split CK2a1, being also found in
nucleus and cytoplasm, as in the case of CK2a1/CK2b1
holoenzyme (Figure 5B). To confirm the presence of the CK2
holoenzyme in the cytoplasm, we perform an alternative approach
by co-transfecting tobacco leaves with CK2a1-GFP and CK2b1
fused to a non-fluorescence tag (Myc). This method allows to verify
that the fluorescence signal detected in the cytoplasm is due to the
presence of CK2a1-GFP in this compartment. Since CK2a1-
GFP/CK2b1-Myc holoenzyme is also located in nucleus and
cytoplasm, we confirmed that CK2b1-Myc is able to modify
CK2a1-GFP localization from nucleus/nucleolus to nucleus and
cytoplasm aggregates. In addition, we have used plants co-
transfected with CK2a1-GFP/CK2b1-Myc to perform subcellular
fractionation and Western blot analysis using anti-GFP antibody
(Figure 5C). In control plants overexpressing CK2a1-GFP alone,
CK2a1 subunit was mainly detected in nuclear soluble fraction (N)
whereas in plants co-transfected with CK2a1-GFP/CK2b1-Myc,
CK2a1 is increased in the insoluble fraction (I), which includes all
insoluble particles from nucleus and cytoplasm. These results
suggest that CK2b1-Myc is able to shift CK2a1-GFP from nuclear
fraction to insoluble aggregates in both nucleus and cytoplasm.
Land plant CK2b subunits show distinctive features from their
eukaryotic counterparts, including the formation of expanded
families, shorter C-terminal domains and longer N-terminal
domains. Preliminary in-silico analysis of the plant specific N-
terminal domain indicates that it presents no homology with other
protein or domains either in sequence or in structure. In addition,
prediction programs were unable to determine a secondary
structure for this domain. In an attempt to understand the role
and functionality of this domain in plants, we performed a
complete sequence analysis. The domain is arranged through
short, conserved motifs, many of them putatively corresponding to
specific kinase phosphorylation sites, including CK2 autophos-
phorylation sites. The domain may have evolved through the gain
and loss of short conserved motifs, resulting in a mosaic pattern.
The occurrence of N-terminal extensions is not exclusive of land
plants, having been also found in fungi, intracellular protozoan
parasites and algae. However, we did not found any protein or
domain outside land plant CK2bs showing significant identity at
the sequence level with the N-terminal domain. Moreover,
phylogenetic analysis shows a separated clade clustering all land
plant CK2b subunits. As expected, green and red algae CK2b
sequences clustered at the base of the land plant clade. However,
branching of the single representative from the red algae
Cyanidioschyzon merolae, which diverged from other photosynthetic
eukaryotes 1.5 billion years ago, was less bootstrap supported .
Also protists and fungi are separated from land plants in both
phylogenetic trees, in accordance to previously reported data that
demonstrate the early diverging evolution of CK2b from plants
. Furthermore, the exon/intron structure of genomic sequenc-
es encoding for the CK2b N-terminal domain was absolutely
conserved in all land plant CK2b genes analyzed. All together,
these results support the independent acquisition of the N-terminal
domain by land plants as a single exon. Further evolutionary
diversification of land plant CK2b would have involved differen-
tial gene family expansion, which may have promoted the
acquisition of additional functional specificities by multiplying
Figure 2. Intra-holoenzyme interactions using yeast two-hybrid system and in vitro CK2 phosphorylation assays using CK2
holoenzymes reconstituted with CK2a1 and regulatory subunits CK2b1 and DNCK2b1. (A) Left, Schematic representation of truncated
versions of maize CK2b1 regulatory subunit used in the assay. Deletion 1, del1 DNCK2b1 (80–276): CK2b1 without N-terminal region, deletion 2 (del2)
(180–276): CK2b1 without N-terminal region and acidic region and deletion 3 (del3) (1–80): N-terminal region alone. Right, Interactions between
truncated versions of CK2b1 subunit and CK2a2/CK2b1 subunits with the two-hybrid system. The indicated transformants were selected in Leu-Trp
plates and replated in selective plates lacking Leu-Trp-His-Ade. (B) Left panel, Gel stained with Coomassie Brillant Blue (CBB) containing the fusion
proteins GST-CK2b1, GST-DNCK2b1 and GST-N-terminal domain (1–80) used in the autophosphorylation and CK2 phosphorylation assays. Middle
panel, Autophosphorylation of reconstituted holoenzymes CK2a1/CK2b1, CK2a1/DNCK2b1 and in vitro phosphorylation of N-terminal domain (1–80)
protein by CK2a1. In the first lane H, high molecular weight protein corresponding to fusion protein GST-CK2b1 (56 kDa) and L, low molecular weight
protein, corresponding to intermediate products of about 30 kDa from fusion protein GST-CK2b1. Right panel, Coomassie Brillant Blue (CBB) and CK2
phosphorylation of GST protein alone (control). Right, Quantification of phosphorylated bands corresponding to CK2a1/CK2b1 and CK2a1/DNCK2b1
holoenzymes. 100% intensity corresponds to autophosphorylation of CK2b1. The data shown are calculated average values 6 SD of three
independent experiments. (C) Quantification of in vitro phosphorylation of b-casein, Rab17 protein and ZIM-like transcription factor by CK2a1/CK2b1
and CK2a1/DNCK2b1 reconstituted holoenzymes. 100% intensity corresponds to the phosphorylation of each protein by CK2a1 alone. The data
shown are calculated average values 6 SD of three independent experiments. (D), Left, In vitro phosphorylation of ZIM-like protein with CK2a1/
DNCK2b1 (lane 1). In lanes 2 to 4 increasing amounts of CK2b1 N-terminal domain (1–80) has been added: 0.2 mg (lane 2), 0.4 mg (lane 3) and 0.8 mg
(lane 4). Right, Relative phosphorylation of ZIM-like with the holoenzyme composed by CK2a1/DNCK2b1 with increasing amounts of CK2a1
N-terminal domain (1–80) (lanes 2–4) compared to phosphorylation of ZIM-like with CK2a1/DNCK2b1 holoenzyme (lane 1, assigned a value of 1). The
data plotted (mean 6SD) represent three independent experiments.
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Figure 3. Subcellular localization of CK2b1-GFP and DNCK2b1-GFP in maize immature embryos (10 DAP) and Agrobacterium-
infiltrated tobacco leaves. (A) Epifluorescence and bright-field images (merged with epiflourescence) (606) of 10 DAP embryos cells transformed
by particle bombardment with the indicated constructs (CK2b1–GFP, DNCK2b1-GFP and GFP alone). (B) Upper, General views (406) of Nicotiana
benthamiana leaves infiltrated with a mixture of Agrobacterium suspensions harbouring the indicated constructs (CK2b1–GFP, DNCK2b1-GFP and
GFP alone) and the gene silencing suppressor HcPro. Bottom, Quantification of cells presenting speckled pattern in cells transformed with CK2b1 and
DNCK2b1. The graphic representation correspond to average for data corresponding to 3 independent experiments 6SD (n=100).
Role of N-terminal of CK2b1 in CK2 Regulation
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Figure 4. Protein degradation of CK2b1 and DNCK2b1. (A) Immunodetection of CK2b1-GFP protein and DNCK2b1-GFP protein in transformed
N. benthamiana leaves using anti-GFP antibody. Protein extracts were incubated at 30uC in an in vitro degradation buffer (see Experimental
procedures) with or without proteasome inhibitor (MG132) for the indicated time (min). 30 mg of total protein was loaded onto gels. 60+I indicates
extracts treated with 100 mM MG132. Each signal strength was measured by Quantity One and plotted in the right panel as the relative amount of
remaining protein. Quantitative data (mean 6SD) represent three independent experiments. (B) General views (406) of Nicotiana benthamiana
leaves infiltrated with CK2b1-GFP and DNCK2b1-GFP with different treatments: control, cycloheximide treatment (CHX, 50 mM), and CHX (50 mM)+
proteasome inhibitor MG132 (100 mM) combination treatment after 4 and 24 hours. The images shown are representative of more than 5
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Figure 5. Subcellular localization of CK2 individual subunits and CK2 holoenzyme in leaf epidemis of N. benthamiana plants.
(A) General views (406) of Nicotiana benthamiana leaves co-infiltrated with a mixture of Agrobacterium suspensions harbouring the indicated
constructs: CK2a1-GFP (left), YFPN-CK2b1/YFPC-CK2b1 (right) together with gene silencing suppressor HcPro. (B) General views (406) of Nicotiana
benthamiana leaves co-infiltrated with Agrobacterium containing the gene silencing suppressor HcPro and the following pair constructs: YFPN-
CK2a1/YFPC-CK2b1 (left), YFPN-CK2a1/YFPC-DNCK2b1 (middle), and CK2a1-GFP/CK2b1-Myc (right). In panel A and B upper correspond to
epifluorescence images and bottom to bright-field images (merged with epiflourescence). (C) Immunodetection of CK2a1-GFP and CK2b1-GFP
proteins using anti-GFP antibody in N. benthamiana leaves transformed with CK2a1-GFP, CK2a1-GFP/CK2b1-Myc and CK2b1-GFP. C corresponds to
cytosolic fraction, N to nuclear fraction and I to insoluble fraction (including nuclear and cytosolic aggregates).
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the number of putative regulatory networks in which they could be
Despite of the elucidation of maize CK2a catalytic structure, no
structure for the plant CK2b regulatory subunit has been reported
to date. Here, by using a two-hybrid approach we show that the
CK2b N-terminal domain did not affect intra-molecular (CK2a/b
or CK2b/b) holoenzyme interactions. These results indicate that
the N-terminal domain is located in the external part of the
holoenzyme, although structural studies such as the crystallization
of the CK2b regulatory subunit would be needed to localize it with
Most animal CK2b subunits are autophosphorylated only at
two highly conserved residues, Ser2and Ser3. This consensus
is only partially conserved in plants: in all plant species Ser2is
conserved as Ser residue (Ser83in maize CK2b1), but Ser3is
replaced in all plant sequences analyzed by acidic residues (Asp or
Glu). In contrast to animals, land plant CK2b subunits present
additional putative autophosphorylation sites (motifs 1 and 5). For
instance, maize CK2b1 has five additional Ser residues at the
motif 1 of the N-terminal domain that might be targets for CK2
autophosphorylation. Motif 1 is rich in Asp and Glu residues and
is one of the most conserved in all plant N-terminal sequences
(Figure 1). In addition, CK2b1 subunits present additional Ser
residues located in the central core of the protein not present in
animal CK2b proteins. Our in vitro phosphorylation assays show
that the holoenzyme reconstituted with CK2a1 and CK2b1 is
higher autophosphorylated than the holoenzyme with CK2a1 and
DNCK2b1 (Figure 2B). Moreover, the N-terminal domain alone is
highly phosphorylated by CK2a1 in vitro. Taken together, these
results suggests that the putative CK2 consensus sites located in the
N-terminal domain are functional and might be involved in
regulating CK2 activity. In vitro phosphorylation assays showed
that when the holoenzyme is reconstituted with CK2a1 and
DNCK2b1 the phosphorylation of several substrates is enhanced.
These results point towards a possible role of the N-terminal
domain of CK2b down-regulating CK2a subunit activity. The
competition assays using the N-terminal fragment support this
hypothesis. The N-terminal extension of the protist Plasmodium
falciparum has also been postulated to act as a down-regulator of
CK2a subunits , even though our analysis supports the
independent origin of the N-terminal domain of land plant CK2b.
The greater efficiency of the maize holoenzyme without the N-
terminal domain is also consistent with the results of our previous
studies comparing human vs. maize holoenzyme, which demon-
strated a high stability and high specific activity of human CK2
holoenzyme (without N-terminal domain) compared to its maize
counterpart . It has been recently reported that a splicing
variant of maize CK2a1 (named CK2a-4) could act as a specific
negative regulator of CK2 activity . Taken together, all these
results suggest that maize CK2 activity could be regulated by
different mechanisms involving both CK2a/b subunits.
Functional studies were performed in order to assess whether the
presence of the N-terminal domain has a role in regulationof CK2b
subcellular localization. Our results show that maize CK2b1 is
highly prone to aggregation in nuclear speckles and the deletion of
N-terminal domain decreases this accumulation of CK2b1 in stable
nuclear aggregates. It has been reported for other proteins such as
mammalian PGC-1a and transcription factor ATF4 that aggrega-
tion in nuclear bodies protects against proteasome degradation
[38,39]. Here we show that maize CK2b1 is also degraded by the
ubiquitin-dependent proteasome pathway as described for Arabi-
dopsis CK2b4 . Interestingly, cell-free degradation assays show
that deletion of the N-terminal domain increases the rate of CK2b1
protein degradation. Our findings indicate a role for the N-terminal
domain in enhancing CK2b1 aggregation in nuclear speckles,
where the protein is assumed to be tightly complexed and less
accessible to degradation machinery. Nevertheless, although the N-
terminal domain can be considered as an ‘‘enhancer’’ of CK2b1
protein aggregation, it is not essential since DNCK2b1 can also
aggregate. Thus, we can consider that aggregation in nuclear
speckles protects CK2b1 against fast degradation by proteasome
even though the protein is eventually degraded.
In human cells, CK2b is normally expressed at a higher level
than CK2a catalytic subunits, allowing part of CK2b to be
incorporated and stabilized into CK2 tetramers, whereas the
excess CK2b is rapidly degraded with a half-life of less than 1 h
. Our results indicate that maize CK2b1 regulatory subunits
are more stable than their animal counterparts probably due to
their aggregation in nuclear speckles. Since the nature of these
aggregates remains unclear, further experiments should be done to
elucidate their composition and functional role.
We have previously demonstrated that different localization of
the individual maize CK2a and CK2b isoforms  but nothing
was known about holoenzyme localization in plants. Here by using
BiFC we show the in vivo localization of CK2 holoenzyme in plant
cells. Whereas individual subunits CK2a1 and CK2b1 present a
nuclear localization, the holoenzyme CK2a1/CK2b1 is assembled
in nucleus and is exported to the cytoplasm, where is complexed in
aggregates. After analyzing the localization of the holoenzyme
reconstituted with DNCK2b1, we conclude that the N-terminal
domain is not involved in this export to the cytoplasm. In
mammals it has recently been reported that CK2b regulatory
subunits are required for the export of the holoenzyme as an
ectokinase bound to the external surface of the cell membrane
. The same authors postulate a role of CK2b exporting not
only CK2a but other CK2 interacting proteins. Our results
implicate CK2b in the shift from nucleus/nucleolus to cytoplasm
of CK2a subunits in plants. Further experiments may elucidate
whether this export mechanism also involves other proteins.
In conclusion, our research shed new light on the regulation of
protein kinase CK2 in plants. The whole amount of data shown in
this work suggests that the plant-specific N-terminal domain of
CK2b subunits was acquired in plants, as a single exon, for
regulatory purposes, particularly in terms of regulation of
holoenzyme activity and stabilization.
Materials and Methods
Plant CK2b regulatory subunits sequence analysis
Search for CK2b protein sequences was performed through
BLAST and HMMER [43,44]. Protein sequences were aligned
using CLUSTALW and MUSCLE and the resulting alignments
further edited through the MEGA 4.0 Alignment Explorer tool
[45–47]. The MEME v. 3.5.7 tool was used to search for repeated
sequence patterns (motifs) conserved across proteins . Settings
were changed to search for short motifs (3–20 aminoacids)
showing any number of repetitions per sequence and position
(p-values,1e-4). Search for functional domain and motifs was
performed through the PROSITE and INTERPRO databases
. NetPhosK v1.0 server was used to predict kinase specific
phosphorylation sites . The location, distribution and phases of
introns at the genomic sequences encoding for the N-terminal
CK2b domain were determined using GENEWISE [50,51].
Phylogenetic analyses performed are detailed in Text S1.
Yeast two-hybrid assays
The Matchmaker two-hybrid system (Clontech) was used to
perform yeast two-hybrid assays. For the two-hybrid assays,
Role of N-terminal of CK2b1 in CK2 Regulation
PLoS ONE | www.plosone.org9 July 2011 | Volume 6 | Issue 7 | e21909
truncated versions of CK2b1 (del1 DNCK2b1 (80–276) del2 (180–
276), and del3 N-terminal domain (1–80)) were generated by PCR
and cloned into pGBT9 or pGBTK7 vectors into EcoRI/SalI
sites. The specific primers used were detailed in Table S5. The
other two-hybrid constructs used in the assays (pGBT9-CK2b1,
pGAD424-CK2b1 and pGAD424-CK2a2) were previously de-
scribed in . Yeast (AH109 strain) transformation was
performed according to the manufacturer’s instructions. Yeast
cells were cotransformed with the different pairs of BD-AD
constructs and transformants were selected on minimal synthetic
dropout medium (SD) -Leu-Trp (SD-LT). To test for protein-
protein positive interaction, independent colonies were transferred
to SD- -Leu-Trp-His-Ade (SD-LTHA).
Recombinant protein expression and purification and in
vitro autophosphorylation and CK2 activity assays
For expression and purification of recombinant CK2b proteins,
the cDNAs of full-length CK2b1, del1 DNCK2b1 (80–276) and
del3 N-terminal domain (1–80) were digested from pGBT9/
pGBTK7 vectors using EcoRI/SalI sites and cloned in expression
vector pGEX-4T-1 in frame to GST protein. The constructs were
transformed into E.coli BL21(DE), and the proteins were expressed
and purified as GST (Glutathione-S-Transferase) fusions as
previously described  and according manufacturer’s manual.
Protein concentration of purified proteins (GST-CK2b1, GST-
DNCK2b1 and GST-N-terminal domain) was determined by
Bioanalyzer methods (Agilent technology) according to the
For the in vitro autophosphorylation assay, the holoenzymes
CK2a1/CK2b1 and CK2a1/DNCK2b1 were reconstituted using
100 ng CK2a1 (Kinase Detect, Denmark) and 400 ng of GST-
CK2b1 or GST-DNCK2b1 in a total volume of 30 ml CK2 buffer
(8.9 mM MgCl2, 0.5 mM EGTA, 27 mM b-glycerol phosphate,
0.5 mM EDTA, 1 mM DTT,
[c-33P]ATP (3000 Ci/mmol). In the case of the CK2 activity
assays, the holoenzymes CK2a1/CK2b1 and CK2a1/DNCK2b1
were reconstituted as described for the autophosphorylation assays
and 0.6 mg of the different substrates tested (b-casein, GST-N-
terminal domain, Rab17 or ZIM-like)were added to the reaction.
In the competition assays, increasing amounts of N-terminal
domain (1–80) (from 0.2 to 0.8 mg) were added to the reaction
containing CK2a1/DNCK2b1 holoenzyme and 0.6 mg of b-
casein, Rab17 or ZIM-like as substrates. In all cases, the samples
were incubated for 30 min at 30uC. Reactions were stopped by
addition of electrophoresis sample buffer, and the phosphorylated
proteins were separated by 12% SDS–PAGE, visualized by
PhosphoImager analysis and the intensity of the phosphorylated
bands obtained was quantified by Quantity One (Bio-Rad)
software according the manufacturer’s suggestions.
0.08 mM ATP, 3 mCi of
Cell-free degradation assays, western blot analysis and
The in vitro cell-free degradation assays was modified from .
0.2 g transformed N. benthamiana leaves with CK2b1-GFP and
DNCK2b1-GFP were ground in liquid nitrogen and resuspended
in buffer A (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM
MgCl2, 5 mM DTT and 5 mM ATP). Equal amounts of extracts
were transferred to individual tubes and incubated at 30uC and
aliquots were taken at 20, 40 and 60 min. One aliquot was
incubated with 100 mM of protein inhibitor MG132 (Enzo, Life
Sciences, Inc.) for 1 h at 30uC. Reactions were stopped by adding
protein gel-loading buffer. For Western blot analysis, proteins were
electrophoresed on 12% SDS-PAGE gels, transferred to immobi-
lon-P membranes (Millipore) and incubated with purchased
antibodies against GFP (Invitrogen). The immunocomplexes were
revealed using the ECL detection kit system (Super Signal West
Femto, Pierce). Subcellular fractionation was done according to
. Briefly, transformed tobacco leaves were excised, ground in
liquid nitrogen and resuspended in hypotonic buffer (10 mM
HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA,
1 mM dithiothreitol, and a protease inhibitor cocktail (1.6 mM
aprotinin, 50 mM leupeptin, 1 mM pepstatin, 10 mM E-64 and
1 mM PMSF)). The extracts were homogenized and centrifuged at
10,000 rpm for 1 min. The supernatant was collected as the
cytosolic fraction (C). The pellet was extracted in a high salt buffer
(20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 1 mM dithiothreitol, and a protease inhibitor cocktail), and
the soluble fraction was collected as nuclear extracts following
another centrifugation (N). The remaining insoluble pellet was
resuspended in SDS lysis buffer (I).
Transient expression of GFP fusions in maize, Nicotiana
benthamiana leaves and onion cells
For transient expression of GFP fusions in maize, tobacco and
onion cells CK2b1 and del1 DNCK2b1 cDNAs were amplified by
PCR usingspecific primers (Table S5)and cloned into binary vector
pCAMBIA1302 under the control of a CamV 35S promoter and
and BglII site for DNCK2b1. Additionally, the cDNA CK2b1 was
amplified by PCR using specific primers (Table S5) and cloned in
pLOLA vector  into BglII site in frame with Myc tag. The
fusion CK2b1-Myc was transferred to pCAMBIA2300 using KpnI
restriction site. For maize transformation immature maize embryos
about 1 mm long were aseptically dissected from ears of field-grown
maize plants (AxBxB73) after 10 days of pollination (10 DAP).
Isolated embryos were placed o/n at 24uC in plates containing MS
medium supplemented with 2.2 mg/L of 2,4D. 4 h before
transformation embryos were moved to MS plates with 16 g/L of
mannitol and were transiently transfected with GFP constructs by
particle bombardment using the Biolistic PDS-1000/He Particle
Delivery System (Bio-Rad). Plasmid DNA containing the different
constructs was precipitated onto gold particles using CaCl2and
spermidine, and 1.5 mg DNA was delivered into intact maize tissue.
After 24 h, the fluorescence of the bombarded cells were viewed
using a FV 1000 confocal microscope (Olympus, http://www.
olympus.com/).The same methodology was used to visualize the
GFP fusion protein in epidermal onion cells. Young, fully expanded
leaves from 5 week old tobacco plants were transiently transfected
with Agrobacterium tumefaciens GV3101/pMP90 transformed with the
GFP construct together with the silencing suppressor HcPro as has
been described in [30,31]. After 3–4 days, infiltrated areas from
leaves were excised and examined by FV 1000 confocal microscopy
(Olympus). For treatment with cycloheximide (CHX) and protease
inhibitor MG132 leaves were excised and placed in sealed Petri
dishes submerged into the solutions containing CHX 50 mM and
MG132 100 mM in 2 ml of phosphate buffer. Treated and control
samples were ground in liquid nitrogen and resuspended in buffer A
(50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 5 mM
DTT, 5 mM ATP, and protease inhibitor cocktail) and analyzed by
Western blot analysis as described above.
Bimolecular fluorescence complementation (BiFC) assays
and DNCK2b1 were cloned in the GATEWAY-compatible vector
pENTRY3C (Invitrogen). The cDNA CK21 was amplified by PCR
using the specificprimersdetailed in TableS5and thePCR fragment
Role of N-terminal of CK2b1 in CK2 Regulation
PLoS ONE | www.plosone.org10July 2011 | Volume 6 | Issue 7 | e21909
and del1 DNCK2b1 (80–276) were digested from pGBT9 vector
using EcoRI/SalI sites and transferred to pENTRY3C. The three
pENTRY3C plasmids were recombined by Gateway reaction into
pYFPN43 and pYFPC43 vectors (kindly provided by A. Ferrando,
University of Valencia, Spain, http://www.ibmcp.upv.es/Ferrando
LabVectors.php.) to produce YFPN-CK2b1, YFPC-CK2b1, YFPN-
CK2a1 and YFPC-DNCK2b1. Transformation of N.benthamiana
leaves and visualization was performed as described above for
transient expression of GFP fusions.
subunits. Phylogenetic analyses were performed on the basis of
amino acid sequence alignments using two independent methods:
Neighbor Joining (NJ) and Maximum Likelihood (ML). NJ
analyses were implemented in MEGA 4.0 using the default
settings  Prior to ML analysis; the best-fitting amino acid
substitution model was selected using the Akaike information
criterion as implemented in ProtTest v1.4 . The resulting
model: JTT with (i) an estimated proportion of invariable sites and
(ii) a heterogeneous distribution of substitution rates across
proteins with eight categories and an estimated shape parameter,
was implemented in PHYML v3.0 to infer ML trees, using the
subtree pruning and regrafting option to optimize tree topology
searching [27–29]. To provide confidence on the resulting tree
topology, a bootstrap analysis with 1,000 and 100 replicates in NJ
and ML analyses, respectively, was performed.
Phylogenetic analysis of CK2b regulatory
CK2b protein kinases.
Summary of genome databases searched for
Sequence identifier refers to the UNIPROT database, excepting
for species examined independently, in which case the accession
from the corresponding database was indicated (Table S1). The *
designs sequence incomplete at its N-terminal end. Some genes
have been identified to encode for alternatively spliced variants. In
such cases, only a single representative protein sequence is shown.
Summary of 34 land plant CK2b sequences.
2 protists CK2b sequences from representative species.
Sequence identifier refers to the UNIPROT database, excepting
for species examined independently, in which case the accession
from the corresponding database was used (Table S1). The *
indicates sequences incomplete at its N-terminal end.
Summary of 7 algae, 14 animal, 12 fungal and
MEME in plant CK2b subunits. Matches of motifs with
specific kinase phosphorylation sites, predicted by NetPhos K v1.0
and PROSITE searches are shown. DNAPK: DNA activated
protein kinase, CDC2: Cell division cycle 2, RSK: 90 kDa
ribosomal S6 kinase, TK: Tyrosine kinase, ATM: Ataxia
Summary of conserved motifs identified by
List of primers used in this study.
tree of CK2b regulatory subunits. The tree is based on the
CLUSTAL alignment of 69 CK2b protein sequences. The clade
clustering land plant CK2b is indicated. Non-land plant CK2b
showing N-terminal extensions are in bold. Bootstrap values are
Unrooted Maximum Likelihood phylogenetic
displayed next to the corresponding nodes. The tree is drawn to
scale, with branch lengths proportional to evolutionary distances.
The scale bar indicates the estimated number of amino acid
substitutions per site.
CK2b regulatory subunits. The tree is based on the CLUSTAL
alignment of 69 CK2b protein sequences. The clade clustering land
plant CK2b is indicated. Non-land plant CK2b showing N-terminal
extensions are in bold. Bootstrap values are displayed next to the
corresponding nodes. The tree is drawn to scale, with branch lengths
proportional to evolutionary distances. The scale bar indicates the
estimated number of amino acid substitutions per site.
Unrooted Neighbor Joiningphylogenetic tree of
phorylation with CK2a1/DNCK2b1 holoenzyme and in-
creasing amounts of CK2b1 N-terminal domain (1–80).
Relative phosphorylation of Rab17 and b-casein with the
holoenzyme composed by CK2a1/DNCK2b1 with increasing
amounts of CK2b1 N-terminal domain (1–80) compared to
phosphorylation of both substrates with CK2a1/DNCK2b1
holoenzyme alone (assigned a value of 1). The data plotted (mean
6SD) represent three independent experiments.
Quantification of Rab17 and b-casein phos-
leaves and onion cells. (A) Upper and middle panels show
detail of fluorescent nucleus (606) of cells from tobacco leaves
infiltrated with a mixture of Agrobacterium suspensions harbouring
the indicated constructs (CK2b1–GFP, DNCK2b1-GFP) and the
gene silencing suppressor HcPro. In upper panel right, a confocal
image of nuclear DAPI staining of cells transformed with CK2b1–
GFP is shown (606). General views (406) of control cells
infiltrated with GFP alone and HcPro are shown in the bottom
of the panels. (B) Detail of fluorescent nucleus (606) of onion cells
transformed with CK2b1–GFP and DNCK2b1-GFP by particle
bombardment. General views of onion cells (406) transformed
with GFP alone are shown on the right. In all cases epifluorescence
and bright-field images (merged with epiflourescence) are shown.
Subcellular localization of CK2b1-GFP and
in Agrobacterium-infiltrated tobacco
DNCK2b1-GFP protein in transformed N. benthamiana
leaves using anti-GFP antibody. (A) Control and Cyclohex-
imide treatment (CHX, 50 mM). Aliquots have taken at different
times (309, 1 h, 2 h and 4 h) (B) Control, Cycloheximide treatment
(CHX, 50 mM) and proteasome inhibitor MG132 (100 mM).
Aliquots have taken at different times (4 h and 8 h). In all analysis,
30 mg of total extracts has been loaded. The hybridation against
Rubisco protein is shown as loading control.
Immunodetection of CK2b1-GFP protein and
We thank Dr A. Ferrando for kindly providing the BiFC GATEWAY-
modified vectors before publication, Dr. J. Lo ´pez-Moya for p35S::HcPro,
M.Capellades for help in maize embryo bombardment and Imma Perez-
Salamo ´ for help in two-hybrid experiments.
Conceived and designed the experiments: MR MP. Performed the
experiments: MR SI ICV-B LC-P. Analyzed the data: MR SI ICV-B
LC-P. Contributed reagents/materials/analysis tools: MR SI ICV-B LC-P.
Wrote the paper: MR VL LC-P MP.
Role of N-terminal of CK2b1 in CK2 Regulation
PLoS ONE | www.plosone.org11July 2011 | Volume 6 | Issue 7 | e21909
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Role of N-terminal of CK2b1 in CK2 Regulation
PLoS ONE | www.plosone.org12 July 2011 | Volume 6 | Issue 7 | e21909