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and promotes its cell-cycle phase-dependent degradation
in yeast
Michaela Angermayr
1,
*, Elisabeth Hochleitner
2,
†, Friedrich Lottspeich
2
and Wolfhard Bandlow
1
1 Department Biologie I, Bereich Genetik, Ludwig-Maximilians-Universita
¨
tMu
¨
nchen, Germany
2 Max-Planck-Institut fu
¨
r Biochemie, Martinsried, Germany
The protein kinase casein kinase 2 (CK2) is ubiquitous
in eukaryotes and is responsible for the Ser ⁄ Thr phos-
phorylation of a large number of protein substrates
[1–3]. The active holoenzyme is most often a hetero-
tetramer composed of two catalytic a subunits, a
(encoded by CKA1) and a¢ (encoded by CKA2), and
two regulatory b subunits, b and b¢ in Saccharomyces
cerevisiae (CKB1 and CKB2). The enzyme occurs in all
possible combinations of a and b subunits [4,5]. In
yeast, deletion of the gene for one of the two catalytic
subunits has little effect, but deletion of both homolo-
gous genes results in loss of viability [6]. To date, more
than 300 endogenous CK2 substrates are known to be
involved in quite d iverse proc esses, e.g. cell proliferation,
signal transduction, transcriptional regulation, transla-
tion and metabolism [3]. Despite this eminent role in
strictly regulated cellular processes and although CK2
is indispensable for cell life, CK2 activity by itself is
apparently unregulated [4,5], although some fluctua-
tion in activity in correlation with cell-cycle progres-
sion has been seen in cultured mammalian cells [7,8].
The physiological effect of substrate phosphorylation
is surprisingly low with almost all targets described to
date [9,10]. In S. cerevisiae, it has been shown using
temperature-sensitive CKA2 alleles that protein kinase
CK2 is required for passage across the G
2
⁄ M bound-
ary and for cell-cycle progression through the G
1
phase [11].
Keywords
atypical protein kinase; casein kinase 2
substrate; cell-cycle phase-dependent
degradation; protein–protein interactions;
Rio1 protein kinase
2
1
Correspondence
M. Angermayr
E-mail: M.Angermayr@lrz.uni-muenchen.de
Present address
*ac-Pharma AG, Oberhaching, Germany
†Wacker Chemie AG, Burghausen, Germany
(Received 16 May 2007, revised 11 July
2007, accepted 16 July 2007)
doi:10.1111/j.1742-4658.2007.05993.x
Using co-immunoprecipitation combined with MS analysis, we identified
the a¢ subunit of casein kinase 2 (CK2) as an interaction partner of the
atypical Rio1 protein kinase in yeast. Co-purification of Rio1p with CK2
from Dcka1 or Dcka2 mutant extracts shows that Rio1p preferentially
interacts with Cka2p in vitro. The C-terminal domain of Rio1p is essential
and sufficient for this interaction. Six C-terminally located clustered serines
were identified as the only CK2 sites present in Rio1p. Replacement of all
six serine residues by aspartate, mimicking constitutive phosphorylation,
stimulates Rio1p kinase activity about twofold in vitro compared with
wild-type or the corresponding (S > A)
6
mutant proteins. Both mutant
alleles (S > A)
6
or (S > D)
6
complement in vivo, however, growth of the
RIO1 (S > A)
6
mutant is greatly retarded and shows a cell-cycle pheno-
type, whereas the behaviour of the RIO1 (S > D)
6
mutant is indistinguish-
able from wild-type. This suggests that phosphorylation by protein kinase
CK2 leads to moderate activation of Rio1p in vivo and promotes cell pro-
liferation. Physiological studies indicate that phosphorylation by CK2 ren-
ders the Rio1 protein kinase susceptible to proteolytic degradation at the
G
1
⁄ S transition in the cell-division cycle, whereas the non-phosphorylated
version is resistant.
Abbreviations
Aky2p, adenylate kinase 2; CK2, casein kinase 2; GST, glutathione S-transferase.
4654 FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS
Rio1p from yeast has been identified as the found-
ing member of a novel family of atypical protein
serine kinases [12–14]. It is essential in yeast and is
only distantly related to previously characterized pro-
tein kinases. The RIO1 gene is transcribed constitu-
tively at an extremely low level [15,16], and Rio1p is
a very low abundant protein [12]. Yeast has only one
such gene, whereas at least two orthologues of Rio1p
are present in higher eukaryotes. The primary struc-
ture of the catalytic domain and the N-terminal
so-called Rio1-family domain are highly conserved
from archaea to man, whereas a great deal of
sequence variation resides in the extreme N- and
C-terminal portions [13].
We found that Rio1p plays a role in cell-cycle pro-
gression. Yeast cells deprived of RIO1 lose minichro-
mosomes at an increased rate relative to wild-type and
arrest either as large G
1
cells (i.e. late in the G
1
phase
of the cell-division cycle) or as large-budded M cells
with a single DNA mass at the bud neck and short
spindles. This indicates that Rio1p is simultaneously
required in the G
1
phase and for the onset of anaphase
(and ⁄ or nuclear division and chromosome segregation)
[12]. Vanrobays et al. [17] obtained evidence from a
synthetic lethal screen with GAR1, an essential gene
required for 18S rRNA maturation, that Rio1p might
be involved in ribosome biogenesis. However, it is fea-
sible that Rio1p has more than one target and plays a
role in several pathways in yeast (as may be deduced
from the fact that two orthologues occur in higher
eukaryotes) [13].
The biological role of Rio1p or even the pathways
in which the Rio1 protein kinase is involved are far
from being understood. Targets or interaction partners
have not been identified as yet. We report here first
attempts to identify interaction partners and found
that the activity and cellular concentration of Rio1p
are regulated by phosphorylation through CK2 in a
cell-cycle-dependent fashion.
Results
Rio1p interacts with Cka2p
In an effort to identify the interaction partners of the
essential Rio1p kinase, we performed co-purification
experiments after overexpression of an N-terminally
myc
3
-tagged version of Rio1p from yeast extracts. Sub-
sequently, proteins were identified by mass spectrome-
try. We found several presumptive Rio1p interaction
partners which co-purified exclusively with full-length
Rio1p but not with a C-terminally truncated version
(M. Angermayr, unpublished). Among them we identi-
fied Cka2p, one of the two a subunits of protein kinase
CK2 using this approach, however, we did not recover
the other a subunit (Cka1p) or any of the b subunits
(Ckb1p or Ckb2p).
To verify interactions between Rio1p and Cka2p,
RIO1 was fused, at its 5¢-end, to a myc
3
tag-encoding
sequence (the RIO1 deletion mutants used are compiled
in Fig. 1), and CKA2 was equipped, at its 5¢-end, with
a nucleotide sequence encoding an HA
3
-tag, both
Fig. 1. Rio1 protein kinase. (A) Domains of the Rio1p kinase are indicated (according to the classification of Hanks et al. [45]); candidates for
CK2 phosphorylation are
7
indicated by an asterisk (*); pos., positions relative to the translational start codon. A, N-Terminal domain; B, Rio1
family-specific domain (R-domain); CK, serine-rich acidic domain (pos. 402–435, CK2 domain); and K, lysine-rich C-terminal domain (K domain,
pos. 436–484). (B) N- and C-terminally truncated versions of the Rio1 protein used in this study; amino acid sequences expressed are
indicated. (C) CK2 phosphorylation sites in the C-terminal domain (CK2 domain) of Rio1p; the respective S residues are emphasized and
positions are indicated (pos. 402–435); the respective CK2 phosphorylation sites are numbered consecutively (1–6).
M. Angermayr et al. Rio1 protein kinase is regulated by CK2
FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4655
transcribed from the inducible GAL10 promoter. Using
yeast extracts, co-immunoprecipitations were performed
once with anti-(myc agarose) to purifiy Rio1p and once
with HA antibodies
3
to purifiy Cka2p. Co-purified
Cka2p or Rio1p was subsequently detected by western
analysis with HA or myc antibodies, respectively
(Fig. 2). Because we presumed that the C-terminal por-
tion of Rio1p was involved in protein–protein inter-
actions and might serve as a substrate for CK2, we also
used a C-terminally truncated version of Rio1p, 1–408
[12] as a control (Fig. 1B). In addition, we investigated
whether a catalytically inactive allele of RIO1, Rio1-
D244N [12] interacts with HA
3
–Cka2p as well (inactive
RIO1 alleles were rescued by the, untagged, genomic
copy of RIO1). When Cka2p was immunoprecipitated
with HA antibodies, we detected the active or inactive
versions of Rio1p in a subsequent western blot by using
myc antibodies, indicating that both active and inactive
Rio1 proteins interact with Cka2p (Fig. 2A,B). This was
also true, when anti-(myc agarose) was applied to pre-
cipitate Rio1p (Fig. 2C,D). The interaction of Rio1p
with CK2 is extremely stable and resistant to extensive
washing (not shown), whereas the C-terminally trun-
cated Rio1p (1–408) displays only weak interactions
with Cka2p (Fig. 2A,C; in C, only a faint signal was
detected).
The above results indicated that the C-terminus of
Rio1p might play a role in the interaction with Cka2p
in yeast cells. To determine domains of Rio1p which are
necessary and sufficient to interact with Cka2p, we pro-
duced a series of RIO1 truncations. Products containing
only amino acids 1–46 or 1–76 turned out to be unstable
in yeast. In order to test the possible importance of the
N–terminus in the interaction with CK2 we could there-
fore use only N-terminal truncations (46–484, 76–484;
Fig. 1) in this experiment. We made another C-terminal
truncation (1–402; in this construct an additional pre-
sumptive CK2 phosphorylation site has been deleted in
comparison with 1–408). In a complementary experi-
ment, we used a construct containing exclusively the
C-terminal part of Rio1p (starting immediately C-termi-
nal adjacent to domain XI, amino acids 335–484).
Co-purification was performed using anti-(myc aga-
rose), and co-purified HA
3
–Cka2p was subsequently
identified by western blot analysis using HA antibodies
(Fig. 2E,F). Interactions between Rio1p and Cka2p
were abolished completely as soon as the 82 C-terminal
amino acids of Rio1p were deleted (constructs 1–402,
46–402 or 76–402). Conversely, Cka2p co-purified with
the C-terminal fragment of Rio1p (amino acids 335–
484). Thus, the C-terminus is necessary and sufficient to
establish this interaction. N)Terminal truncations
behave like full-length Rio1p in this respect and do not
affect Rio1p–Cka2p complex formation, demonstrating
that the N-terminus of Rio1p has no bearing on the
interactions between Rio1p and Cka2p.
Rio1p is a target of CK2
The above results indicated that Rio1p and Cka2p
interact with one another and that the interaction
Fig. 2. Co-immunopurification experiments and identification of
domains essential for Rio1p–Cka2p interactions. (A) HA
3
-tagged
Cka2p was immunoprecipitated using HA antibodies and protein
A–Sepharose; co-purified myc
3
-tagged Rio1p was subsequently
detected by immunodetection with myc antibodies. (B) Control
detection of total immunoprecipitated HA
3
-tagged Cka2p using HA
antibodies. (C) Myc
3
-tagged Rio1 proteins were immunoprecipitated
using anti-(myc agarose). Co-purified HA
3
-tagged Cka2p was subse-
quently detected by immunodecoration with HA antibodies. (D)
Control detection of total immunoprecipitated myc
3
-tagged Rio1
proteins using myc antibodies. (E) Myc
3
-tagged Rio1 proteins were
immunoprecipitated with anti-(myc agarose). Co-purified Cka2p was
subsequently detected by HA antibodies in a western blot. (F)
Immunoprecipitation efficiencies of the Rio1 proteins were con-
trolled by immunodetection with myc antibodies. Cka2p, HA
3
-Cka2p
which was still immunoreactive during the second detection on the
same blot in (F). p1, p2, control plasmids containing exclusively the
GAL10 promoter and the myc
3
-orHA
3
-tag, respectively. fl, full-
length Rio1p; 1–408p, C-terminally truncated Rio1p.
Rio1 protein kinase is regulated by CK2 M. Angermayr et al.
4656 FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS
domain might involve the C-terminal segment of
Rio1p. To analyse whether Rio1p and Cka2p interact
directly, i.e. without bridging factors from yeast, we
purified recombinant glutathione S-transferase (GST)–
Rio1p from Escherichia coli. Rio1 wild-type protein
has autophosphorylation activity, but GST-fused
Rio1p is enzymatically inactive, as observed with sev-
eral kinases. Therefore, this fusion protein is a suitable
substrate to unambiguously test whether Rio1p is a
target of CK2. GST–Rio1p was incubated without and
with recombinant human CK2 holoenzyme in the pres-
ence of [
32
P]ATP[cP] (Fig. 3A). GST served as an
additional negative control in the kinase assays. No
autophosphorylation (absence of CK2) was detected
corroborating that GST–Rio1p is inactive. GST–Rio1p
phosphorylation signals were detected only after incu-
bation with CK2. C-Terminally truncated GST–Rio1p
(1–408p) was poorly phosphorylated by CK2 when the
signal strengths of precipitated GST–Rio1p and GST-
1–408p were compared (Coomassie Brilliant Blue-
stained gel, Fig. 3B). No phosphate incorporation was
detected when amino acids 1–402 of Rio1p served as a
substrate for CK2 (Fig. 3C), suggesting the absence of
CK2 sites N-terminal of position 402 in the catalytic
domain and the presence of several phosphorylation
sites for CK2 in the C-terminal portion of Rio1p, one
(or more) of them in the segment between positions
402 and 408. In the complementary experiment, the
C-terminal fragment of Rio1p (335–484p) was heavily
phosphorylated (Fig. 3C).
These results show that: (a) Rio1p and CK2 interact
directly in vitro, because both proteins are of recombi-
nant origin; (b) recombinant CK2 holoenzyme has the
capacity to phosphorylate Rio1p; and (c) the CK2
phosphorylation sites of Rio1p lie within a region
between amino acid 402 and the C-terminus at position
484.
To provide evidence that Rio1p is also a target of
CK2 in vivo, we examined the extent of Rio1p phos-
phorylation from extracts of a Dcka1 or Dcka2 yeast
mutant, respectively. As controls we used an inactive
allele of RIO1, Rio1-D244N, and the truncated Rio1(1–
402-D244N) mutant, the latter is both inactive and not
phosphorylated by Cka2p (Fig. 4). Using yeast extracts
obtained from cells wild-type for CKA1 and CKA2,
tagged versions of Rio1p or Rio1-D244Np were heavily
phosphorylated, whereas only a weak signal was
detected with Rio1(1–402-D244N)p (Fig. 4A). (This
residual phosphorylation is independent of both Rio1p
and CK2 kinase activities and, likely attributable to the
action of still another protein kinase; M. Angermayr,
unpublished). However, in the Dcka1 or Dcka2 genetic
backgrounds, phosphate incorporation dropped to
40% (Dcka1) or 25% (Dcka2) with either Rio1p or
Rio1(D244N)p (Fig. 4C) indicating that Rio1p is phos-
phorylated mainly by a heterotetramer containing both
Fig. 3. Rio1p is a target of CK2. (A) Affinity-purified recombinant inactive GST-tagged Rio1 proteins were incubated with (+) or without (–)
recombinant CK2 in the presence of [
32
P]ATP[cP], electrophoresed and autoradiographed. (B) Coomassie Brilliant Blue-staining of the respec-
tive gel. GST served as a negative control in in vitro kinase assays. *, degradation product of the recombinant full-length version of Rio1p
(present in lanes 2 and 3). (C) Different recombinant GST-tagged Rio1 protein full-length or truncated versions were affinity-purified, incu-
bated with (+) or without (–) recombinant CK2 in the presence of [
32
P]ATP[cP], and detected by autoradiography after SDS ⁄ PAGE. * denotes
degradation products of full-length Rio1p and the C-terminal portion (amino acids 335–484). (D) Coomassie Brilliant Blue-stained gel of (C).
M. Angermayr et al. Rio1 protein kinase is regulated by CK2
FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4657
Cka1p and Cka2p less efficiently by a heterotetramer
composed of two Cka2p subunits, and only poorly by
the two Cka1p catalytic subunits-comprising holoen-
zyme. The observed differences in phosphate incorpora-
tion ( 20%) between Rio1 wild-type and mutant
(D244N) proteins are likely attributable to simulta-
neous autophosphorylation of Rio1p and ⁄ or the action
of still another protein kinase (M. Angermayr,
unpublished observations).
The above results indicate that Rio1p–CK2 inter-
actions are not restricted to Cka2p, but might be
exerted via Cka1p as well. To test the capacity of
tagged versions of Cka1p or Cka2p to compete with
the respective residual version of CKA in either a
Dcka1 or Dcka2 genetic background, we performed
co-immunoprecipitation experiments using an HA
3
-
tagged version of Cka1p (Fig. 5). Cka1p interacts with
Rio1p, although to a much lesser extent than Cka2p.
Quantification of co-immunoprecipitates in the Dcka1
or Dcka2 genetic background showed that Rio1p binds
with higher affinity to Cka2p, corroborating the results
obtained with in vitro phosphorylation experiments.
Protein kinase CK2 phosphorylates six clustered
serine residues of Rio1p
Computational analyses (http://scansite.mit.edu/
motifscan_seq.phtml) indicated that several (four to six,
depending on the stringency set) high- and low-affinity
Fig. 4. Phosphorylation of Rio1p by CK2 after Co-purification from
yeast cellular extracts. (A) Myc
3
-tagged Rio1 proteins were immu-
noprecipitated with anti-(myc agarose) using yeast extracts from
wild-type-, Dcka1-, and Dcka2 yeast strains; immunoprecipitates
were incubated in the presence of [
32
P]ATP[cP], and detected by
autoradiography after SDS ⁄ PAGE. fl, full-length Rio1p. (B) Coomas-
sie Brilliant Blue-stained gel; IgG, antibody heavy chain; fl, full-
length proteins. (C) Quantitative evaluation of phosphate incorpora-
tion into the respective Rio1 proteins with respect to the different
genetic backgrounds (wild-type, Dcka1, Dcka2).
Fig. 5. Interaction of Rio1p with Cka1p or Cka2p. (A) Myc
3
-Rio1p
[myc3-RIO1] was immunoprecipitated from yeast extracts from dif-
ferent genetic backgrounds (wild-type, Dcka1, Dcka2), coexpressing
either HA
3
-Cka1p, [HA3-CKA1], or HA
3
-Cka2p, [HA3-CKA2], respec-
tively. Co-purified HA
3
-Cka1p or HA
3
-Cka2p was subsequently iden-
tified by immunodetection in a western blot using HA antibodies.
IgG, antibody heavy chain. (B) Control of immunoprecipitation effi-
ciencies of myc
3
-Rio1p by western blot. (C) Quantitative evaluation
(normalized to the strongest signal in 5B) of relative interaction effi-
ciencies between Rio1p and Cka1p or Rio1p and Cka2p, respec-
tively, in yeast strains disrupted for either CKA1 or CKA2.
Genotypes are indicated below (C), and alleles in brackets denote
the tagged (and immunoprecipitated) isozyme of CK2. (Quantitative
evaluation is only shown for the respective Dcka1 or Dcka2 genetic
background, respectively).
Rio1 protein kinase is regulated by CK2 M. Angermayr et al.
4658 FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS
phosphorylation sites for CK2 might exist exclusively
in the C-terminal part of Rio1p. To determine whether
these sites are functional, we changed the candidate Ser
residues one by one to Ala using site-directed in vitro
mutagenesis. In vitro kinase assays with recombinant
CK2 holoenzyme and the respective (enzymatically
inactive) recombinant GST-fused Rio1 mutant proteins
as substrates revealed a total of six tightly clustered ser-
ine residues as CK2 phosphorylation sites [S402 (S1),
S403 (S2), S409 (S3), S416 (S4), S417 (S5), S419 (S6)]
consecutively numbered 1–6; cf. Fig. 1C (Fig. 6). The
total number of CK2 phosphorylation sites of Rio1p
was deduced from experiments with several single, dou-
ble, and triple S to A mutations; not all combinations
are shown. Recombinant (inactive) GST-fused RIO1
mutant alleles in which all six presumptive phosphory-
lation sites for CK2 had been mutated exhibited no
residual phosphorylation signal at all after incubation
with recombinant CK2 proving that all CK2 recogni-
tion sites within the Rio1p kinase had been destroyed.
Quantitative evaluation of phosphate incorporation
indicated that CK2 displays different affinities towards
the respective serine residues (Fig. 6C).
Phosphorylation by CK2 stimulates Rio1p kinase
activity
To investigate the possible physiological importance of
Rio1p phosphorylation by CK2, we changed all six
CK2 phosphorylation sites from S to either A (S > A)
6
,
or D (S > D)
6
, respectively. N-Terminally His
6
-tagged
wild-type and mutant proteins were purified from E. coli
and analysed in vitro by kinase assays using histone
H2B [12] as a substrate (Fig. 7). Quantification revealed
comparable rates of phosphate incorporation into the
heterologous substrate by the Rio1 wild-type and
(S > A)
6
mutant proteins, as expected for recombinant
proteins lacking modifications (e.g. unphosphorylated
by CK2). However, when the CK2 sites were changed to
D(S>D)
6
(mimicking permanently CK2-phosphory-
lated Rio1p), Rio1 mutant protein kinase activity was
stimulated approximately twofold.
To prove the functionality of the respective CK2
phosphorylation sites in yeast, we incubated immuno-
precipitated myc
3
-tagged Rio1 wild-type, (S > A)
6
,or
(S > D)
6
mutant proteins from yeast in the presence of
[
32
P]ATP[cP] (Fig. 8). Quantification of phosphate
incorporation into the respective Rio1p versions
showed that the Rio1 wild-type protein was heavily
phosphorylated (Fig. 8C). However, when the six CK2
phosphorylation sites were mutated to either alanine or
aspartate, phosphate incorporation dropped to 20 or
40%, respectively, which reflects autophosphorylation
of Rio1p and ⁄ or the presence of a site for another as
yet unidentified kinase which co-immunoprecipitated
together with Rio1p in addition to CK2.
Biological implications of phosphorylation of
Rio1p by CK2
In order to examine the possible biological importance
of Rio1p phosphorylation in vivo, we tested whether
substitution of all six CK2 phosphorylation sites in
Rio1p by either A or D (mimicking unphosphorylated
or permanently phosphorylated Rio1p, respectively) has
any consequences on yeast viability or growth rate. For
this purpose the respective mutant alleles were brought
into the genuine genomic context (i.e. at the RIO1
locus). Gene-shuffling experiments showed that the
(S > A)
6
as well as the (S > D)
6
mutant alleles comple-
mented the deletion of the RIO1 wild-type copy. How-
ever, growth rates of yeast cells harbouring the
(S > A)
6
mutant allele were significantly reduced
Fig. 6. Mutational analyses of the CK2-sites of Rio1p. (A) Recombi-
nant GST-fused Rio1 mutant proteins were incubated without (–) or
with (+) recombinant CK2 in the presence of [
32
P]ATP[cP], sepa-
rated by SDS ⁄ PAGE and autoradiographed. (B) Coomassie Brilliant
Blue-stained gel. (C) Quantification of phosphate incorporation
(relative to GST–Rio1p precipitated amounts) into the respective
Rio1p mutant proteins by protein kinase CK2; * denotes degrada-
tion products.
M. Angermayr et al. Rio1 protein kinase is regulated by CK2
FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4659
(Fig. 9A), indicating that the respective serine residues
must be phosphorylated in vivo to give a biologically
fully active Rio1p kinase. The yeast strain carrying the
mutant (S > D)
6
allele behaved indistinguishably from
wild-type, suggesting that the Rio1p kinase phosphory-
lated by CK2 is the fully functional form of the enzyme
in vivo. These findings obtained with the respective
RIO1 mutant alleles corroborate the results obtained
in vitro, i.e. that the Rio1p kinase is moderately
activated by CK2 phosphorylation also in vivo in
the wild-type and that this activation accelerates cell
proliferation. These observations imply that lack of
phosphorylation is disadvantageous for cell prolifera-
tion.
One possible reason for the slow growth of the non-
phosphorylatable (S > A)
6
mutant could be that these
cells are impeded in entering or exiting from a certain
Fig. 8. Functionality of the CK2 phosphorylation sites in yeast.
(A) Myc
3
-tagged Rio1 wild-type or mutant proteins were immuno-
precipitated from yeast extracts with anti-(myc agarose) and incu-
bated in the presence of [
32
P]ATP[cP], separated by SDS ⁄ PAGE
and autoradiographed. (B) Coomassie Brilliant Blue-stained gel. (C)
Quantitative evaluation of phosphate incorporation into the respec-
tive Rio1 proteins. Values represent the average of three indepen-
dent experiments, SD bars are given in the figure.
Fig. 7. CK2 phosphorylation stimulates Rio1p kinase activity.
(A) In vitro kinase assays were performed using recombinant affin-
ity-purified His
6
-tagged wild-type or mutant Rio1p proteins (S > A)
6
or (S > D)
6
, with histone H2B as a substrate (autoradiograph). (B)
Coomassie Brilliant Blue stain of Rio1p input. (C) Quantitative evalu-
ation of phosphate incorporation into H2B (normalized to H2B input
and to input of Rio1 proteins). Values represent the average of
three independent experiments (three different purifications from
E. coli), SD bars are given in the figure.
Rio1 protein kinase is regulated by CK2 M. Angermayr et al.
4660 FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS
phase of the cell-division cycle. Therefore, we tested,
using light microscopy and after DAPI staining, whether
the slow growth yields a cell-cycle phenotype. Cells of
the logarithmically growing wild-type or the (S > A)
6
mutant strain were photographed and cells assigned to
the respective phase of the cell-division cycle (similarly
as described previously) [12]. In the (S > A)
6
mutant,
the number of cells in the S phase having a small bud
was drastically diminished to almost one third relative
to wild-type or the (S > D)
6
mutant, i.e. 8% in the
(S > A)
6
mutant versus 22% in the wild-type, and the
number of G
1
cells was increased accordingly – 39% in
the (S > A)
6
mutant versus 29% in the wild-type. By
contrast, G
2
plus M phase cells were not affected signifi-
cantly – 53% in the (S > A)
6
mutant versus 49% in the
wild-type (Fig. 9B). However, we found a slight imbal-
ance with respect to the distribution of G
2
⁄ M cells: the
number of metaphase cells with a single DNA mass at
the bud neck and the number of anaphase cells were
increased slightly in the mutant (metaphase cells: 30.2%
in the mutant versus 24% in the wild-type; anaphase
cells: 4.5% in the mutant versus 3.2% in the wild-type),
whereas the number of telophase cells was decreased
slightly (18.3% in the mutant versus 21.8% in the
wild-type). These slight imbalances are considered
insignificant, in contrast to the differences observed with
the distribution of G
1
and S phase cells. These obser-
vations suggest that (S > A)
6
mutant cells, that fail
to become phosphorylated, mainly have difficulties
entering the S phase.
The reason for the accumulation of cells in the
G
1
phase may be due to the slightly lower kinase activity
of the (S > A)
6
mutant Rio1p kinase during the G
1
phase relative to wild-type or the (S > D)
6
mutant,
thereby retarding entry into the S phase. However, we
obtained direct evidence that a different mechanism
may play a role. A first hint to this mechanism came
from quantitative analysis of Rio1 protein concentra-
tions in the two mutants. In logarithmically growing
cells of the (S > A)
6
mutant the concentration of the
respective Rio1 protein generally exceeded (approxi-
mately fivefold) that of the wild-type or the (S > D)
6
mutant proteins (see Fig. 10; log ¼ cycling cells). One
possible explanation for this finding, which was further
Fig. 9. Growth rates of RIO1 wild-type and mutant yeast strains
and cell-cycle phase distribution. (A) A yeast strain carrying a single
genomic copy of the RIO1 (S > A)
6
mutant allele is hampered in
growth rate; the respective (S > D)
6
mutant yeast strain is indistin-
guishable from the wild-type. Two independent clones were tested
in all cases. Maximum deviations are indicated by bars. (B) Loga-
rithmically growing cells were stained with DAPI, photographed
and evaluated according to the stages of the cell-division cycle as
indicated below the diagram. A total of 546 wild-type cells or 239
mutant (S > A)
6
mutant cells have been analysed.
Fig. 10. Phosphorylation of Rio1p by CK2 renders the protein sus-
ceptible to proteolysis.RIO1 wild-type, (S > A)
6
,or(S>D)
6
mutant
cells were arrested with a-factor, hydroxyurea (HU), or nocodazole
(Noc) or left untreated (log) as a control in the presence of galactose
as a carbon source (for details please refer to Experimental proce-
dures). Cellular extracts were separated by SDS ⁄ PAGE, and the
respective proteins were detected by western blotting. (A) Immuno-
precipitated myc
3
-tagged versions of Rio1p were detected by myc
antibodies after SDS ⁄ PAGE in a western blot. (B) Loading control.
The stable protein Aky2p served as an input control and was analo-
gously detected by Aky2p antibodies derived from hen egg yolk.
M. Angermayr et al. Rio1 protein kinase is regulated by CK2
FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4661
pursued, is that the unphosphorylated version is stable,
whereas the (S > D)
6
mutant protein, mimicking the
phosphorylated form (and obviously also wild-type
Rio1p) is proteolytically degraded. Generally increased
proteolytic instability of Rio1p wild-type and the
(S > D)
6
mutant proteins relative to the (S > A)
6
mutant was, however, not observed in unsynchronized
cells (not shown). Therefore, we tested whether Rio1p
and Rio1(S > D)
6
mutant proteins are degraded at a
certain stage of the cell cycle. Such temporary instability
may explain the lower steady-state concentration of
the Rio1 mutant protein in the (S > D)
6
p version and
wild-type Rio1p relative to (S > A)
6
p in asynchronous
cells. To test whether phosphorylated Rio1p is, in fact,
degraded at a certain stage of the cell-division cycle,
whereas the unphosphorylated form is not, we per-
formed cell-cycle arrest experiments (see Experimental
procedures). Logarithmically growing cells were induced
by galactose (RIO1 alleles under the control of the
GAL10 promoter) and simultaneously arrested by treat-
ment with either a-factor (arrest before the G
1
⁄ S transi-
tion), hydroxyurea (S phase), or nocodazole (before
onset of anaphase), respectively. Cellular concentrations
of Rio1p were measured relative to adenylate kinase 2
(Aky2p), which is a constitutively expressed, stable pro-
tein [18] as a loading control (see Experimental proce-
dures). Our results indicate that in the RIO1 wild-type
and the (S > D)
6
mutant the level of Rio1 protein is
low to undetectable in the S phase but normal in G
1
and
during mitosis compared with cycling cells (Fig. 10;
traces of material detected after arrest with hydroxyurea
may be attributable to nonarrested cells; 10–15%).
However, the level of Rio1p is surprisingly high and
constant in the (S > A)
6
mutant and, most notably, not
at all affected by the stage of the cell-division cycle
(Fig. 10), thus displaying significant resistance to pro-
teolytic degradation. These findings demonstrate that
Rio1p and the (S > D)
6
mutant proteins, mimicking
the phosphorylated version, are degraded at the G
1
⁄ S
transition, whereas the nonphosphorylatable (S > A)
6
version is not.
Discussion
The Rio1p kinase from yeast is essential and highly
conserved from archaea to man and, thus, likely to
serve an evolutionarily ancient, highly important, as yet
unknown function [12–14,17,19]. Therefore, it is of gen-
eral interest to identify interaction partners or sub-
strates and, as a consequence, the pathway(s) this
kinase is involved in. We have found that the catalytic
a subunits of protein kinase CK2, Cka2p and to a les-
ser extent Cka1p, specifically interact with Rio1p. We
have shown that the C-terminus of Rio1p is essential
and sufficient for this interaction. We have also shown
that Rio1p is a substrate for CK2 holoenzyme in vitro
and, most likely, also in vivo.
Several combinations of serine mutations in the
C-terminal portion of Rio1p proved the presence of six
clustered CK2 phosphorylation sites with different
affinities. Neither the (S > A)
6
mutant nor the 1–402
C-terminally truncated protein served as a substrate for
CK2 (excluding the presence of additional CK2 sites
N-terminal of position 402), whereas the C-terminal
fragment Rio1-335–484p alone displayed strong wild-
type-like phosphorylation by CK2.
The C-terminal portion of yeast Rio1p displays a
striking two-partite primary structure. The part C-ter-
minally adjacent to the catalytic domain is rich in
serines and acidic residues (referred to as CK2 domain,
positions 402–435), whereas the extreme C-terminus
(positions 436–484) lacks serines and is highly posi-
tively charged (mainly lysines, referred to as K-domain,
Fig. 1A).
It is noteworthy that the C-terminal domain of
Rio1p is least conserved in evolution. Archaea, that do
not have CK2, lack the CK2- and K-domains com-
pletely, but also among higher eukaryotic Rio1p ortho-
logues high sequence divergence is observed in the
C-terminal part. Higher eukaryotes harbour two ortho-
logues of Rio1p named the SUDD-type and the
ad 034-type according to their first identification [13].
The SUDD proteins have only a short stretch of basic
amino acid sequences lacking C-terminal CK2 sites.
ad 034 proteins are more closely related to yeast Rio1p
and have both a CK2- and a K-domain, although the
direct sequence similarity is low. We have cloned both
types of human cDNAs, ad 034 and SUDD, as myc
3
-
tagged version and expressed them in yeast and E. coli.
Neither, alone or together, complements RIO1 defi-
ciency in yeast. Nevertheless, both recombinant orthol-
ogous proteins are heavily phosphorylated by CK2
in vitro (M. Angermayr, unpublished results).
In vitro kinase assays with recombinant Rio1 pro-
teins from yeast have revealed that the (S > D)
6
muta-
tion, mimicking permanent phosphorylation by CK2,
stimulates Rio1p kinase activity about twofold with
H2B as a heterologous substrate. The same is true
when the extent of Rio1p autophosphorylation of the
respective mutant proteins isolated from yeast is com-
pared, suggesting that phosphorylation of Rio1p by
CK2 has only a minor, presumably modulating effect
on the kinase activity of Rio1p.
Also with other substrates of CK2 that have been
described in detail in the literature, very small physio-
logical effects of phosphorylation by CK2 have been
Rio1 protein kinase is regulated by CK2 M. Angermayr et al.
4662 FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS
reported. Many of the targets of CK2 have been found
to serve essential functions. For example, CK2
increases the efficiency of transcription of the tRNA
and 5S rRNA genes by RNA polymerase III due to
phosphorylation of TBP within TFIIIB [20]. Eukary-
otic topoisomerase II is another target of protein
kinase CK2 which is essential for viability. However,
the importance of CK2 phosporylation of topoisomer-
ase II is not well understood, because mutation of the
respective CK2 phosphorylation sites does not cause
an obvious phenotype in yeast [21,22]. Cyclin-depen-
dent protein kinase from S. cerevisiae, Cdc28p, is
phosphorylated at a single serine residue by protein
kinase CK2 [23]. Lack of phosphorylation at this site
affects neither Cdc28p kinase activity in vitro nor yeast
growth rate, but leads to a slightly decreased cell size
during the G
1
phase [9,23]; by contrast, S > E muta-
tion of this residue stimulates Cdc28p twofold, at least
in vitro [9]. Sic1p, the cyclin ⁄ CDC28 cell-cycle kinase
inhibitor that prevents premature entrance into the
S phase, is another interesting substrate of CK2, but
phosphorylation by CK2 has little influence on its
physiological function [10,24,25]. The essential transla-
tional initiation factors, eIF2a (encoded by SUI2) [26]
and eIF5 (encoded by TIF5) [27], are additional tar-
gets of CK2, but phosphorylation by CK2 of either
eIF2a or eIF5 by CK2 is not essential for their respec-
tive functions. A seeming exception of a low effect of
CK2 site mutation is constituted by Cdc37p, a kinase-
associated molecular chaperone required in concert
with Hsp90p in the regulation of the activity of several
signalling protein kinases. Mutation of the single CK2
site on Cdc37p is not lethal but severely impedes
growth, presumably because of the additive negative
effects on several important protein kinases [28,29].
Taken together, there are many examples of proteins
which serve important functions that are substrates of
CK2, but mutational alteration of the respective CK2
phosphorylation sites has little effect or, at least, is not
deleterious to cell viability. This is more surprising as
deletion of CK2 (in yeast the double deletion of CKA1
and CKA2) leads to inviability [6]. However, the pleio-
tropy of CK2 may explain its indispensability. Failure
in CK2 deletion mutants of modulating a plethora of
processes, although within narrow limits, is likely to be
detrimental to cell life. Thus, moderate activation of the
Rio1p kinase upon phosphorylation by CK2 is in the
same range as observed with most other substrates.
We have shown that mutant RIO1 alleles, in which
all six CK2 phosphorylation sites have been mutated to
alanine or aspartate, respectively, sustain yeast viability.
The (S > D)
6
mutant behaves indistinguishably from
wild-type indicating that Rio1p is heavily phosphory-
lated by CK2 in vivo. In contrast, yeast cells harbouring
exclusively the (S > A)
6
mutant allele, a substrate that
does not become phosphorylated by CK2, are severely
hampered in growth rate. In addition, we have observed
a cell-cycle phenotype with this mutant. Cytological
approaches investigating cell-cycle stages reveal that
this mutant yeast strain accumulates G
1
cells, whereas
the number of S phase cells is drastically diminished. It
may be noteworthy that we detected a slight imbalance
of metaphase, anaphase and telophase cells as well.
Most significantly, this is in accordance with our previ-
ous observation that either depletion of Rio1p in the
cell or the use of a weak D244E mutant allele leads to
increased loss of minichromosomes and to the accumu-
lation of both, large-budded M cells with a single DNA
mass at the bud neck and large G
1
cells, indicating that
Rio1p is required for exit from mitosis and during G1
phase, but obviously not during S phase [12]. However,
in contrast to our previous Rio1p depletion experiments
or the use of the weak active site mutant of Rio1
(D244E) in which inhibition of the entrance of ana-
phase was the most significant effect, we describe here
with the nonphosphorylatable (S > A)
6
mutant that
the exit from the G
1
phase is more pronouncedly
retarded than the arrest in mitosis. These findings indi-
cate that Rio1p phosphorylation by CK2 mainly plays
a role in the G
1
phase conceivably by slightly increasing
Rio1p kinase activity, but is less (or not) important
during mitosis.
What might be the physiological basis of the moder-
ate G
1
arrest phenotype? By comparing the cellular con-
centrations of Rio1p and Rio1 mutant proteins, we were
able to exclude that Rio1p or Rio1 (S > D)
6
p are pro-
teolytically less stable per se than the (S > A)
6
mutant
protein. This means that phosphorylation by CK2
causes no general signal for the degradation of Rio1p.
Rather, we observed that the cellular concentration of
Rio1p and (S > D)
6
p is extremely low to undetectable
in the S phase, indicating that phosphorylated Rio1p
and the (S > D)
6
protein are destined for degradation
at the G
1
to S boundary, whereas the (S > A)
6
protein
is not. In this context, it may be relevant that the CK2
phosphorylation sites of Rio1p overlap a bona fide
destruction box, an amino acid sequence rich in P, E, S
and T residues which has been implied to be involved in
the degradation of the respective protein in a ubiquitin-
and proteasome-dependent manner [30]. In the case
of Rio1pk, this potential degradation signal may be
activated upon phosphorylation to act as a phospho-
degron [31] and to effect cell-cycle phase-dependent
proteolysis of Rio1p before entrance into the S phase.
This presumably occurs at the same time when other
important G
1
-specific proteins (e.g. Sic1p, Cln1p,
M. Angermayr et al. Rio1 protein kinase is regulated by CK2
FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4663
Cln2p) [32]; are polyphosphorylated and destined for
degradation through the ubiquitin-condensing E3-com-
plex, SCF and the proteasome in order to establish
commitment for progression to the S phase. This would
imply that Rio1p phosphorylation by CK2 constitutes a
signal for its specific degradation upon G
1
⁄ S transition
and that the absence of Rio1p during the S phase is con-
ducive to cell proliferation, conceivably by accelerating
passage through the G
1
⁄ S boundary. The absence of
wild-type Rio1p and, more obviously, of the (S > D)
6
mutant protein during the S phase may be taken
as evidence in favour of this interpretation. In contrast,
the concentration of the (S > A)
6
mutant protein
mimicking the unphosphorylated version is higher than
wild-type (see Fig. 10). The fact that growth is simulta-
neously impeded in the (S > A)
6
mutant (see Fig. 9)
suggests that ridding of Rio1p before start of a new
S phase might constitute a biologically important event
and that the phosphorylated form of Rio1p is the more
active and physiologically relevant.
In summary, we have described Rio1p as a substrate
of protein kinase CK2 and provide evidence that phos-
phorylation of Rio1p by CK2 generates a signal for its
degradation in a cell-cycle phase-specific manner.
Although proteolytic degradation of Rio1p is not a
stringent prerequisite, the absence of Rio1p during the
S phase promotes cell proliferation presumably by
accelerating G
1
to S transition. Altogether, we believe
that phosphorylation of Rio1p by CK2 has at least
two biologically relevant consequences: (a) it increases
Rio1 kinase activity and, conceivably, also controls
interaction with other proteins (presumably during the
G
1
phase); and (b) it provides a signal for commitment
for degradation of Rio1p before entrance of the
S phase. We have shown for the first time that the
cellular concentration and activity of constitutively
expressed Rio1p [15,16] are likely regulated through
phosphorylation by CK2.
Experimental procedures
Plasmids and strains
E. coli expression vectors pQE32 (Qiagen, Hilden, Germany)
or pGEX-4T-2 (Amersham Biosciences, Freiburg, Germany)
were used to produce His
6
- or GST-tagged versions of Rio1p.
E. coli strain BL21-Codon Plus-RIL (Stratagene, Heidelberg,
Germany), which had been additionally transformed with the
chaperonin-harbouring plasmid pREP4-groESL [33], served
for recombinant expression of Rio1 proteins. YEp351 or
YEp352 [34] were used for protein expression in yeast strains
WCG-4a (obtained from D. H. Wolf, University of Stuttgart,
Germany) or BY4741 (Mat a, his3D1, leu2D0, met15D0,
ura3D0) [35] (obtained from EUROSCARF, Frankfurt,
Germany). Deletion strains (isogenic to BY4741) Y01428
(Dcka1)(Mat a, his3D1, leu2D0, met15D0, ura3D0, YIL035c::
kanMX4) and Y01837 (Dcka2)(Mat a, his3D1, leu2D0,
met15D0, ura3D0, YOR061w::kanMX4) were obtained from
EUROSCARF. Integration plasmid pRS306 [36] was used to
integrate myc
3
-tagged RIO1 alleles under the control of the
GAL10 promoter into the genome at the URA3 locus. Yeast
strains carrying exclusively the RIO1 (S > A)
6
or (S > D)
6
mutant alleles in the genuine genomic context (i.e. at the
RIO1 locus under the control of the RIO1 promoter) were
generated using yeast strain YMA69 (Mat a, rio1::HIS3,
ade2–1, his3–11, 15, leu2–3, 112, trp1–1, ura3–1, can1–100
[pMA 221]). The haploid rio1-deletion strain was rescued by
plasmid pMA221 which carried a RIO1 wild-type copy in the
vector pGBDU-C1 [37]. The coding regions of the (S > A)
6
or (S > D)
6
mutant alleles were amplified by PCR. YMA69
was cotransformed with the respective PCR fragments and
pFL36 [38] to have a selectable marker. Transformants were
replica-plated onto 5-flouroorotate-containing plates [39] to
select against pMA221. Candidates were analysed by PCR
and DNA sequencing.
In vitro mutagenesis and recombinant DNA
methods
Truncated versions of RIO1 were produced by PCR
(Fig. 1). RIO1 constructs were N-terminally equipped with
a myc
3
-tag and expressed under the control of the GAL10
promoter from YEp351 in yeast strain WCG-4a. CKA2 or
CKA1, respectively, were amplified from genomic DNA by
PCR, N-terminally fused to an HA
3
-tag, and expressed
under the control of the GAL10 promoter from YEp352.
Presumptive CK2 phosphorylation sites were mutated from
serine to alanine or aspartate, respectively (QuikChange
Site-Directed Mutagenesis Kit, Stratagene) (Fig. 1).
Co-purification experiments
Yeast cells were disrupted by vortexing with glass beads in
lysis buffer (50 mm Hepes-KOH, pH 7.25, 15% glycerol,
10 mm MgCl
2
, 0.1% NP-40, 1 mm NaF, 1 mm Na
3
VO
4
,
1mm phenylmethylsulfonyl fluoride, 1 lgÆmL
)1
each apro-
tinin, leupeptin and pepstatin). Myc
3
-orHA
3
-tagged pro-
teins were immunoprecipitated with anti-(myc agarose) or
HA antibodies (both from Santa Cruz Biotechnology,
Santa Cruz, CA), respectively, or protein A–Sepharose
(Sigma, Deisenhofen, Germany) in lysis buffer adjusted to
150 mm NaCl (final concentration). Immunoprecipitates
were washed three times with 50 mm Hepes-KOH, pH 7.25,
15% glycerol, 150 mm NaCl, 10 mm MgCl
2
, 0.1% NP-40,
1mm NaF, 1 mm Na
3
VO
4
,1mm phenylmethylsulfonyl
fluoride, 1 lgÆmL
)1
each aprotinin, leupeptin and pepstatin,
and twice with 50 mm Hepes-KOH, pH 7.25, 8 mm MgCl
2
,
Rio1 protein kinase is regulated by CK2 M. Angermayr et al.
4664 FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS
1.5 mm MnCl
2
. Samples were subjected to SDS ⁄ PAGE
and analysed by MS or western blotting using antibodies
directed against the myc- or HA-tag, respectively (Santa
Cruz Biotechnology).
Protein identification
The Coomassie Brilliant Blue-stained protein spots of co-
precipitates were excised from the gels and placed in a Mul-
tiscreen 96-well filter plate (Millipore, Bedford, MA) posi-
tioned on top of a vacuum manifold integrated into a
Multiprobe II Ex robotic liquid-handling system (Perkin–
Elmer, Wellesley, MA). The gel pieces were destained with
alternating washing steps using 50 mm NH
4
HCO
3
buffer
and acetonitrile. A vacuum was applied to drain the wells
after each washing step. Subsequently, trypsin (Roche
Diagnostics, Mannheim, Germany) dissolved in 50 mm
NH
4
HCO
3
was added (enzyme to substrate ratio approxi-
mately 1 : 10), and the plate was placed into an incubator at
36 °C overnight. To elute the peptides, a 96-well receiver
plate was positioned at the bottom of the Multiscreen filter
plate in the vacuum manifold. Gel pieces were incubated
with acetonitrile for 5 min, and the supernatant was eluted
into the receiver plate under vacuum. This elution step was
repeated with 10% formic acid and acetonitrile. The com-
bined supernatants were spotted onto a MALDI target. An
aliquot (0.5 lL) of the sample was mixed on the target with
0.5 lL of the matrix solution (5 mgÆmL
)1
of a-cyano-4-hy-
droxycinnamic acid dissolved in 50% acetonitrile, 0.1% tri-
fluoroacetic acid) and dried at room temperature. Mass
analysis was performed using a positive reflector mode with
a deflection cut off range of m ⁄ z 800 on a 4700 Proteomics
Analyser (Applied Biosystems, Framingham, MA) equipped
with an Nd-YAG laser that produces pulsed power at
355 nm at pulse rates of 200 Hz. One thousand laser shots
were accumulated to produce one single spectrum. Subse-
quently, high-energy MALDI-TOF ⁄ TOF CID spectra were
recorded on selected ions from the same sample spot. The
collision energy was 1 kV. Air was used as collision gas.
The peptide mass fingerprints were submitted to a search at
the NCBI protein database using mascot
4
(http://www.
matrixscience.com). For unambiguous identification of the
proteins, tandem MS analysis was performed on one or two
peptides of the peptide mass fingerprints followed by a
search of the NCBI protein database using mascot.
Purification of recombinant Rio1p from E. coli
His
6
-tagged Rio1p was expressed in E. coli BL21 and puri-
fied as described [12,13]. Expression of GST–Rio1p in
E. coli was induced by 1 mm isopropyl thio-b-d-galactoside
at 30 °C for 3.5 h. Cells were harvested, incubated on ice in
50 mm Tris-Cl, pH 8.0, 15% glycerol, 15 mm KCl, 5 mm
MgCl
2
,1mm NaF, 1 mm Na
3
VO
4
,1mm phenylmethylsul-
fonyl fluoride, 1 lgÆmL
)1
each aprotinin, leupeptin and
pepstatin, and 1 mgÆmL
)1
lysozyme for 30 min and lysed
by sonication. The 15 000 g supernatant was incubated
with glutathione agarose (Sigma). Sedimented complexes
were washed three times with 50 mm Tris-Cl, pH 7.5,
100 mm KCl, 0.1% NP-40, 5 mm MgCl
2
,1mm NaF, 1 mm
Na
3
VO
4
,1mm phenylmethylsulfonyl fluoride, 1 lgÆmL
)1
each aprotinin, leupeptin and pepstatin, twice in kinase
buffer, and used for in vitro kinase assays.
In vitro kinase assays
Recombinant human protein kinase CK2 holoenzyme
(0.5 U, corresponding to 1 ng; New England Biolabs, Frank-
furt am Main, Germany) was incubated with purified recom-
binant enzymatically inactive GST–Rio1p as substrate in
20 mm Tris-Cl, pH 7.5, 50 mm KCl, 10 mm MgCl
2
in the
presence of 5 lCi [
32
P]ATP[cP] (10 CiÆmmol
)1
; final ATP
concentration 16.7 lm)at30°C for 30 min.
Myc
3
-tagged
5
Rio1 proteins were purified from yeast
wild-type, Dcka1-, or Dcka2-genomic backgrounds and
incubated in 50 mm Hepes-KOH, pH 7.25, 10 mm MgCl
2
in the presence of 5 lCi [
32
P]ATP[cP] (10 CiÆmmol
)1
; final
ATP concentration 16.7 lm)at30°C for 30 min. Reactions
were terminated by addition of gel-loading buffer and run
on SDS ⁄ PAGE. Gels were stained and dried for autoradio-
graphy.
Recombinant wild-type or mutant Rio1 proteins were
incubated with 15 lg histone H2B (Roche) as a substrate in
50 mm Hepes-KOH, 5 mm MgCl
2
,5mm MnCl
2
in the
presence of 5 lCi [
32
P]ATP[cP] (10 CiÆmmol
)1
; final ATP
concentration 16.7 lm)at30°C for 30 min.
Cell-cycle arrest experiments to test Rio1p
stability
Yeast strains carrying the respective myc
3
-tagged RIO1-
alleles under the control of the GAL10 promoter in the
genomic context (the respective alleles were integrated at the
URA3 locus with the help of the integration plasmid
pRS306) were cultured on 2% glucose-rich medium, shifted
to medium containing 2% raffinose as a carbon source for
two generations and then 2% galactose (final concentration)
was added. At this point the respective yeast cultures were
divided into aliquots and treated with a-factor (3 lgÆmL
)1
;
with further addition of 1.5 lgÆmL
)1
after 1.5 and 2.25 h to
ensure a stable arrest), 150 mm hydroxyurea or 15 lgÆmL
)1
nocodazole, respectively. One untreated aliquot served as
the control (cycling cells). Cells were pelleted, washed once
in H
2
O and frozen in liquid nitrogen. Subsequently, yeast
cells were disrupted by vortexing with glass beads in the
same buffer as described for the co-immunopurification
experiments, protein contents were determined, and the
samples subjected to SDS ⁄ PAGE and analysed by western
blotting using myc antibodies (Santa Cruz Biotechnology).
As a loading control, we used antibodies to detect Aky2p
M. Angermayr et al. Rio1 protein kinase is regulated by CK2
FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4665
[18], a protein which is stable throughout the cell cycle and
highly resistant to proteolytic degradation [40].
Miscellaneous procedures
Yeast were grown on standard media [41] and transformed
as described by Gietz et al. [42]. Microscopy and cell cycle
analyses were performed as described [12]. Protein contents
were determined by the method published by Bradford [43].
Other molecular methods were performed according to
standard procedures [44] or as recommended by the manu-
facturers.
6
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