Protein kinase CK2 activates the atypical Rio1p kinase
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

*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-

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

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).

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

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–

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 ( />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

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).

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

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)

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

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

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

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).

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

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

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)

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

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

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

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

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

growth causes oscillations in casein kinase II activity.
J Biol Chem 264, 7345–7348.
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