From the
Saccharomyces cerevisiae casein kinase II (CKII)
contains two distinct catalytic (
Casein kinase II (CKII)
The physiological role
of the catalytic subunit has been explored by molecular-genetic
analysis in budding and fission yeast. In S. cerevisiae,
simultaneous disruption of the CKA1 and CKA2 genes
encoding the
The
role of the regulatory subunit has remained enigmatic. Comparisons of
the free catalytic subunit with native or reconstituted holoenzyme
in vitro have established several biochemical functions of
this polypeptide. First, the
Roussou and Draetta (1994) have recently reported the
isolation and disruption of the ckb1
We report here the isolation of the CKB1 gene
encoding the 38-kDa
Sensitivity of stationary phase cultures to nitrogen
starvation or heat shock was determined as described by Broek et
al. (1987).
Plasmids pJCR12 and pJCR14, which contain the
CKB2 gene in pRS316 and pRS426, respectively, have been
described (Reed et al., 1994). Plasmid pDH3 containing a cDNA
encoding the
A cDNA
encoding the D. melanogaster
Strains YAPB3 and YAPB7 were mated to create the homozygous
ckb1/ckb1 diploid, YAPB9 (). Quantitative analysis
of the mating mixture revealed no effect on the frequency of
diploidization compared to control strains (data not shown), indicating
that CKB1 is not required for signal transduction by the
mating pheromone pathway. Furthermore, the diploid YAPB9 sporulated
efficiently, and each tetrad gave rise to four viable haploid progeny
(data not shown), indicating that CKB1 is not required for
either meiosis or spore germination. Similar results have been observed
with the CKB2 gene (Reed et al., 1994).
In order
to isolate strains lacking both CKB1 and CKB2, two
differently marked CKB1/ckb1 CKB2/ckb2 diploids, YAPB10 and
YAPB13, were constructed (). Sporulation of either diploid
yielded four viable spores/tetrad, with approximately 25% of the
progeny carrying markers indicative of the simultaneous disruption of
both CKB1 and CKB2. This result established that
S. cerevisiae lacking both
ckb1, ckb2, and ckb1 ckb2 haploids grew
identically to their isogenic parent on both fermentable and
non-fermentable carbon sources, exhibited normal resistance to nitrogen
starvation and heat shock, and were neither temperature- nor
cold-sensitive over the range from 15 to 37 °C (data not shown and
Reed et al., 1994).
We report here the isolation and sequencing of the gene
CKB1 encoding the 38-kDa
Sc Ckb1 contains
all of the conserved elements previously identified in the
Two distinct
Phenotypic analysis of ckb1 ckb2 haploids and ckb1/ckb1
ckb2/ckb2 diploids indicates that the regulatory subunit of Sc
CKII is not required for viability, normal growth on either fermentable
or nonfermentable carbon sources, mating, sporulation, germination, or
resistance to nitrogen starvation or heat shock. Loss of regulatory
subunit function also exerts no (or marginal) effect on checkpoint
controls involved in responding to incomplete DNA replication and DNA
damage. While these results rule out a specific requirement for
CKB1 and CKB2 in each of these processes, we
emphasize that they do not preclude a requirement for CKII activity.
For example, CKII activity is known to be essential for viability in
S. cerevisiae and can be limiting for growth (Padmanabha
et al., 1990). The viability and normal growth rate of cells
lacking either or both
The only
clear phenotype we were able to identify in ckb1,
ckb2, and ckb1 ckb2 strains is a significant
attenuation of growth rate in the presence of NaCl or LiCl. The
specificity for Na
The fact that disruption of either CKB1 or CKB2 alone is sufficient to cause a salt-sensitive phenotype has
interesting implications. The sensitivity of such strains cannot be due
to the 50% reduction in gene dosage, since in both cases the phenotype
can be suppressed by a single copy of the disrupted gene but not a
second copy of the intact gene. The data thus imply that the two genes
are not functionally redundant with regard to salt tolerance. One
interpretation of this observation is that Sc
The modest conditional phenotype of S. cerevisiae ckb1 ckb2 strains is surprising given the dramatic phenotypic deficiencies
of S. pombe ckb1 strains. We consider it unlikely that there
exist additional, functionally redundant
The absence of a phenotype upon overexpression
of CKB1 in S. cerevisiae similarly contrasts with the
dramatic effects obtained in S. pombe. As discussed by Reed
et al. (1994) for CKB2, the absence of a phenotype in
S. cerevisiae may result from inadequate expression levels or
again from differences in regulatory subunit function in the two
species. To this we add the possibility that simultaneous
overexpression of both subunits may be required, consistent with the
arguments presented above that the regulatory subunit may be a
heterodimer of
The availability of genes
encoding the
The
nucleotide sequence(s) reported in this paper has been submitted to the
GenBank
We thank M. Snyder for the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
`) and regulatory
(
and
`) subunits. We report here the isolation and
disruption of the gene, CKB1, encoding the 38-kDa
subunit. The predicted Ckb1 sequence includes the N-terminal
autophosphorylation site, internal acidic domain, and potential metal
binding motif (CPX
C-X
-CPXC) present in other
subunits but is unique in that it contains two additional
autophosphorylation sites as well as a 30-amino-acid acidic insert.
CKB1 is located on the left arm of chromosome VII,
approximately 33 kilobases from the centromere and does not correspond
to any previously characterized genetic locus. Haploid and diploid
strains lacking either or both
subunit genes are viable,
demonstrating that the regulatory subunit of CKII is dispensable in
S. cerevisiae. Such strains exhibit wild type behavior with
regard to growth on both fermentable and nonfermentable carbon sources,
mating, sporulation, spore germination, and resistance to heat-shock
and nitrogen starvation, but are salt-sensitive. Salt sensitivity is
specific for NaCl and LiCl and is not observed with KCl or agents which
increase osmotic pressure alone. These data suggest a role for CKII in
ion homeostasis in S. cerevisiae.
(
)
is a
messenger-independent Ser-Thr protein kinase which is ubiquitous among
eukaryotes (Issinger, 1993; Pinna, 1990; Tuazon and Traugh, 1991). The
CKII holoenzyme is a constitutively active
heterotetramer composed of catalytic (
) and regulatory
(
) subunits. Two distinct
subunits encoded by separate genes
are present in most organisms, although only a single
subunit
isoform has so far been identified in Drosophila melanogaster (Glover et al., 1983), Caenorhabditis elegans (Hu and Rubin, 1990a), and Schzosaccharomyces pombe (Roussou and Draetta, 1994). Saccharomyces cerevisiae (Bidwai et al., 1994) and Arabidopsis thaliana (Collinge and Walker, 1994) also contain two distinct
subunits. A second
-like polypeptide, the product of the
Stellate locus, has been identified in D. melanogaster (Livak, 1990). Stellate is expressed exclusively in the testis,
but its biological function and relevance to CKII are unknown. CKII
phosphorylates a Ser or Thr followed by a stretch of acidic residues,
and numerous endogenous substrates have been identified, including
diverse transcription factors, nuclear oncoproteins, and components of
signal transduction cascades (Pinna, 1990). In vitro, CKII
activity is stimulated by polybasic compounds, such as polylysine and
protamine (Meggio et al., 1987), and inhibited by polyacidic
compounds, such as polyaspartate and polyglutamate (Meggio et
al., 1983), but the relevance of these compounds to in vivo regulation of CKII activity is unknown.
and
` subunits is lethal (Padmanabha et
al., 1990). Arrested cells are enlarged and exhibit morphologies
reminiscent of mutations which affect the cell division cycle. Analysis
of a strain carrying a temperature-sensitive cka2 allele
indicates that CKII is required for normal cell cycle progression in
both G
and G
/M (Hanna, 1991). Depletion of CKII
activity also results in Ca
-dependent cell-cell
aggregation or flocculation, suggesting alteration in the structure or
function of the cell wall (Padmanabha et al., 1990). In S.
pombe, the catalytic subunit appears to be encoded by a single
gene, cka1
, which has been isolated both by
the polymerase chain reaction (Roussou and Draetta, 1994) and as a
temperature-sensitive recessive lethal mutation, orb5, which
confers a spherical morphology at the non-permissive temperature (Snell
and Nurse, 1994). Genetic and cytological analysis of orb5 strains indicates a role for CKII in the reinitiation of polarized
growth following cytokinesis (Snell and Nurse, 1994). The available
data strongly suggest that CKII performs an essential function in both
yeasts, but the rather different phenotypes observed make it unclear to
what extent the physiological role of the enzyme is conserved.
subunit stabilizes the catalytic
subunit against proteolytic degradation and denaturation by heat or
urea treatment (Issinger et al., 1992; Meggio et al.,
1992b). Second, it serves as a general activator of the catalytic
subunit, stimulating it approximately 5-fold against most substrates at
physiological ionic strength (Bidwai et al., 1993; Hu and
Rubin, 1990b; Lin et al., 1991; Meggio et al.,
1992b). Third, it modulates CKII specificity by inhibiting the
phosphorylation of a subset of potential substrates, including
calmodulin (Bidwai et al., 1993; Meggio et al.,
1992b). Fourth, it plays an important role in mediating the activation
of the catalytic subunit by polybasic effectors (Bidwai et
al., 1993; Hu and Rubin, 1990b; Lin et al., 1991; Meggio
et al., 1992a). The
subunit also undergoes
autophosphorylation, at Ser2 in the mammalian enzyme (Boldyreff et
al., 1993a), but the functional significance of this reaction is
not known.
gene
encoding the
subunit of S. pombe CKII. Loss of
ckb1
function is not lethal but results in
slow growth, cell-cell aggregation, and cold sensitivity. ckb1 cells exhibit an abnormal, rounded morphology, reminiscent of the
orb5 mutation and display intense and irregular staining with
Calcofluor, suggesting abberant structure and/or composition of the
cell wall. Overexpression of ckb1
results in
slow growth and inhibits cytokinesis, leading to the accumulation of
multiseptated cells. Purified CKII from S. cerevisiae contains
two distinct regulatory subunits,
and
` (Bidwai et
al., 1994). Disruption of the CKB2 gene, encoding the
32-kDa
` subunit, has no overt phenotypic effect in a wild type
background but leads to slow growth and flocculation in a cka1 or cka2 background (Reed et al., 1994). While
the occurrence of some shared phenotypes in the two systems is
intriguing, the interpretation of the S. cerevisiae results is
obviously hampered by the potential functional redundancy of the
subunit.
subunit of Sc CKII. In contrast to S.
pombe ckb1 strains, S. cerevisiae strains lacking both
regulatory subunits exhibit essentially wild type behavior on normal
media. However, we find that strains lacking either or both
subunits are specifically sensitive to high concentrations of
Na
or Li
. Mutations in at least two
other protein kinases and the protein phosphatase, calcineurin, also
affect salt tolerance in S. cerevisiae, suggesting that CKII
may function in a complex pathway regulating ion homeostasis in this
organism.
Yeast Strains and Media
The strains of S.
cerevisiae used in this study are listed in . Unless
indicated, all strains were grown at 29 °C in YPD or complete
dropout medium as described (Ausubel et al., 1987). YPGal,
super YPD/YPGal (YPD/YPGal supplemented with all amino acids, adenine,
and uracil at concentrations as listed), and sporulation medium were
prepared as described (Ausubel et al., 1987). Escherichia
coli DH5 (Clonetech laboratories), XL1-Blue (Stratagene, La
Jolla, CA), and Y1090 (Young and Davis, 1983) were grown in Luria broth
containing 150 µg/ml ampicillin and/or 15 µg/ml tetracycline as
appropriate. Media components were purchased from Difco Laboratories
with amino acids, and other supplements were from Sigma.
Oligonucleotide Probes Specific for Sc CKII
We previously determined the sequence of several internal
tryptic peptides derived from the 38-kDa subunit (Bidwai et
al., 1994). The sequence of one of these peptides,
FGHEYFCQVPTEFIEDDFN, was used to design oligonucleotide probes specific
for the 38-kDa polypeptide (the Cys and second Asp of this sequence,
which were not identified in the original report, were assigned based
on homology with other
subunits and a weak signal in the
sequencing run, respectively). Two non-overlapping, 64-fold degenerate,
antisense probes were synthesized: 5`-AC (C/T)TG (A/G)CA (A/G)AA
(A/G)TA (C/T)TC (A/G)TG-3` (CKB1-P3), and 5`-TT (A/G)AA (A/G)TC (A/G)TC
(C/T)TC IAT (A/G)AA (C/T)TC-3` (CKB1-P4), where I indicates inosine.
Oligonucleotides were synthesized at the University of Georgia
Molecular Genetics Instrumentation Facility on an Applied Biosystems
model 394 DNA-RNA synthesizer and purified according to the
manufacturer's instructions.
Genomic Southern Analysis
S. cerevisiae genomic DNA was isolated as described (Sherman et al.,
1986) and digested with a set of 6-base ( EcoRI,
BamHI, HindIII, and PvuII) and 4-base
( HaeIII and MspI) restriction enzymes. Digested DNA
(7 µg/lane) was electrophoresed in 0.8% agarose gels in 1
TBE (89 mM Tris base, 89 mM boric acid, 2 mM EDTA) and transferred to nitrocellulose filters (Schleicher and
Schuell) as described (Ausubel et al., 1987). Probes CKB1-P3
and CKB1-P4 were 5` end-labeled using
[
-
P]ATP and T4 polynucleotide kinase
(Ausubel et al., 1987) and used to screen duplicate blots of
genomic DNA. Both probes were hybridized in 50 mM potassium
phosphate, pH 7.0, 6
SSC, 5
Denhardt's, 0.2% SDS
at 48 °C for 48 h. The filters were washed three times for 5 min
each in 2
SSC, 0.1% SDS at 23 °C and finally in 6
SSC, 0.1% SDS for 1 min at 58 °C prior to autoradiography.
Genomic Library Screening
A gt11 library of
random-sheared, EcoRI-linkered S. cerevisiae genomic
DNA (Mike Snyder, Yale University) was plated on E. coli Y1090, transferred in duplicate to nitrocellulose, and screened
with CKB1-P3 and CKB1-P4 under the conditions described above for
genomic Southern analysis. A total of 150,000 clones were screened, and
clones hybridizing with both probes were identified and plaque
purified. Seven clones, CKB1-1 through CKB1-7, were
obtained and restriction-mapped (Fig. 1 C).
Figure 1:
Isolation, chromosomal localization,
and sequencing of the CKB1 gene. A, schematic
representation of S. cerevisiae chromosome VII. The expanded
region corresponds to two overlapping ATCC clones that hybridize with
CKB1. B, schematic representation of ATCC clones no.
70669 and 70830. Ellipses indicate that the exact end points
of these clones have not been defined. The direction of transcription
of CKB1 is indicated by an arrow and the CKB1 open reading frame by an unshaded box. The approximate
location of the TRP5 gene, which hybridizes to clone no.
70669, was determined by comparing the ATCC map of clone no. 70669 with
the restriction map of TRP5 (Zalkin and Yanofsky, 1982).
Neither the exact location nor the orientation of the gene can be
deduced from the available data, but the TRP5 open reading
frame ( solid line) must lie within the region indicated by the
dotted line. Restriction sites are designated as: E,
EcoRI; H, HindIII; *, either EcoRI
or HindIII. Additional EcoRI and HindIII
sites exist on both clones but are not shown because their positions
have not been defined. C, schematic representation of seven
independent gt11 clones containing CKB1 ( CKB1-1 to CKB1-7). Restriction sites are indicated below
CKB1-7 and are abbreviated as follows: E,
EcoRI; H, HindIII; K,
KpnI; S, SacI; Xb, XbaI;
Xh, XhoI. D, sequencing strategy used to
obtain the sequence of the 1.6-kb EcoRI fragment of clone
CKB1-7. ATG and TAA indicate the start and stop
codons, respectively, of the CKB1 open reading
frame.
DNA Sequencing
The 1.6-kb EcoRI fragment
common to all seven clones (Fig. 1 C) was isolated from
CKB1-7 and subcloned into pBluescript II KS and
KS
(Stratagene, La Jolla, CA) to generate plasmids,
pAPB10 and pAPB12, respectively. The CKB1 open reading frame
is oriented opposite to that of lacZ in both constructions,
the other orientation being not well tolerated in E. coli.
Nested deletions of the 1.6-kb insert were constructed using the
ExoIII/Mung Enhanced Deletion Kit (Stratagene, La Jolla, CA). A 5`
deletion series was constructed using
SmaI+ SacI-digested pAPB10 and a 3` series using
ApaI+ EcoRV-digested pAPB12. Single-stranded DNA
from the deletion clones was synthesized using helper phage R408 at a
multiplicity of infection of 20:1. Sequencing was achieved by
dideoxy-chain termination using [
-
S]dATP
and Sequenase 2.0 (United States Biochemical Corp.) using a universal
primer or, when necessary, custom primers. Portions of the sequence
were confirmed by double-stranded sequencing on an Applied Biosystems
DNA Sequencer, 373A.
Physical Mapping
The location of CKB1 on
the physical map of the yeast genome was determined by hybridization to
an ordered array of overlapping and cosmid clones representing
most of the genome of S. cerevisiae AB972 (Olson et
al., 1986; American Type Culture Collection, catalog no. 76269).
The 1.6-kb EcoRI insert of pAPB10 was labeled by random primer
labeling (Boehringer Mannheim), and unincorporated label was removed
with an Insta-Prep column (5 Prime-3 Prime, Boulder, CO). Hybridization
was carried out in 50 mM potassium phosphate, pH 7.0, 50%
formamide, 5
SSC, 5
Denhardt's, and 0.2% SDS for
24 h at 42 °C. After hybridization, membranes were washed in 0.5
SSC, 0.1% SDS for 1 min at 63 °C and autoradiographed.
Disruption of CKB1
Disruptions of the CKB1 gene were achieved by -integration (Sikorski and Hieter,
1989). A 491-bp 5` fragment of the gene (containing 450 bp of
5`-untranslated sequence and 41 bp encoding the 14 N-terminal amino
acids of the open reading frame) was isolated by digestion of pAPB10
DNA with DraII, end-repair with the Klenow fragment of E.
coli DNA polymerase I, and digestion with EcoRI. A 485-bp
3` fragment (containing 211 bp encoding the C-terminal 70 amino acids
plus 274 bp of 3`-untranslated sequence) was isolated by digestion of
pAPB10 with HindIII plus EcoRI. Both fragments were
simultaneously ligated into
SmaI+ HindIII-digested pRS306 and used to
transformed E. coli DH5
. Plasmid DNA was isolated and
restriction mapped to verify the presence and correct orientation of
the two fragments, and one transformant, pAPB15, was chosen. In order
to convert the plasmid-associated marker from URA3 (pRS306) to
HIS3 (pRS303), the insert of pAPB15 was isolated by digestion
in the polylinker region with XhoI plus SacI. The
1060-bp fragment was ligated into
XhoI+ SacI-digested pRS303 and used to transform
E. coli DH5
. Plasmid DNA was prepared and restriction
mapped, and one transformant, pAPB17, was chosen. Both constructions,
pAPB15 and pAPB17, delete the coding region of CKB1 from S15
to T208. pAPB15 and pAPB17 were linearized by digestion with
EcoRI and used to transform yeast strains YPH499, YPH500, and
YPH501 by electroporation (Bio-Rad Gene Pulser). Uracil and histidine
prototrophs, respectively, were isolated on selective medium and
genomic DNA isolated from 5-ml cultures as described (Sherman et
al., 1986). Targeting of the two constructs to the CKB1 locus was verified by Southern blots of DNA digested with either
EcoRI or KpnI+ SacI using the 1.6-kb
EcoRI fragment of pAPB10 as a probe. Isolates displaying the
specific pattern of bands indicative of integration at the CKB1 locus (data not shown) were chosen for further analysis.
Genetic Analysis
Diploid products of mating
reactions were positively selected via complementation of nutritional
markers (Guthrie and Fink, 1991). Diploids were sporulated as described
(Guthrie and Fink, 1991) except that asci were digested in 0.5 mg/ml
zymolyase T-100 (ICN) in 1 M sorbitol for 15-30 min at
23 °C and dissected on super YPD containing 1 M sorbitol.
Phenotypic Analysis
Sensitivity to ultraviolet
light (UV) was determined as follows. Cells were grown in YPD and
diluted in water to a final density of 1 10
cells/ml. A 25-ml sample of diluted culture was placed in a 100
15-mm Petri dish on a shaking table and exposed to a calibrated
UV lamp (Sylvania shortwave germicidal bulb G8T5, 8 watts) at 1 J
m
s
. Aliquots were removed at
20-s intervals, and viable cell number was determined by plating cells
in triplicate on YPD. Percent survival was calculated by dividing the
viable count at each time point by that obtained just prior to UV
exposure.
Expression Plasmids
To construct low and high copy
plasmids carrying the CKB1 gene, a 1.6-kb
XhoI+ SacI fragment was isolated from pAPB10.
This fragment contains the entire protein-coding region of the CKB1 gene plus 450 bp of 5` and 271 bp of 3`-flanking sequences. The
purified fragment was ligated into
XhoI+ SacI-digested centromere vector, pRS316
(Sikorski and Hieter, 1989), and high copy, 2 µm vector, pRS426
(Christianson et al., 1992), to generate plasmids pAPB16 and
pAPB18, respectively.
subunit of D. melanogaster CKII under
transcriptional control of GAL10 in pBM272 has also been
described previously (Padmanabha et al., 1990).
-like protein, Stellate, was
amplified from plasmid pOG1.24 (Livak, 1990) by the polymerase chain
reaction, using upstream primer 5`-GCGAATTCTCGAGGTGAACTGGCAACATGTC-3`
and downstream primer 5`-GCGAATTCTCTAGATACATTGATGCGCCTGAC-3`. The
amplified DNA of 650 bp was digested with EcoRI and ligated
into the EcoRI site of plasmid pBM272. The orientation of the
insert was determined by restriction mapping. Clone pAPB19 contains the
Stellate cDNA in the correct orientation to allow transcription from
the GAL10 promoter. The sequence of the amplified fragment was
confirmed by DNA sequencing.
Suppression of Salt Sensitivity
Strains YAPB6 and
JCR7 () were transformed with plasmids pBM272, pDH3,
pAPB19, pRS316, pAPB16, pJCR12, pRS426, pAPB18, and pJCR14 by
electroporation. Transformants prototrophic for uracil were isolated
and analyzed for growth on super YPGal and super YPGal containing 0.7
M NaCl. The use of galactose instead of dextrose was necessary
since the expression of D. melanogaster and Stellate is
under the control of the inducible promoter, GAL10. Strains
were grown overnight in YPGal, adjusted to 1
10
cells/ml in YPGal, and 10-fold serially diluted to 1
10
cells/ml. Aliquots of 5 µl each, containing 5, 50,
and 500 cells, were spotted and the plates incubated at 29 °C for
4-8 days.
Isolation of CKB1
We employed the sequence of a
tryptic peptide derived from the 38-kDa subunit of Sc CKII to
design two degenerate, non-overlapping, antisense oligonucleotide
probes specific for this subunit (see ``Experimental
Procedures''). Genomic Southern analysis with both probes yielded
an identical set of hybridizing fragments (data not shown). The two
probes were then used to screen an S. cerevisiae genomic
library in
gt11, and seven clones (CKB1-1 through
CKB1-7) were isolated (Fig. 1 C). Restriction
mapping indicated that all seven clones contained identical or
overlapping inserts which shared a common EcoRI fragment of
1.6 kb (Fig. 1 C). This fragment from clone CKB1-7
was subcloned into pBluescript II and sequenced as described under
``Experimental Procedures'' and outlined in
Fig. 1D.
Nucleotide Sequence Analysis of CKB1
Fig. 2
presents the nucleotide sequence of the 1.6-kb
EcoRI fragment and its deduced amino acid sequence. The
sequence contains a single long open reading frame of 837 nucleotides,
which is preceded by an in-frame stop codon located 18 bp upstream of
the putative initiating methionine. Conceptual translation of the open
reading frame yields a predicted polypeptide of 278 amino acids with a
calculated molecular mass of 32,475 Da. The perfect agreement of the
deduced sequence (Fig. 2) with the available peptide sequence
data (Bidwai et al., 1994) confirms that the gene encodes the
38-kDa subunit. The calculated molecular mass of 32,475 Da is
significantly lower than that determined by SDS-polyacrylamide gel
electrophoresis (38,000-41,000 Da, depending upon the gel
conditions; Bidwai et al., 1994). This discrepancy may reflect
either secondary modification of the protein ( e.g. phosphorylation) or simply an anomolous electrophoretic mobility.
Figure 2:
Nucleotide sequence and deduced amino acid
sequence of CKB1. The nucleotide sequence of the 1.6-kb
EcoRI fragment of Fig. 1 D is shown above the deduced
amino acid sequence of the Ckb1 polypeptide (shown in one-letter
code). Underlined nucleotides correspond to genomic
EcoRI sites. An in-frame stop codon 15 nucleotides upstream of
the initiating methionine is indicated by double underline,
and the asterisk denotes the end of the open reading frame.
Underlined amino acids correspond to residues identified by
Edman degradation of tryptic peptides derived from the 38-kDa
subunit (Bidwai et al., 1994). A dotted underline indicates a blank or ambiguous cycle in these sequencing runs. The
square brackets indicate the region of the sequence deleted in
the ckb1-
1 allele.
Relationship to Other
An
alignment of Sc Ckb1 with other Subunit Sequences
subunits and D. melanogaster Stellate is shown in Fig. 3. Sc Ckb1 exhibits all of the
major features conserved in the
subunits of other organisms (Reed
et al., 1994), including an N-terminal autophosphorylation
motif, internal acidic region (in the vicinity of residue 60), and
potential metal binding motif,
C
PX
C-X
-CPXC
. Sc
Ckb1 resembles other fungal and plant
subunits in that it
contains an N-terminal extension relative to the invariant N terminus
of metazoan
subunits. Features unique to the Sc
subunit
include a 30 amino acid insertion (following residue 68), an unusually
long acidic region (which includes the N-terminal portion of the 30
residue insert), and two additional autophosphorylation motifs
(S
DEEEDEDD within the acidic domain and
S
TEEDWEE near the C terminus).
Figure 3:
Alignment of CKII subunits and
D. melanogaster Stellate. Sequences shown and their GenBank
accession numbers are S. cerevisiae Ckb1 (U21283), S.
cerevisiae Ckb2 (U08849), S. pombe Ckb1 (X74274), A.
thaliana Ckb1 (L22563) and Ckb2 (U03984), C. elegans
(M73827), D. melanogaster Stellate (X15899), D.
melanogaster
(M16535), and Homosapiens
(X16312). Because the
subunits from Gallus gallus (M59458) and Mus musculus (X52959) are 100% identical to
H. sapiens
, and Xenopus laevis
(X62376)
contains a single amino acid replacement, only the human sequence has
been included in the alignment. The numbering above the aligned
sequences follows the human sequence throughout. An asterisk indicates the C terminus, and a dash indicates a gap
introduced to maintain alignment. Invariant residues are boxed and shaded. Potential autophosphorylation sites and the
internal acidic region of Sc Ckb1 are indicated in bold face,
as is the potential metal-binding motif in each
protein.
Pairwise comparisons
among the sequences shown in Fig. 3reveal that Sc Ckb1 is not a
close relative of any previously described subunit. In
particular, it is as distantly related to Sc Ckb2 (45% identity) as it
is to the
subunits of other species (45-49% identity). In
order to analyze more rigorously the relationships among the available
subunits and Stellate, we used a distance matrix algorithm to
construct the evolutionary tree shown in Fig. 4. Except for minor
alterations in the topology near the center of this diagram, similar
results were obtained by neighbor joining and maximum parsimony methods
(data not shown). In general, the divergence among the fungal and plant
species is quite high compared to that among metazoans. The gene
duplication event giving rise to Sc Ckb1 and 2 appears to be quite
ancient, that giving rise to A. thaliana Ckb1 and 2 relatively
recent. The Stellate protein presumably represents a special case and
is considered under ``Discussion.''
Figure 4:
Unrooted evolutionary tree of the
subunits. The deduced amino acid sequences of the
subunits from
H. sapiens, M. musculus, G. gallus, X.
laevis, D. melanogaster, C. elegans, A.
thaliana, S. pombe, S. cerevisiae, and D.
melanogaster Stellate (see Fig. 3) were aligned using the PILEUP
program (Genetics Computer Group, Madison, WI) with a gap weight of 3.0
and a gap length weight of 0.1. Residues N-terminal to the initiating
methionine of the human sequence or C-terminal to the last residue of
the A. thaliana sequences were eliminated from the alignment.
PHYLIP 3.5 (Phylogenetic Inference Package, version 3.5; Felsenstein,
1982) was then used to compute evolutionary distances using the
PROTDIST subroutine and the Pam-Dayhoff matrix and to obtain an
unrooted tree via the algorithm of Fitch and Margoliash. Evolutionary
distance is plotted in units of ``expected number of amino acid
replacements/site.''
Chromosomal Mapping of CKB1
In order to determine
the chromosomal location and orientation of the CKB1 gene, the
1.6-kb EcoRI fragment (Fig. 1 D) was used to
screen an array of ordered lambda and cosmid clones representing
approximately 96% of the yeast genome. Two -clones, ATCC 70669 and
70830, were found to hybridize with the probe, and these contained
overlapping inserts derived from the left arm of chromosome VII
(Fig. 1, A and B). A comparison of the
EcoRI plus HindIII restriction map of the seven
gt11 clones (Fig. 1 C) with that of 70669 and 70830
(Fig. 1 B) indicated that CKB1 is located near
the proximal end of 70669 (consistent with the weaker hybridization of
this clone), approximately 33 kb from the centromere, and that the
direction of transcription is toward the centromere. The only genetic
locus which has been correlated to this region of the physical map is
TRP5, which is located distal to CKB1 on clone 70669.
The absence of known genes immediately centromere proximal to TRP5 (Mortimer et al., 1992) suggests that the CKB1 locus has not been identified genetically prior to this study.
Construction and Analysis of ckb1 and ckb1 ckb2
Strains
In order to determine the phenotype of cells lacking the
subunit, the CKB1 gene was disrupted by integrative
transformation as described under ``Experimental
Procedures.'' The constructions employed result in the deletion of
70% of the CKB1 open reading frame from S15 to T208, including
the N-terminal autophosphorylation site(s), acidic cluster, and the
cysteine-rich motif (Figs. 2 and 3). Two disruption plasmids, pAPB15
and pAPB17, containing different selectable markers, URA3 and
HIS3, respectively, were linearized with EcoRI and
used to transform yeast strains YPH499, YPH500, and YPH501
(). With either plasmid, homologous integration events were
readily obtained in the two haploid as well as the diploid strain,
indicating that CKB1 is not essential for viability.
subunits are viable. The
ckb1 ckb2 haploids, YAPB10-2c and YAPB13-8d, were
next mated to generate a ckb1/ckb1 ckb2/ckb2 diploid, YAPB14
(). Once again, quantitative analysis of the mating mixture
indicated no change in the frequency of diploidization relative to
control strains (data not shown). Finally, sporulation and dissection
of YAPB14 yielded four viable haploids. These results indicate that, at
least in S. cerevisiae, the regulatory subunit of CKII is not
essential for the mating response, meiosis, or spore germination.
Checkpoint Control in Strains Lacking the
Teitz et al. (1990) have shown that expression
of the human
Subunits
subunit partially suppresses the UV sensitivity of
cell lines from patients with xeroderma pigmentosum, complementation
groups C and D (XP-C and XP-D). The available data suggest that this
suppression is indirect, raising the possibility that CKII might
function in checkpoint control in response to DNA damage (Teitz et
al., 1990). For this reason we analyzed the survival of S.
cerevisiae strains lacking either or both
subunit genes
following UV-mediated DNA damage (Fig. 5). We observed that such
strains exhibit a modest (1.5-3-fold) increase in UV sensitivity
relative to the isogenic parent. In contrast, a 500-fold decrease in
survival was observed in a strain lacking the RAD9 gene and
thus defective in sensing damaged DNA (Weinert and Hartwell, 1988).
These results do not support a critical role for CKB1 and
CKB2 in checkpoint control mechanisms monitoring DNA damage in
S. cerevisiae. Because UV sensitivity varies with cell cycle
position, the modest increases in UV sensitivity observed could reflect
a shift in the proportion of cells at various points in the cycle. A
similar explanation has been offered to explain the partial suppression
of the UV sensitivity of XP cells upon
subunit overexpression
(Teitz et al., 1990).
Figure 5:
Effect of disruption of CKB1 and/or CKB2 on the viability of S. cerevisiae following DNA damage. Percent survival in response to UV-induced
DNA damage was determined as described under ``Experimental
Procedures.'' Strains analyzed were YPH499 ( wild type),
YAPB6 ( ckb1), JCR7 ( ckb2), YAPB10-2c ( ckb1
ckb2), and TWY398 ( rad9). The latter strain serves as a
positive control for UV sensitivity.
The possibility that the regulatory
subunit might be involved in monitoring completion of DNA synthesis was
also examined. Deletion of either or both subunit genes had no
effect on cell survival following inhibition of DNA synthesis with
hydroxyurea, and both mutant and wild type strains accumulated as
large-budded cells, consistent with S phase arrest (data not shown). In
contrast, a strain carrying a mutation in the MEC1 gene, which
is required for sensing both DNA damage and completion of DNA synthesis
(Weinert et al., 1994), failed to arrest as large budded cells
and exhibited a greatly enhanced sensitivity to hydroxyurea.
Salt Sensitivity of ckb1, ckb2, and ckb1 ckb2 Strains and
Suppression by
YCK1 and
YCK2 are an essential gene pair encoding two functionally
redundant isoforms of S. cerevisae CKI (Robinson et
al., 1994). Multicopy plasmids harboring either gene reduce the
sensitivity of wild type cells to high salt stress, and YCK2 was originally isolated by virtue of this phenotype (Robinson
et al., 1994). Since CKI and CKII exhibit some overlap with
regard to substrate specificity (Tuazon and Traugh, 1991), we tested
whether disruption of CKB1 and/or CKB2 might yield a
salt-sensitive phenotype. We found that this is indeed the case. As
shown in Fig. 6, strains lacking either Subunits and Stellate
subunit gene
exhibited a 2-fold reduction in growth rate relative to the isogenic
control in the presence of 0.7 M NaCl. Interestingly, the
growth rate of the double mutant was identical to that of the two
single mutants. Salt sensitivity was observed with NaCl (0.3-1.5
M) and LiCl (0.1-0.6 M) but not KCl
(0.9-2.0 M). Agents which alter osmotic pressure without
affecting ionic strength, such as glucose, sorbitol, or glycerol
(0.6-2.5 M), had no preferential effect on the growth
rate of mutant versus wild type strains (data not shown).
Figure 6:
Salt sensitivity of ckb1,
ckb2, and ckb1 ckb2 strains. Salt sensitivity of
YPH499 ( wild type), YAPB6 ( ckb1), JCR7
( ckb2), and YAPB10-2c ( ckb1 ckb2) was tested in
super YPD containing 0.7 M NaCl. Aliquots were removed at the
indicated times and fixed with 3.7% formaldehyde. Cell number was
determined by counting in a hemacytometer.
The existence of the salt-sensitive phenotype provides an assay to
explore the functional relationships among Ckb1, Ckb2, and the
subunits of other organisms. To this end, we tested whether the NaCl
sensitivity of ckb1 and ckb2 strains could be
suppressed by single copy or multicopy expression of CKB1 or
CKB2 or by galactose-induced expression of D. melanogaster
or Stellate. As shown in Fig. 7, expression of
CKB1 or CKB2, either from a CEN or multicopy
plasmid, completely suppressed the NaCl sensitivity of strains carrying
a deletion of the corresponding chromosomal gene, confirming that the
phenotype is indeed due to loss of CKB1/2 and that the cloned
fragments contain the signals necessary for expression. Overexpression
of CKB2 from a multicopy plasmid yielded partial suppression
of the NaCl sensitivity of a ckb1 strain, but overexpression
of CKB1 was unable to suppress the NaCl sensitivity of a
ckb2 strain. Galactose-induced expression of D.
melanogaster
partially suppressed the salt sensitivity of
both ckb1 and ckb2 strains, implying some degree of
functional conservation of the regulatory subunit, analogous to that
observed for the catalytic subunit (Padmanabha et al., 1990).
In contrast, expression of the Stellate polypeptide was without effect.
While this result could be due to a low level of Stellate expression or
an inability of Stellate to interact with the heterologous yeast
catalytic subunits, at face value the data suggest that Stellate does
not function as a
subunit. Boldyreff et al. (1993a) have
reached a similar conclusion based on their inability to demonstrate an
interaction in vitro between bacterially expressed Stellate
and human
.
Figure 7:
Suppression of NaCl sensitivity of
ckb1 and ckb2 strains. S. cerevisiae strains
YAPB6 ( ckb1) and JCR7 ( ckb2)were transformed with
CKB1 or CKB2 on a CEN plasmid (pRS316) or a 2-µm
plasmid (pRS426) as well as with D. melanogaster or
Stellate cDNAs (under control of the galactose inducible promoter,
GAL10) on a CEN plasmid (pBM272). Strains transformed with
empty vector and the untransformed isogenic parent strain, YPH499, were
included as controls. Strains were grown overnight in YPGal and cell
number determined by spectrophotometry at 600 nm. Each culture was
adjusted to 1
10
/ml in YPGal and then serially
diluted in water. Aliquots corresponding to 500, 50, and 5 cells of
each strain were spotted onto YPGal and YPGal + 0.7 M
NaCl and incubated for 5 and 10 days, respectively, at 29 °C prior
to photography.
Overexpression of Ckb1
Roussou and Draetta (1994)
have reported that overexpression of the subunit in S. pombe results in slow growth and the accumulation of multiseptated
cells. The suppression studies just described suggest that
overexpression of the regulatory subunit in S. cerevisiae does
not have comparable phenotypic effects. To investigate this more
rigorously, we transformed the wild type strain, YPH499, with pAPB18
carrying the CKB1 gene on the 2 µm-based vector, pRS426.
Relative to cells harboring the vector alone, cells harboring this
plasmid grew normally and displayed no morphological defects as assayed
by phase contrast microscopy (data not shown). Similar results have
been obtained with the CKB2 gene (Reed et al., 1994).
subunit of Sc CKII. The
isolation of CKB1 (this paper) and CKB2 (Reed et
al., 1994) confirms our earlier conclusion that Sc CKII contains
two distinct regulatory subunits,
and
` (Bidwai et
al., 1994). Alternative interpretations of the regulatory subunit
composition of purified Sc CKII have been ascribed to strain
differences (Issinger, 1993). We consider this unlikely given that the
peptide sequences of the
and
` subunits of CKII from
commercial yeast (Bidwai et al., 1994) match the deduced amino
acid sequences of the corresponding genes from a defined laboratory
strain (Reed et al., 1994; this paper).
subunits of other species (Reed et al., 1994), including the
N-terminal autophosphorylation site(s), internal acidic region, and
potential metal binding motif, CPX
C-X
-CPXC.
However, the protein also exhibits several features not present in the
subunits of other species. Perhaps most remarkable is a
30-amino-acid insertion located just downstream of the internal acidic
region. This insertion occurs in a region of length heterogeneity among
subunits generally, consistent with a probable location within a
solvent-exposed turn or loop at the surface of the protein. Because of
the strongly acidic character of its N-terminal half, the presence of
this insert approximately doubles the size of the internal acidic
region found in other
subunits. The acidic region is thought to
function as a negative regulator of the basal activity of the catalytic
subunit and to play a role in mediating stimulation by polybasic
effectors (Boldyreff et al., 1993b). This region is also
required for efficient N-terminal autophosphorylation (Boldyreff et
al., 1994). It remains to be determined whether the longer acidic
region in Ckb1 enhances or otherwise modulates these various effects.
In addition to the N-terminal autophosphorylation region, Ckb1 contains
two additional strong CKII recognition motifs, S
DEEEDEDD
and S
TEEDWEE. Ckb1, like Ckb2, is subject to
autophosphorylation in vitro (Bidwai et al., 1994),
but whether the novel sites are utilized will require mapping of in
vivo and in vitro phosphorylation sites.
(or
-like) polypeptides have been reported in three species,
S. cerevisiae, A. thaliana, and D.
melanogaster. Given the occurrence of two
isoforms in all
three eukaryotic kingdoms, we were intrigued by the possibility that
these isoforms might represent two conserved subfamilies, possibly with
distinct functions. However, the phylogenetic analysis shown in
Fig. 4
offers no support for this hypothesis, as clustering of the
six isoforms into two subfamilies is not observed. On the contrary, the
data suggest three independent gene duplication events, an ancient
duplication in the line leading to S. cerevisiae, a recent
duplication in the line leading to A. thaliana, and a third
duplication giving rise to the Stellate protein. The extensive
divergence of the S. cerevisiae subunits (45% identity) is
comparable to that between
subunits of different kingdoms
(45-49%) and may reflect functional specialization of the two
polypeptides (see below). The divergence of the A. thaliana subunits (93% identity) is modest by comparison, and the
duplication event in this case may reflect a need for differential
regulation (Collinge and Walker, 1994). The testis-specific Stellate
protein appears to represent a special case. At face value, the strong
divergence of Dm
and Stellate (40% identity) suggests an ancient
gene duplication. However, the failure to detect Stellate homologous
sequences in distantly related Drosophila species (Livak,
1990) and the absence of a Stellate-like polypeptide in CKII purified
from bovine testis (Litchfield et al., 1990) argue that the
duplication event is more recent, possibly very recent, and that the
Stellate protein has undergone rapid evolution since its appearance.
One possibility is that Stellate lost the ability to associate with the
catalytic subunit (see above) and acquired a novel function. It appears
likely that Stellate retains the basic
subunit fold, as evidenced
by retention of the Cys-Pro motif and other invariant residues, but the
remainder of the protein may have rapidly evolved under new or no
constraints. Taken together, the available data indicate that
subunit heterogeneity is idiosyncratic from species to species, and
many or even most species may contain only a single isoform.
subunit genes imply that such cells must
retain a substantial level of catalytic subunit function.
and Li
distinguishes this phenotype from generalized osmosensitivity,
such as observed in some mutations that perturb the function of the
actin cytoskeleton (Chowdhury et al., 1992; Welch et
al., 1993), and focuses attention on mechanisms regulating ion
homeostasis. Na
homeostasis in S. cerevisiae is mediated via the coordinate regulation of Na
influx and efflux across the plasma membrane (Mendoza et
al., 1994). Influx of Na
and other alkali metals
occurs via the K
uptake system in response to the
existing electrochemical gradient. Under Na
stress,
the K
uptake system is converted to a higher affinity
form which discriminates more efficiently against Na
and other alkali metals. This conversion requires the product of
the TRK1 gene, and trk1 mutants exhibit Na
and Li
sensitivity. Na
efflux
is dependent upon a putative P-type ATPase encoded by the
ENA1-4 gene cluster, and disruption of these genes also
results in specific sensitivity to Na
and
Li
. A role for protein phosphorylation in the
regulation of both systems is suggested by recent studies of protein
phosphatase 2B or calcineurin (Cyert et al., 1991; Cyert and
Thorner, 1992). Mutation of either the catalytic or regulatory subunit
of calcineurin causes Na
and Li
sensitivity (Mendoza et al., 1994; Nakamura et
al., 1993), and this sensitivity appears to result from both
reduced expression of ENA1 and failure to switch to the high
affinity form of the K
uptake system (Mendoza et
al., 1994). Based on this precedent, it is plausible to speculate
that CKII may also regulate the expression or activity of one or both
of these systems. Because loss of function mutations in a protein
kinase and a protein phosphatase confer the same phenotype, something
more than simple phosphorylation-dephosphorylation of a single site is
required to explain the data. Salt tolerance is also affected by other
protein kinase genes, YCK1 and 2, which encode
redundant isoforms of CKI (Robinson et al., 1994), and open
reading frame YCR101, which encodes a putative but
uncharacterized protein kinase (Skala et al., 1991),
suggesting that a complex network may be involved. A priori, the NaCl
sensitivity of ckb1 and/or ckb2 strains could be due
either to qualitative changes in enzyme targeting or specificity or
simply to decreased kinase activity. We cannot at present distinguish
these two alternatives but note that disruption of either CKA1 or CKA2 also results in Na
and
Li
sensitivity.
(
)
Salt
sensitivity has not previously been described in connection with CKII
and indicates additional complexity in the physiological role of this
kinase.
and
` form an
obligatory heterodimer (within the holoenzyme) which is inactivated by
loss of either polypeptide. The identical salt sensitivity of ckb1 and ckb2 strains and the absence of an additive effect in
the double mutant are consistent with this scenario, as is the high
degree of sequence divergence between Sc Ckb1 and Ckb2. The partial
suppression achieved upon overexpression of Ckb2 in a ckb1 strain could be explained by weak association of
` subunits
at high concentration or by increased function of monomeric
` at
high concentration. We have previously proposed that Sc CKII may be an
obligatory heterotetramer of all four subunits (Reed et al.,
1994), and the data described here are consistent with this idea.
subunit genes in S.
cerevisiae since purified Sc CKII appears to contain only
and
`, and the sequences of all available tryptic- and V-8-derived
peptides from these two polypeptides can be unambiguously assigned to
the deduced sequences of Ckb1 and Ckb2. Genomic Southern analysis and
ATCC library screens also fail to support the existence of additional
genes. Perhaps the simplest explanation is that the catalytic subunit
of S. pombe CKII has unusually low activity in the absence of
. In support of this idea, extracts of S. pombe ckb1 strains contain no measurable CKII activity, and overexpression of
cka1
causes an increase in CKII activity only
when coexpressed with ckb1
(Roussou and
Draetta, 1994). An alternative but related explanation is that S.
pombe is more sensitive to depression of CKII activity than is
S. cerevisiae, either because of a higher requirement for this
activity or a smaller pool of it. Finally, it is possible that the
function of the regulatory subunit and/or of CKII itself may differ in
the two species. Additional studies in both systems will be required to
clarify these issues.
and
` in S. cerevisiae. The latter
possibility is currently being tested.
and
` subunits of Sc CKII will facilitate
further biochemical and genetic analysis of this enzyme. The salt
sensitivity of strains lacking either or both
subunits provides
additional insight into the physiological role of the enzyme and also
provides an in vivo assay for regulatory subunit function in
S. cerevisiae. This phenotype should facilitate both
structure-function studies of autophosphorylation sites, metal-binding
motif, etc., as well as genetic screens for interacting loci.
/EMBL Data Bank with accession number(s) U21283.
gt11 yeast genomic
library, K. Livak for plasmid pOG1.24, and T. Weinert for strain
TWY398. We thank A. Rethinaswamy for assistance in constructing the
Stellate expression plasmid, J. Keswani for assistance with the
phylogenetic analysis, and M. Cyert for helpful discussions on
calcineurin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.