©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Disruption of CKB1, the Gene Encoding the 38-kDa Subunit of Saccharomyces cerevisiae Casein Kinase II (CKII)
DELETION OF CKII REGULATORY SUBUNITS ELICITS A SALT-SENSITIVE PHENOTYPE (*)

Ashok P. Bidwai , J. Craig Reed , Claiborne V. C. Glover (§)

From the (1) Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, Georgia 30602-7229

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Saccharomyces cerevisiae casein kinase II (CKII) contains two distinct catalytic ( 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 (CPXC-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.


INTRODUCTION

Casein kinase II (CKII)() 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.

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 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.

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 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.

Roussou and Draetta (1994) have recently reported the isolation and disruption of the ckb1 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.

We report here the isolation of the CKB1 gene encoding the 38-kDa 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.


EXPERIMENTAL PROCEDURES

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.

Sensitivity of stationary phase cultures to nitrogen starvation or heat shock was determined as described by Broek et al. (1987).

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.

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 subunit of D. melanogaster CKII under transcriptional control of GAL10 in pBM272 has also been described previously (Padmanabha et al., 1990).

A cDNA encoding the D. melanogaster -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.


RESULTS

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 Subunit Sequences

An alignment of Sc Ckb1 with other 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, CPXC-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 (SDEEEDEDD within the acidic domain and STEEDWEE 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.

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 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.

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).

Checkpoint Control in Strains Lacking the Subunits

Teitz et al. (1990) have shown that expression of the human 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 Subunits and Stellate

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 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).


DISCUSSION

We report here the isolation and sequencing of the gene CKB1 encoding the 38-kDa 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).

Sc Ckb1 contains all of the conserved elements previously identified in the subunits of other species (Reed et al., 1994), including the N-terminal autophosphorylation site(s), internal acidic region, and potential metal binding motif, CPXC-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, SDEEEDEDD and STEEDWEE. 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.

Two distinct (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.

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 subunit genes imply that such cells must retain a substantial level of catalytic subunit function.

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 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.

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 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.

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 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.

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 and ` in S. cerevisiae. The latter possibility is currently being tested.

The availability of genes encoding the 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.

  
Table: S. cerevisiae strains



FOOTNOTES

*
This work was supported by American Cancer Society Grant VM-19 and National Institutes of Health Grant GM33237 (to C. V. C. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U21283.

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Life Sciences Bldg., University of Georgia, Athens, GA 30602-7229; Tel.: 706-542-1769; Fax: 706-542-1738; E mail: glover@bscr.uga.edu.

The abbreviations used are: CKII, casein kinase II; Sc, Saccharomyces cerevisiae; kb, kilobase(s); bp, base pair(s).

A. P. Bidwai, J. C. Reed, and C. V. C. Glover, unpublished results.


ACKNOWLEDGEMENTS

We thank M. Snyder for the 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.


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