©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Casein Kinase I Subfamily
MOLECULAR CLONING, EXPRESSION, AND CHARACTERIZATION OF THREE MAMMALIAN ISOFORMS AND COMPLEMENTATION OF DEFECTS IN THE SACCHAROMYCES CEREVISIAE YCK GENES (*)

Lanmin Zhai , Paul R. Graves , Lucy C. Robinson (1), Michelle Italiano (2), Michael R. Culbertson (2), Joie Rowles (3), Melanie H. Cobb (3), Anna A. DePaoli-Roach , Peter J. Roach

From the (1) Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, the Department of Biochemistry and Molecular Biology, Louisiana State University, School of Medicine, Shreveport, Louisiana 71130-3932, the (2) Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, and the (3) Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Casein kinase I, one of the first protein kinases identified biochemically, is known to exist in multiple isoforms in mammals. Using a partial cDNA fragment corresponding to an isoform termed CK1, three full-length rat testis cDNAs were cloned that defined three separate members of this subfamily. The isoforms, designated CK11, CK12, and CK13, have predicted molecular masses of 43,000, 45,500, and 49,700. CK13 may also exist in an alternatively spliced form. The proteins are more than 90% identical to each other within the protein kinase domain but only 51-59% identical to other casein kinase I isoforms within this region. Messages for CK11 (2 kilobases (kb)), CK12 (1.5 and 2.4 kb), and CK13 (2.8 kb) were detected by Northern hybridization of testis RNA. Message for CK13 was also observed in brain, heart, kidney, lung, liver, and muscle whereas CK11 and CK12 messages were restricted to testis. All three CK1 isoforms were expressed as active enzymes in Escherichia coli and partially purified. The enzymes phosphorylated typical in vitro casein kinase I substrates such as casein, phosvitin, and a synthetic peptide, D4. Phosphorylation of the D4 peptide was activated by heparin whereas phosphorylation of the protein substrates was inhibited. The known casein kinase I inhibitor CK1-7 also inhibited the CK1s although less effectively than the CK1 or CK1 isoforms. All three CK1s underwent autophosphorylation when incubated with ATP and Mg. The YCK1 and YCK2 genes in Saccharomyces cerevisiae encode casein kinase I homologs, defects in which lead to aberrant morphology and growth arrest. Expression of mammalian CK11 or CK13 restored growth and normal morphology to a yeast mutant carrying a disruption of YCK1 and a temperature-sensitive allele of YCK2, suggesting overlap of function between the yeast Yck proteins and these CK1 isoforms.


INTRODUCTION

The term casein kinase I (CK1)() has been used to describe a ubiquitous protein kinase characterized by, among other properties, a monomeric structure and a preference for acidic substrates (for reviews, see Refs. 1-3). Although originally considered to be a protein Ser/Thr kinase, a recent report describes the ability of several yeast CK1 homologs to autophosphorylate also on tyrosine residues, a characteristic of the so-called dual-specificity enzymes (4) . The widespread distribution of CK1, in different cell types and in different subcellular compartments including cytosol, nucleus, and membrane, has long suggested important regulatory roles for this enzyme. One form of mammalian casein kinase I has been reported to be associated with nuclear spindles, with a potential role in cell cycle controls (5) . Although a long list of in vitro protein substrates for CK1 has been accumulated (1, 2, 3) , the physiological relevance has been established in fewer instances. CK1 phosphorylates rabbit muscle glycogen synthase at Ser-10, in vitro(6) and probably in vivo(7) . This site is thought to be important for hormonal controls (8) . The other well established substrate is the simian virus 40 large T antigen whose activity is altered by CK1 phosphorylation at sites thought to be relevant in vivo(9, 10) . The p53 tumor-suppressor protein is also phosphorylated by CK1 at NH-terminal sites, some of which are modified in vivo(11) . This result is provocative because of the linkage of some CK1 isoforms to DNA repair processes, but p53 is not yet confirmed as a physiological CK1 substrate.

It is now clear that casein kinase I actually represents a multigene family. Rowles et al.(12) first described mammalian cDNAs encoding multiple CK1 isoforms. Two full-length bovine brain cDNAs were cloned that corresponded to isoforms designated CK1 and CK1, of predicted M 37,600 and 38,700, respectively. A partial cDNA was isolated that would encode a third form, designated CK1, as well as a small PCR product predicting the existence of yet another species, termed CK1. Independently, Robinson et al.(13) obtained a PCR product corresponding to a fragment of rabbit testis casein kinase I that could later be classified as CK1, and Graves et al.(14) isolated a full-length cDNA for CK1 from rat testis. CK1 has predicted M of 49,000. Multiple forms of CK1 have also been identified in yeasts. Saccharomyces cerevisiae has four genes encoding protein kinases with kinase domains similar to mammalian CK1. One, HRR25(15) , is a gene implicated in DNA repair and meiosis, a finding of particular interest in light of the high level of the CK1 message found in testis (14) and the in vitro phosphorylation of p53 by CK1 noted above (11) . Two other yeast CK1 genes are YCK1 and YCK2(13, 16) . YCK1 was first isolated as a suppressor of a defective Snf1 kinase activity in a snf4 strain and YCK2 by its ability to enhance tolerance to salt. This essential gene pair encodes plasma membrane-associated CK1 proteins (13, 16, 17) . The functions of the Yck proteins include role(s) in cellular morphogenesis. A temperature-sensitive allele of YCK2 in combination with deletion of YCK1 results in arrest with multiple elongated buds and multiple nuclei at the restrictive temperature for growth (18). In Schizosaccharomyces pombe, four CK1 genes, cki1+, cki2+, hhp1+, and hhp2+, have been characterized (19, 20, 21) . Of these, hhp1+ and hhp2+ appear to be functionally related to hrr25 and are implicated in DNA repair processes (20, 21) .

Enzymological studies of mammalian CK1 had typically described a monomeric enzyme with M 36,000, but values for M over the range 25,000-55,000 have been recorded (1, 2, 3) . In retrospect, it is likely that some of the variability in properties is related to the existence of isoforms. To complicate matters further, some forms of CK1 are susceptible to partial proteolysis (14, 22) . As important potential functions for mammalian CK1 are slowly emerging, it is clear that more powerful and specific probes will be needed to identify the specific CK1 isoform involved, and a thorough knowledge of the members of the CK1 family is needed. In this paper, we describe the identification of three isoforms, CK11, CK12, and CK13, that form a distinct subfamily within the CK1s. Although over 90% homologous to each other, CK1s are only 51-59% identical within the protein kinase domain to other CK1 isoforms. CK11 and CK13 were also found to complement defects in yeast cells defective in the YCK genes.


EXPERIMENTAL PROCEDURES

Cloning of CKI1 2, and 3 cDNAs

A partial bovine cDNA of 900 bp which encodes for a CK1 isoform termed CK1 (12) was used as a probe to screen a rat testis oligo(dT) primed cDNA library (approximately 2 10 independent recombinants) in the Lambda Zap II vector. The probe, which was excised from a pBluescript SK vector by digestion with EcoRI, was labeled by random priming (23) and hybridized to duplicate filters containing approximately 5 10 recombinants. Prehybridization was performed at 58 °C in a solution of 10 Denhardt's solution (1 Denhardt's solution: 0.02% (w/v) each of Ficoll, polyvinylpyrrolidone, and gelatin), 6 SSPE (1 SSPE: 0.15 M sodium chloride, 10 mM sodium phosphate, and 1 mM EDTA, pH 7.4), 0.05% NaPP, 0.1% SDS, and 0.1 mg/ml of Torula RNA. Hybridization was performed under identical conditions to those used for prehybidization except for addition of the radiolabeled probe 2 10 cpm/ml. Nitrocellulose filters were washed in 6 SSC (1 SSC: 0.15 M sodium chloride and 15 mM sodium citrate, pH 7), 0.05% NaPP, and 0.1% SDS twice for 15 min at room temperature and once for 30 min at 58 °C. Following autoradiography of the filters using DuPont Quanta III intensifying screens, 11 positive clones were identified and plaque-purified. Each cDNA insert was rescued in pBluescript by co-infection with R408 helper phage according to the manufacturer's instructions. The cDNAs were sequenced using the dideoxy method of Sanger et al.(24) . For CK1 and CK2 double-stranded sequencing was performed in pBluescript using T7, T3, and sequence-specific primers. The CK13 cDNA was subcloned into M13mp19 and single-stranded sequencing of both strands utilized the universal and sequence-specific primers.

Northern Analysis

Total RNA from rat testis, heart, brain, skeletal muscle, liver, kidney, and lung was isolated by the method of Chomczynski and Sacchi (25) . Poly(A) RNA from rat testis was isolated using an mRNA purification kit (Pharmacia). Ten to 20 µg of total rat RNA and 2 µg of rat testis Poly(A) RNA was denatured with formaldehyde/formamide and was resolved on a 1.5% agarose gel containing 6% formaldehyde and transfered to nitrocellulose filter in 20 SSC (26) . The filters were baked at 80 °C under vacuum for 2 h and prehybridized and hybridized as described above using radiolabeled probe at 1.5-3 10 cpm/ml at 65 °C. cDNA probes from rat testis CK1 clones to coding and noncoding regions were labeled by random priming with [-P]ATP (23) . The probes used were as follows: for CK11, a 361-bp EcoRI-StuI fragment, position 1280-1641, composed of 50% coding and 50% 3`-untranslated sequences; for CK12, a 159-bp EcoRI-PstI fragment, position 1430-1589, from the 3`-untranslated region; for CK13, a 268-bp EcoRI-EcoRI fragment, position 2286-2554, from the 3`-untranslated region. Following hybridization, the filters were washed twice with 6 SSC, 0.1% SDS, and 0.05% NaPP for 30 min at room temperature and once for 30 min at the hybridization temperature. The filters were subjected to autoradiography using DuPont Quanta III intensifying screens.

Expression Vector Construction

CK11, 2, and 3 cDNAs were subcloned into pET 8c, 3c, and 8c, respectively, as follows.() The 1.688-bp cDNA of CK11 was digested with EcoRI, blunt-ended, and then partially digested with NcoI producing a 1.352-kb fragment. This fragment contains 1.047 kb of coding region and 305 bp of 3`-noncoding region. The fragment was then subcloned into the pET-8c vector which was previously digested with BamHI, blunt-ended, and then digested with NcoI, to form pETCK11.

From the 1,582-bp cDNA of CK12, part of the coding region (from 249-542 bp) was amplified by PCR using the following primers: CAGGCATATGTCCAAAACCGGCA (sense) and GCACCATGGCGTTGTACTTCC (antisense) to give a product of 293 bp. The underlined portion of each oligo indicates an NdeI site at 249 bp and an NcoI at 542 bp, respectively. The PCR product was subcloned into the TA vector (Invitrogen) and sequenced. Following digestion of the PCR product in the TA vector with KpnI and NcoI, a 40-bp fragment was obtained and subcloned into pBluescriptSK which contained the original CK12 cDNA previously cut with KpnI and NcoI. Next, pBluescript was digested with NdeI and BamHI, generating a 1.36-kb fragment which was subcloned into the expression vector pET-3c (previously digested with NdeI and BamHI) to form pETCK12. Two complementary oligonucleotides of 41 and 45 bp were synthesized corresponding to the CK13 cDNA coding region 810-846 bp. The sense oligo was: GACACCATGGCACGGCCCAGTGGTCGGTCAGGGCACAGCAC and the antisense oligo: TCGAGTGCTGTGCCCTGACCGACCACTGGGCCGTGCCATGGTGTC. Annealing of these two oligonucleotides formed a DNA fragment containing an NcoI and XhoI site indicated by the underlined regions, respectively. The fragment was ligated to the CK13 cDNA which was digested with EcoRI, blunt-ended, and then digested with XhoI generating a 1.44-kb fragment. After ligation, the 1,476-bp product was treated with NcoI and inserted into the expression vector pET-8c previously cut with BamHI, blunt-ended, and then cut with NcoI to form pETCK13.

The yeast expression vector pYcDE2 has the yeast ADH1 promoter and the CYC1 termination sequences flanking a EcoRI cloning site (27) . EcoRI fragments containing the CK11 and CK13 coding sequences were excised from pBluescript plasmids and inserted into the pYcDE2 plasmid. The resulting plasmids pMG1b and pMG3b contain CK11 and CK13, respectively, in the correct orientation. The plasmids pMG1a and pMG3a have CK11 and CK13 sequences in the antisense orientation.

Expression of CK11, 2, and 3 in Escherichia coli

The E. coli strain BL21/DE3, which contains the T7 RNA polymerase gene under control of the lacUV5 promoter, was transformed with pETCK11, pETCK12, or pETCK13. Cells were grown at 37 °C until an OD of 0.8-1 was obtained, and then induced with 100 µM isopropyl-1-thio--D-galactopyranoside and grown for an additional 10 h at 30 °C. Cells were harvested by centrifugation at 5,000 g for 10 min and the pellet resuspended in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 50 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mMN-p-tosyl-L-lysine-chloromethyl ketone and 2 mM benzamidine. After one pass through a French press at 900 pounds/square inch, extract was centrifuged at 11,000 g for 15 min to remove cell debris. The resultant supernatant was applied to an S-Sepharose column (14) previously equilibrated with buffer A containing 100 mM NaCl (buffer A; 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol) until no protein was eluted. CK11, 2, or 3 were step eluted, respectively, in buffer A containing 0.6, 0.4-0.5, and 0.6 M NaCl, respectively. In some cases, following S-Sepharose chromatography, a further casein-agarose column was used.

Protein Kinase Assays and Autophosphorylation

CK1 activity was measured by incubation for 20 min at 30 °C in 25 µl of reaction mixture containing 75 mM Tris-HCl, pH 7.5, 6 mM magnesium acetate, 1 mM EDTA, 0.4 mM EGTA, 1 mM -mercaptoethanol, 0.25 mM [-P]ATP (specific activity 300-1500 cpm/pmol), and 1 mM D4 peptide (DDDDVASLPGLRRR). When -casein or phosvitin were substrates, they were present at 1 mg/ml. The reaction was terminated by the addition of 20 mM EDTA and 1.5 mM adenosine. The P incorporation into the peptide was determined by depositing an aliquot of the reaction mixture onto p81 paper, followed by washing in 0.5% phosphoric acid as described previously (28) . For incorporation into protein, aliquots were placed on Whatman 31 ET paper previously spotted with 20% trichloroacetic acid and then washed in trichloroacetic acid (29). For analysis of inhibition by CK1-7, the ATP concentration was reduced to 50 µM. For analysis of autophosphorylation, enzyme was incubated in 83 mM Tris-HCl, pH 7.5, 0.47 mM [-P]ATP (300-1000 cpm/pmol), 6.6 mM magnesium acetate, 1 mM -mercaptoethanol. After reaction, samples were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.

A slight variation of the above assay was used to compare CK1 activities in yeast protein extracts (13) . Total protein extracts were prepared essentially as described by Garrett et al.(30) except that 0.1% (v/v) Nonidet P-40 was included. Cytosolic proteins were separated from a large particulate fraction by microcentrifugation. Membrane-enriched fractions were separated from soluble proteins using the method of Casperson et al.(31) except that lysis was carried out using a tissue grinder. Immunoblot analysis with Yck-specific antisera showed that full-length, plasma membrane-localized Yck2 protein is detected almost exclusively in such membrane-enriched fractions (17) .() CK1 assays were carried out using either 25 or 50 µg of cytosolic or soluble protein fractions, respectively, or 25 µg of membrane-enriched or particulate fraction. Assays were at 37 °C for 15 min, the elevated temperature decreasing background activity due to the yck2-2 protein whose activity is unstable at the higher temperature.

Yeast Strains and Methods

The yeast strains used were LRB519 (MAT his3 leu2 trp1 ura3 yck1-1 yck2-2) and LRB576 (MAT a his3 leu2 ura3 trp1 yck2-1::HIS3). The diploid strain LRB600 was generated by a cross of these two strains, and the CK1 plasmids were introduced into this diploid strain by transformation. The transformant strains were sporulated for tetrad analysis, and tetrads were dissected and incubated on synthetic medium lacking Trp to provide constant selection for the plasmids. The optimal growth temperature for strains containing these plasmids was 30 °C. Rich and synthetic yeast growth media and procedures for alkali cation transformation and standard genetic analysis have been described (32) .

Other Materials and Methods

The rat testis cDNA library, R408 helper phage and XL1-Blue cells were obtained from Stratagene. The pET expression system was obtained fom the Department of Biology, Brookhaven National Laboratory. Sequencing and random primed DNA labeling kits were from United States Biochemical Corp. The mRNA purification kit was from Pharmacia LKB Biotechnology Restriction enzymes, T4 DNA ligase, and M13 vector were from Bethesda Research Laboratories or New England Biolabs. CKI-7 was from Seikagaku America, Inc. All other common chemicals were purchased from Sigma and Boehringer Mannheim. The TA cloning kit was from Invitrogen CK1 and a COOH terminally truncated form of CK1 was as described by Graves et al.(14) . Standard recombinant DNA methods, bacterial culture, and transformation were as described by Sambrook et al.(33) .


RESULTS

Cloning of cDNAs Encoding CKI1, CKI2, and CKI3

From screening a rat testis library using the bovine partial CK1 cDNA as a probe, 11 positives were identified. Three full-length clones were analyzed (Fig. 1). The predicted protein sequences were 90-93% identical to each other over the protein kinase domain and 69-78% identical over their entire lengths. The protein kinase domains were 51-59% identical to other mammalian CK1s. These protein kinases, though clearly in the CK1 family, are most like each other and form a subfamily. Therefore, the proteins were designated CK11, CK12, and CK13 in the order in which the corresponding cDNAs were characterized. The partial bovine CK1 sequence was most similar, 97% identical, to that of CK13. Of the 11 positives, two corresponded to CK11, seven to CK12, and two to CK13. CK11, CK12, and CK13 proteins would have predicted molecular masses of 45,075, 47,425, and 51,370 and would contain 390, 414, and 448 residues, respectively (Fig. 2). One partial clone corresponding to CK13 (designated CK13L) had identical sequence in the region of overlap with the sequence reported in Fig. 2except that it contained an 24-bp insert such that Lys-423 is changed to Asn followed by the sequence CQKVLNMW before returning in-frame to the CK13 sequence beginning at Cys-424.


Figure 1: Restriction maps of cDNA clones encoding CK11, CK12, and CK13. The coding sequences are shown as filled boxes; the inserted sequence in one CK13 clone is labeled CK13L. The regions of probes used for Northern hybridization are indicated as A-C.




Figure 2: Alignment of predicted protein sequences of CK1, CK11, CK12, and CK13.



Tissue Distribution of CKI Messages

Northern analyses of total RNA from rat testis, brain, heart, kidney, lung, liver, skeletal muscle, and spleen was used to assess the distribution of CK1 messages. Poly(A) RNA from testis was also analyzed. For the CK1 enzymes, isoform-specific probes were based primarily on non-coding sequences (see Fig. 1). CK11 message was scarcely detectable in total RNA from any of the tissues but was observed as a species of 2 kb in the poly(A) RNA from testis ( Fig. 3and 4). The CK12 probe hybridized two species of 1.5 and 2.4 kb in total and poly(A) RNA from testis but no signal was seen in the RNA from other tissues. The CK13 probe hybridized to a 2.8-kb message that was present in all but spleen and heart of the tissues analyzed. In other experiments using a larger fragment of CK13 cDNA that included coding sequences, a second signal, corresponding to 4.2 kb, was observed in RNA from testis, brain, kidney, and lung (not shown). A similar larger message had also been seen in previous work using probes that included coding sequences (12, 14) . Using a non-coding probe for rat CK1, a species of 1.8 kb was detected, only in testis, essentially in agreement with previous studies (14) . We also analyzed for mRNA corresponding to CK1 and CK1. For CK1, we used a probe corresponding to the coding region of the rat enzyme and in these experiments only observed a signal in testis RNA (data not shown). This species was approximately 1.7 kb. However, Rowles et al.(12) had previously detected CK1 message in bovine brain and thymus. For CK1, the probe was a fragment of the bovine brain cDNA. Two species, of 1.6 and 2.6 kb, hybridized in RNA from several tissues; the larger message was present in testis, brain, heart, kidney, muscle, and spleen, with smaller message detected in testis, brain, and heart (data not shown).


Figure 3: Tissue distribution of CK13 messages. Total RNA from the indicated tissue was separated by electrophoresis, transferred to nitrocellulose, and hybridized with a CK13 probe as described under ``Experimental Procedures.'' An autoradiogram is shown.



Expression and Characterization of CK11, CK12, and CK13

The three CK1 isoforms could be expressed as active proteins in E. coli. and were partially purified. Incubation with ATP and Mg led to autophosphorylation of all three enzymes (Fig. 5). All three CK1 isoforms phosphorylated typical CK1 substrates such as casein and phosvitin as well as the specific D4 peptide substrate that we have used in other studies (Fig. 6). Phosphorylation of the D4 peptide was enhanced by the presence of heparin, as had been described for CK1 (14). Under the conditions used, the activity of CK12 and CK13 was stimulated over 10-fold, compared with about 6-fold activation for CK1. The CK11 isoform, however, was stimulated only about 2-fold by heparin. The truncated CK1, which is not affected by heparin, served as a negative control. With phosvitin as substrate, heparin inhibited CK12 and CK13, as had been observed for CK1 (14) . The CK11 isoform was activated by low heparin levels but was ultimately inhibited at high heparin concentration, behavior similar to that of CK1 (34) . With casein as a substrate, all three CK1s were inhibited by the glucosaminoglycan. CK1-7 (35) , a known inhibitor of other CK1 forms, was also tested on the CK1 enzymes (Fig. 7). Although some inhibition was achieved, CK1-7 was a much less effective inhibitor of this group of enzymes than of CK1, which was run as a control. Under the conditions of the assay, 50% inhibition of truncated CK1 occurred around 10 µM, similar to the value of 12 µM reported for the full-length enzyme (14) . Corresponding values for CK11 and CK12 were 200 and 60 µM, respectively. CK13 was the least affected by CK1-7, with only 30% inhibition at 300 µM inhibitor, the highest concentration tested.


Figure 5: Autophosphorylation of CK11, CK12, and CK13. Purified protein kinase was autophosphorylated as described under ``Experimental Procedures,'' analyzed by SDS-polyacrylamide gel electrophoresis, and an autoradiogram prepared. Track 1, CK11; track 2, CK12; track 3, CK13.




Figure 6: Effects of heparin on CK11, CK12, and CK13 activity. Protein kinase activity of CK11 (squares), CK12 (triangles), or CK13 (inverted triangles)was measured in the presence of the indicated concentration of heparin with either D4 peptide (panel A), phosvitin (panel B), or casein (panel C) as a substrate. With the D4 peptide, CK1 (diamonds) and truncated CK1 (circles) were also analyzed.




Figure 7: Inhibition of CK11, CK12, and CK13 by CKI-7. Protein kinase assays with D4 peptide as a substrate were performed in the presence of the indicated concentration of inhibitor.



Complementation of YCK Mutants in S. cerevisiae by CK1 Proteins

Little is known regarding the biological functions of any of the known CK1 enzymes. To determine whether the CK1 proteins possess functional similarity with the S. cerevisiae Yck proteins, we first expressed the CK11 and 3 coding sequences in the strain LRB519, which is temperature sensitive for Yck activity. This strain, which carries a deletion of YCK1 and the yck2-2 allele, grows at 23 °C but ceases growth at 37 °C. Introduction of pMG1b or pMG3b, carrying the CK11 and CK13 genes, respectively, in the sense orientation, restored both growth at 37 °C and normal morphology to the yck strain (Fig. 8). The change in phenotype was due to expression of CK1, since transformants of this strain with the control plasmids pMG1a and pMG3a (containing the CK11 and CK13 coding sequences, respectively, in antisense orientation) resembled transformants with the vector pYCDE-2 with respect both to restrictive temperature for growth as well as the altered morphology characteristic of strains lacking Yck activity.


Figure 8: pMG1b and pMG3b allow growth of yck cells at nonpermissive temperature and restore normal morphology. Two transformants of the yck strain LRB519 with each plasmid were grown overnight in liquid synthetic media lacking Trp and drops (5 µl) were placed onto plates incubated at the indicated temperatures. Photographs were taken at 48 h after plating. The cells shown in the micrographs to the right ( 600 magnification) were taken from the plate shown.



To confirm that active CK1 proteins were produced in strains carrying pMG1b and pMG3b, we assayed CK1 activity in extracts from the strains described above using the D4 peptide substrate. A significant elevation in CK1 activity was observed in protein preparations from strains carrying either pMG1b or pMG3b (Fig. 9). Activity was detected in total extracts but was greatly increased in particulate (Fig. 9) as well as membrane-enriched (data not shown) fractions, suggesting that, like the Yck proteins, the rat CK1 proteins may be membrane-associated.


Figure 9: Phosphorylation of D4 peptide in extracts from yck yeast cells expressing rat CKI isoforms. Protein fractions from two transformants of the yck strain LRB519 with each CK1 plasmid were assayed for activity on the D4 peptide and compared to the control strain that does not express CK1. Activity is picomoles of P incorporated into D4 peptide/milligram of protein in the assay mixture/minute assay time. Particulate and soluble protein fractions were prepared and assays were carried out as described under ``Experimental Procedures.''



Although expression of the CK1 proteins restores normal morphology to yck strains at restrictive temperature, the restoration of growth at 37 °C is not to wild-type rates. The activity of the yck2-2 protein is significantly impaired but not completely abolished at 37 °C. Thus, the partial complementation could reflect the fact that the rat proteins carry out a subset of Yck functions. Therefore, we tested whether the rat proteins could support growth of a strain with both YCK genes deleted. We introduced the pMG1b and pMG3b plasmids into the diploid strain LRB600 (yck1-1/yck1-1 yck2-2/yck2-1::HIS3) and sporulated the resulting transformant strains for tetrad analysis. If the CK1 plasmids support growth of yck1 yck2 double deletion strains, meiotic progeny of such diploids should include His (yck1 yck2-1::HIS3) strains. This proved to be the case for both pMG1b and pMG3b diploids. Whereas no haploid His strains were recovered from control diploids, haploid His strains were recovered from diploid strains carrying either pMG1b or pMG3b, and these His strains were always Trp, i.e. carried the plasmid. These strains were extremely slow growing but showed normal morphology (data not shown).

The growth tests shown in Fig. 10 illustrate the partial nature of complementation of the growth defects of yck mutants by the rat CK1 clones. These tests, comparing the growth rates of yck, yck1 yck2, and YCK strains carrying pMG1b and 3b, were photographed at 4 days and 9 days of incubation. The contrast in growth rate between the YCK and yck strains carrying pMG1b or pMG3b is most evident at 4 days of incubation (Fig. 10, top rowsversusbottom rows). The extremely slow growth of the double yck deletion strains is clear at 4 days, but growth clearly continues over the next 5 days.


Figure 10: Partial complementation of yck1 yck2 lethality by expression of CK I proteins. Strains with YCK genotypes as follows: open circles, yck; shaded circles, yck1 yck2; filled circles, YCK, and carrying the indicated plasmid were grown overnight in rich medium. Drops (5 µl) of each culture at 10 (left) or 10 (right) cells/ml were placed onto synthetic medium lacking Trp and incubated at 30 °C. Photographs were taken at the indicated times after plating.



The partial biological complementation suggests that the rat proteins carry out a subset of Yck functions. Although function(s) required for optimal growth are not provided, functions essential for viability as well as for normal morphogenesis are met. This idea is supported by the observation that the slow growth provides strong selective pressure for spontaneous revertants. One strain with pMG3b (Fig. 10, middle row) gave rise to two such mutants during growth of the culture that was used for the growth test. The revertant colonies showed wild-type growth rate. Genetic characterization of each revealed that they contain two different genomic mutations, and each alone partially eliminates the requirement for Yck proteins. However, only in combination with pMG1b or pMG3b was wild-type growth rate and morphology restored by either mutation. These synergistic effects confirm that the CK1 proteins provide essential Yck function(s) as well as functions required for morphogenesis but that these functions represent a subset of the Yck functions required for optimal growth.


DISCUSSION

In the present work, we define a novel subfamily of at least three enzymes, CK11, CK12, and CK13, within the CK1 family of protein kinases. One cDNA for CK13L had a sequence that included an 8-amino-acid insert suggestive of an alternately spliced form although this has not been rigorously proven; evidence has been presented for alternate splicing of CK1 (12) . That the CK1s, identified by cDNA cloning, be considered CK1s rests on several factors. First, the CK1s have a clear sequence relationship to the CK1, CK1, and CK1 isoforms, although it should be noted that they are the most remote of the group, having only 51-59% identity to the other isoforms within the protein kinase domain. However, the CK1s carry several signature sequences that are characteristic of other CK1s, such as LLGPSLEDLF, HIPXR, EQSRRDD, and LPWQGLKA. Secondly, biochemical analysis of the three CK1s revealed properties consistent with other CK1 isoforms. The CK1 proteins expressed in E. coli were active protein kinases that phosphorylated typical acidic CK1 substrates such as casein and phosvitin as well as, perhaps more tellingly, the D4 peptide which has been shown to be relatively specific for CK1 (29) . In addition, all CK1 isoforms were activated by heparin when the D4 peptide was substrate, a property shared with CK1 (14) . With protein substrates, heparin was inhibitory as is true for CK1 and CK1 (14, 34) . Therefore, by several criteria, the CK1s can be classified as casein kinase I and, even if this historical name is imperfect, it is probably wise to retain this familiar nomenclature until better functional definition of the enzymes is available.

The justification for classing the CK1s as a subfamily rests primarily on amino acid sequence comparisons. Within the kinase domain, the degree of identity is over 90% within the CK1 subfamily and is 69-78% over the entire proteins. As a consequence, in 55 locations there are residues conserved in CK1 and not present in the other CK1s. In some instances, the other CK1s have a different but common residue. The S. cerevisiae Yck proteins are also divergent from the previously identified CK1 isoforms, showing 50-60% identity with other CK1s (). Although there is no more significant overall identity between the Yck proteins and the CK1 isoforms, there are several features in common. At 19 of the 55 residues conserved in CK1 isoforms but different among the other CK1 isoforms, both Yck proteins have the CK1-specific residue, whereas at nine locations the Ycks have a residue common to CK1, CK1, and CK1. In addition, the CK1s have a 2-amino-acid insertion (after residue 146 of CK1), a feature in common with the yeast Yck1 and Yck2 proteins. CK12 and CK13 also have considerable sequence identity in the COOH terminus, in which both have Cys-Cys-Cys sequences followed by a region in which 8 of 12 residues are Arg or Lys. The Cys-Cys-Cys motif is rather striking but whether it has any special significance is not known. It is also interesting that the insert in CK13L is immediately preceding the first of these Cys residues. The Cys residues are not disposed properly to form a CAAX box (36) . The yeast Yck1 and Yck2 proteins are alone among the casein kinase 1 isoforms characterized to date in containing the COOH-terminal Cys-Cys sequence motifs that are necessary for isoprenylation and membrane localization of the Ypt1 and Sec4 proteins (37) . The Yck proteins are associated with the plasma membrane, and alteration of the isoprenylation sequence motif abolishes membrane localization (17) .

As judged by Northern analysis, the CK13 isoform is the most widely distributed, with corresponding message readily detected in most tissues analyzed. The same was true for CK1 in our studies. Although we succeeded in detecting CK1 message only in testis, it seems likely that this isoform is more widely distributed since the original clone was from a brain library (12) . Of the tissues analyzed, testis was the only one in which we found message for CK11 and CK12. Thus, testis is the only tissue expressing mRNAs for all the known CK1s and three isoforms, CK11, CK12, and CK1, if not actually testis specific, are most highly expressed in this tissue. A larger message, of 4.2 kb, was detected in several tissues when probes including coding sequences were used. Since no signal was observed with more specific non-coding probes, the large message may correspond to yet another isoform. Another form called CK1 has been identified that shows strongest similarity to CK1 but is a distinct isoform.() The large message could encode CK1.

As noted in the Introduction, current understanding of CK1 functions is limited, and assignment of isoform-specific roles is even less developed. We initiated a different approach to analyzing CK1 function by testing for complementation of defects caused by mutation in the yeast YCK genes. Such complementation would allow both structure-function studies of the CK1 proteins and genetic studies to begin to identify the biological substrates shared by these protein kinases. The results presented, suppression of the yck mutant and partial complementation of yck1 yck2 phenotypes, provide a basis for such studies with CK1 genes. The partial complementation observed suggests that a subset of multiple independent Yck functions is provided by the rat proteins. This idea is supported by the fact that the morphogenesis defect of the yck mutants is genetically separable from the viability defect. Extragenic suppressors of the yck mutant, as well as the mutants described briefly here as augmenting complementation by CK1 proteins, allow growth without restoring normal morphology. Thus, multiple independent pathways require Yck activity, and some of these are accessible to and modified by the rat CK1 proteins.

There are notable similarities between the CK1 and Yck proteins that could suggest functional similarity in vivo. For example, as described previously these proteins share several sequence features that are not shared by other CK1 isoforms. Also, the relatively low response of the CK1 proteins to the CK1-specific inhibitor is more like that of the Yck proteins (Vancura et al., 1993) than the other CK1 isoforms. However, it is probable that the rat CK1 proteins do not represent complete functional counterparts of the yeast Yck proteins since the complementation of the yck mutants by the highly expressed rat proteins is partial. The rat CK1 proteins may be more specialized derivatives of the Yck proteins (more highly evolved for specific functions) or the localization of the rat proteins is similar but not identical to that of the Yck proteins, and thus only certain substrates are accessible to the rat proteins.

  
Table: Amino acid identities among CKI family members

The identities were computed over the kinase domain, residues 15-312 of casein kinase I .



FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK27221 (to P. J. R.), GM26217 (to M. R. C), and GM44140 (to M. H. C.). 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) U22296, U22297, and U22321.

The abbreviations used are: CK1, casein kinase I; kb, kilobase(s); PCR, polymerase chain reaction; bp, base pair(s); cpm, counts/min.

For these experiments, the chosen start codon was based on that proposed in the original partial CK1 sequence analysis (12); further inspection of the predicted sequences for the three CK1 forms, though, shows an upstream, in-frame ATG present in all three isoforms, and this is most likely the true start site. Thus, the proteins that we first expressed in E. coli started at Met-18, Met-18, and Met15 for CK11, CK12, and CK13, respectively, in relation to the protein sequences reported in this paper. Subsequently, we have also expressed full-length CK13 and found no major differences in properties as compared with the shorter form described herein (L. Zhai and P. J. Roach, unpublished results).

L. C. Robinson, unpublished results.

D. Virshup, unpublished data.


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