©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of Human Casein Kinase I (CKI), a Novel Member of the CKI Gene Family (*)

Kimberly J. Fish (1) (2), Aleksandra Cegielska (1) (2), Michael E. Getman (4) (5), Gregory M. Landes (4), David M. Virshup (1) (2) (3)(§)

From the (1)Program in Human Molecular Biology and Genetics, (2)Department of Oncological Science, Division of Molecular Biology and Genetics, and (3)Department of Pediatrics, University of Utah, Salt Lake City, Utah 84112, (4)Department of Human Genetics, Integrated Genetics, Inc., Framingham, Massachusetts 01701, and the (5)Worcester Polytechnic Institute, Worcester, Massachusetts 01609

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The casein kinase I (CKI) gene family is a rapidly enlarging group whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. We report here the cloning and characterization of a novel isoform of CKI from a human placental cDNA library. The cDNA for this isoform, hCKI, predicts a basic polypeptide of 416 amino acids and a molecular mass of 47.3 kDa. It encodes a core kinase domain of 285 amino acids and a carboxyl-terminal tail of 123 amino acids. The kinase domain is 53-98% identical to the kinase domains of other CKI family members and is most closely related to the isoform. Localization of the hCKI gene to chromosome 22q12-13 and the hCKI gene to chromosome 17q25 confirms that these are distinct genes in the CKI family. Northern blot analysis shows that hCKI is expressed in multiple human cell lines. Recombinant hCKI is an active enzyme that phosphorylates known CKI substrates including a CKI-specific peptide substrate and is inhibited by CKI-7, a CKI-specific inhibitor. A budding yeast isoform of CKI, HRR25, has been implicated in DNA repair responses. Expression of hCKI but not hCKI rescued the slow-growth phenotype of a Saccharomyces cerevisiae strain with a deletion of HRR25. Human CKI is a novel CKI isoform with properties that overlap those of previously described CKI isoforms.


INTRODUCTION

Casein kinase I (CKI)()was among the first serine/threonine protein kinases described. CKI is a ubiquitous monomeric enzyme ranging in size from 25 to 55 kDa and is present in membranes, nucleus, cytoplasm, and cytoskeleton of eukaryotic cells, and on mitotic spindles of mammalian cells (Tuazon and Traugh, 1991; Brockman et al., 1992). Its substrate specificity is determined both by acidic or phosphoryl groups three to four residues amino-terminal to the target residue (Flotow et al., 1990; Flotow and Roach, 1991), and by the tertiary structure of the substrate (Cegielska et al., 1994). Protein substrates for CKI identified in vitro encompass both nuclear and cytosolic proteins including glycogen synthase, RNA polymerases I and II, p53, and simian virus 40 (SV40) large T antigen. However, in only a few instances has phosphorylation by CKI been shown to correlate with changes in function of the substrate. For example, phosphorylation of large T antigen by CKI inhibits its ability to initiate viral DNA replication (Cegielska and Virshup, 1993; Cegielska et al., 1994). Similarly, glycogen synthase activity is significantly inhibited by synergistic phosphorylation by cyclic AMP-dependent protein kinase and CKI (Flotow and Roach, 1989).

cDNA cloning has revealed that CKI comprises an ever-growing family of highly related proteins. In recent years a number of isoforms of casein kinase I have been identified from a variety of sources. Four mammalian isoforms, , , , and , were first defined in a bovine brain cDNA library (Rowles et al., 1991). A full-length isoform, CKI (both protein and cDNA), was subsequently isolated from rat testes (Graves et al., 1993). In budding yeast, at least two membrane-associated isoforms, CKI1 and CKI2, are required for vegetative growth (Robinson et al., 1992, 1993; Wang et al., 1992). In Schizosaccharomyces pombe, two homologs, cki1 and cki2, encode distinct but related cytoplasmic enzymes (Wang et al., 1994). A number of the yeast casein kinase I isoforms have tyrosine kinase activity (Hoekstra et al., 1994).

CKI homologs in budding and fission yeast appear to regulate aspects of cellular DNA metabolism. HRR25, a Saccharomyces cerevisiae homolog of CKI, was identified in a screen for mutants sensitive to DNA double-strand breaks (Hoekstra et al., 1991; DeMaggio et al., 1992). Yeast with mutations in HRR25 grow very slowly, and exhibit sensitivity to endonuclease cleavage, alkylating agents, and x-irradiation. Cells with null mutations of HRR25 accumulate in the G phase of the cell cycle. A pair of related CKI homologs, hhp1 and hhp2, play a similar role in DNA repair in S. pombe (Dhillon and Hoekstra, 1994).

To better understand the role of CKI in cellular metabolism we have characterized human members of the gene family. Here we report the cloning, sequence, genomic localization, and activity of a novel member of the CKI family. This isoform, which we have termed human CKI epsilon (hCKI), is most closely related to CKI. Unlike CKI, hCKI contains a carboxyl-terminal extension of 123 amino acids that is similar in size (but not sequence) to those present in yeast CKI family members. Expression of hCKI (but not hCKI) rescued the slow growth phenotype of budding yeast deleted for the HRR25 gene. These findings demonstrate that human and yeast CKI family members share both sequence and functional similarities and suggest that the isoform may be involved in mammalian DNA metabolism.


MATERIALS AND METHODS

Molecular Cloning and Sequencing of hCKI cDNA Clones

Polymerase chain reaction primers were designed using the sequence of bovine CKI as a guide. Forward primer (5`-AGA CCG CGG CTG GGG AAG AAG GGC AAC TTG GT) containing a SacII site and reverse primer (5`-GGA AGA TCT GGT GGG TGC GGG CGT CCC) containing a BglII site were used to amplify a 94-base pair (bp) fragment from a human placental cDNA library in ZapII (Stratagene). The polymerase chain reaction product was sequenced and used as a probe (Probe 1, Fig. 1A) to screen the library. Positive clones were plaque-purified, and plasmids were isolated from the recombinant phage according to the manufacturers instructions (Stratagene). The library was probed a second time with a 760-bp EcoRI fragment of the 10.1 (hCKI) clone (Probe 2, Fig. 1A) isolated in the first screen (Fig. 1A). cDNAs were sequenced on both strands using the Sequenase 2.0 sequencing kit (U. S. Biochemical Corp.).


Figure 1: Map and sequence of human CKI. A, map of clone 10.1 encoding hCKI. Clone 10.1 is depicted schematically with the deduced protein coding sequence shown as a box and the 5`- and 3`-untranslated regions shown as a single line. Probes 1 and 2 are defined under ``Materials and Methods.'' B, nucleotide and predicted amino acid sequence of human cDNA encoding hCKI. Amino acids are indicated in single-letter code. The stop codon is indicated by a star. The EcoRI linkers on either end of the library cDNA are in italics.



Human CKI cDNAs were isolated from the same library using a 1.1-kilobase pair bovine CKI cDNA clone (provided by Melanie Cobb, University of Texas, Southwestern) as a probe. One positive cDNA clone, 2.2.1, was sequenced on both strands. The insert contains a 204-nucleotide 5`-untranslated region, an open reading frame of 1011 nucleotides encoding a protein of 337 amino acids, and 170 nucleotides in the 3`-untranslated region. The deduced amino acid sequence of the 2.2.1 cDNA indicates extremely high conservation between human and bovine CKI with 100% sequence identity at the amino acid level. It lacks the 84-bp insert found in the bovine L form (Rowles et al., 1991) but contains a novel 12-amino acid insertion at the carboxyl terminus. Because a human CKI clone without the 12-residue insertion has been isolated previously,()we refer to our clone as hCKI2. hCKI2 appears to be an additional splice variant of CKI. This sequence has been deposited in GenBank with accession number L37042. A cDNA clone similar to the one described here has recently been reported (Tapia et al., 1994). That clone differs from our hCKI2 clone at 27 nucleotides, 12 of which lie in the coding region and are responsible for mismatches at 4 of the amino acids predicted by the primary sequence.

Northern Blot Analysis

Total RNA was isolated from human tissue culture cell lines 293, HeLa, HL60, MCF-7, NMB, HCT116, HCT15, and DLD1 by the acid-phenol extraction method. Poly(A)-selected RNA was isolated from HeLa cells by CsCl purification and Oligotex-dT matrix (Qiagen). RNA from both sources was separated by denaturing gel electrophoresis in 1% agarose and transferred to supported nitrocellulose (Hybond C-super, Amersham) essentially as described (Ausubel et al.(1994), and manufacturers instructions). The filters were probed with nick-translated P-labeled full-length hCKI2, hCKI, and hCKI cDNAs or with a 0.9-kb PstI fragment of mouse actin cDNA.

Isolation of hCKI Genomic Clones

Genomic clones for hCKI, hCKI, and hCKI were isolated from a P1 human genomic library (Shepherd et al., 1994) using the corresponding full-length cDNAs. Single clones A115C12 and E14G10 were obtained for hCKI2 and hCKI, respectively, while two clones, D46B1 and D76C5, were obtained for hCKI. By restriction mapping, P1 clones D46B1 and D76C5 both had overlapping fragments that aligned with fragments present on genomic Southern blots probed with hCKI. Additionally, two overlapping clones, cos3.3 and cos4.2, were isolated from a total human placental cosmid library (Stratagene) probed with hCKI.

Fluorescent in Situ Hybridization

Metaphase spreads were prepared from normal cultured 46, XY lymphoblasts by standard procedures. Probe preparation and fluorescence in situ hybridization were performed essentially as described (Lichter et al., 1988; Curran et al., 1992). In addition, Cot1 and salmon sperm DNA were both present in the hybridization mixture at 5 µg/ml. Biotinylated probes were visualized using fluorescein isothiocyanate-avidin (5 µg/ml, Vector Laboratories, Burlingame, CA) or Cy3-steptavidin (2 µg/ml, Jackson ImmunoResearch Laboratories, West Grove, PA). Digoxigenin-labeled probes were detected using fluorescein isothiocyanate-anti-digoxigenin (0.5 µg/ml, Boehringer Mannheim). Preparations were examined using a Zeiss Axioskop microscope equipped for epifluorescence with the appropriate filter sets. Photographs were taken directly from the microscope onto Kodak Gold 400 film. Digitized images of hybridized preparations were obtained using a PXL cooled CCD camera (Photometrics, Tucson, AZ) mounted on a Zeiss Axioskop microscope. Fractional lengths (Lichter et al., 1990) were determined as described previously (Landes et al., 1995) except that digitized images were analyzed by a chromosome mapping algorithm developed by Biological Detection Systems (BDS, Pittsburgh, PA). For each genomic clone, 10 hybridized metaphase chromosomes were analyzed and the FLpter% determined. The high and low values were eliminated and the remaining values averaged and the standard deviation (S.D.) determined. Localizations are denoted as FLpter% ± 2 S.D.

Bacterial Expression of hCKI

The hCKI clone 10.1 in the pBluescript polylinker was digested with NcoI to completion and partially with SalI. The 1333-bp product was inserted into NcoI/XhoI-digested pET16b (a T7-based expression vector (Novagen)). The pET-hCKI expression construct (pKF115) contains 1248 bp of coding sequence followed by 55 bp of 3`-untranslated sequence and 30 bp of pBluescript sequence. This construct removes the hexahistidine sequence present in the pET16b vector downstream of the NcoI site. The construct was transformed into Escherichia coli strain BL21(DE3) pLysS, which contains the T7 RNA polymerase gene under the control of the lacUV5 promoter and a plasmid encoding T7 lysozyme (Studier et al., 1990; Studier, 1991). Bacteria were grown at 37 °C in L broth containing 50 µg/ml carbenicillin and 34 µg/ml chloramphenicol to an A between 0.7 and 0.85. Isopropyl-1-thio--D-galactoside was then added to a final concentration of 0.4 mM and the cultures were grown for an additional 3 h at 37 °C. Cells were collected by centrifugation, snap-frozen in liquid nitrogen, and lysed by thawing on ice and resuspending in 1/30 the original volume of buffer B consisting of 20 mM HEPES, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 10% sucrose, 0.02% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, with the addition of 200 µg/ml DNase I. The extracts were incubated on ice for 60 min with occasional mixing and clarified by centrifugation in a microcentrifuge at 12,000 g for 15 min. The supernatants were used either directly or after immunoprecipitation for kinase assays.

Kinase reactions were performed in buffer containing 20 µM ATP, 30 mM HEPES, pH 7.5, 7 mM MgCl, 0.5 mM dithiothreitol, and 1-5 µCi of [-P]ATP in a final volume of 20 µl, and incubated for 30 min at 37 °C. Peptide phosphorylation reactions contained 1 mM D4 peptide (DDDDVASLPGLRRR (Flotow and Roach, 1991)) and were quantitated by spotting trichloroacetic acid-treated reactions on P81 phosphocellulose filters (Casnellie, 1991). Other reactions were stopped by the addition of SDS-PAGE sample buffer and were analyzed by SDS-PAGE and autoradiography as described previously (Cegielska and Virshup, 1993; Cegielska et al., 1994).

Anti-peptide Antibodies Against hCKI and CKI

To generate antibodies against hCKI, the synthetic peptide MELRVGNKYRLGC, consisting of the first 12 amino-terminal amino acids of hCKI (Fig. 1B) with a cysteine residue at the carboxyl terminus was coupled to m-maleimidobenzoyl-n-hydroxysuccinimide ester-activated bovine serum albumin (Harlow and Lane, 1988). To generate antibodies against hCKI, the synthetic peptide CSGQGQQAQTPTGF, corresponding to the last 13 amino acid residues of hCKI with an amino-terminal cysteine, was similarly coupled to m-maleimidobenzoyl-n-hydroxysuccinimide ester-activated keyhole limpet hemocyanin. Rabbits were injected with conjugated peptides at HRP, Inc., Denver, PA. Antiserum against the hCKI peptide (expected to recognize both CKI and , see Fig. 2) is from rabbits UT31 and UT32, while rabbits UT3 and UT4 were immunized with the CKI peptide-KLH conjugate.


Figure 2: Alignment of selected casein kinase I family members. Alignment of hCKI, hCKI, hCKI2, and HRR25, generated by the PILEUP program of the GCG package (Devereux et al., 1984). Amino acid identities are indicated with a hyphen (-), a star marks the first and last residues of the kinase domain, deletions are indicated by a dot, and # marks the putative nuclear localization signal. HRR25 is truncated at residue 421. The sequences used to generate antisera are underlined.



Extracts from E. coli expressing hCKI were subjected to 12% SDS-PAGE and transferred to a nitrocellulose filter in 12.5 mM Tris, pH 8.5, 86 mM glycine, 0.1% SDS, 20% methanol. After a 15-min fixation in 0.5% glutaraldehyde in phosphate-buffered saline, the filter was blocked with 3% nonfat dry milk and then incubated with a 1:1000 dilution of UT31 antiserum. Bound antibody was detected using a alkaline phosphatase-conjugated goat-antirabbit IgG (Bio-Rad) followed by incubation with bromochloroindolyl phosphate and nitro blue tetrazolium (Harlow and Lane, 1988).

Immunoprecipitation of Recombinant hCKI

Recombinant hCKI protein was immunoprecipitated from E. coli extracts (100 µg of protein) by the addition of 10 µl of rabbit antiserum UT31, followed 30 min later by 30 µl of a 50% slurry of protein A-agarose (Pierce). After an overnight incubation at 4 °C, the immunoprecipitates were washed three times in 50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, once in the same buffer without SDS, and once in buffer B. Kinase reactions were performed as described above. To determine kinase autophosphorylation and phosphorylation of protein substrates, the reactions were stopped by boiling in sample buffer and analyzed on SDS-polyacrylamide gels. To determine peptide phosphorylation, protein A-agarose beads were removed by centrifugation, and acetic acid-treated supernatants were spotted onto P81 filters.

Mutagenesis of hCKI and hCKI2

To generate an inactive form of hCKI, lysine 38 was mutated to arginine (hCKI)(pDV35) by the unique site elimination method (Deng and Nickoloff, 1992) using the oligonucleotide C ACA CTC CAG CCG GAT GGC GAC TTC. Mutations in hCKI2 were created using the pAlter-1 Altered Sites system (Promega). To facilitate subsequent cloning, an NcoI site (CCATGG) was created at the initiating ATG of hCKI2(pELV3) using the oligonucleotide CT CTT CGT CTC TCA CCA TGG CGA GTA GC. hCKI2(pELV6) contains an NcoI site at the initiating ATG and a mutation at codon 46 in which lysine was changed to alanine using the oligonucleotide GG AAG TGG CAG TGG CGC TAG AAT CTC AG. All mutations were confirmed by DNA sequencing.

Yeast Manipulation

S. cerevisiae strain hrr25 (ura3-1, trp1-1, leu2-3, 112, his3-11, 15, can1-100, ade2-1, hrr25 ) was provided by Merl Hoekstra (Icos Corp.). Standard methods and media for yeast propagation and transformation were employed (Sherman et al., 1986; Schiestl and Gietz, 1989).

Expression of Galactose-inducible CKI Expression Vectors

To obtain galactose-inducible expression of CKI in S. cerevisiae, we utilized a derivative of pRS305 (pRS305 2µ Gal1-10) that contains the 2µ origin of replication and the Gal1-10 promoter followed by a polylinker including NcoI and HindIII sites (provided by M. Hoekstra). hCKI and hCKI were excised from pBluescript SK(-) with NcoI and HindIII and inserted into the corresponding site of pRS305 2µ Gal1-10 (pDV49 and pKF119, respectively). hCKI2 and hCKI2 were excised from pAlter by a HindIII/partial NcoI digest and similarly ligated into the expression vector (pKF120 and pKF121, respectively). As a positive control, a construct containing HRR25, likewise cloned into the NcoI site of pRS305 2µ Gal1-10, was obtained from M. Hoekstra. The expression plasmids were transformed into hrr25 cells and single transformants were selected and grown to saturation in synthetic complete media lacking leucine.

To ascertain whether the CKI2 construct indeed produced active kinase, duplicate transformants of hCKI2 and hCKI2 were grown in 10-ml synthetic medium with 2% raffinose as the carbon source to an A of 0.2. Kinase production was induced by the addition of galactose to a final concentration of 2%. After a 4-h induction at room temperature, the culture was harvested for soluble protein by glass bead disruption. Aliquots of extract containing 100 µg of protein were incubated with UT3 antiserum and protein A-agarose beads and the resulting immunoprecipitates used for a peptide kinase assay as described previously.

Figures

Polaroid photographs, autoradiographs and SDS-PAGE gels were scanned on a Scanmaker IIXE (MicroTek) at 300 dots per inch and assembled in Photoshop 2.5.1 (Adobe). Labels were applied in Canvas 3.5 (Deneba Software).


RESULTS

Isolation and Characterization of a Human Casein Kinase I cDNA Clone

A human placental cDNA library was screened using a 94-bp polymerase chain reaction product (Probe 1, Fig. 1A; see ``Materials and Methods''). The two clones obtained appeared to be identical by restriction digest and partial sequence analysis. The cDNA insert in one of these clones, clone 10.1, was sequenced to completion. The clone 10.1 insert is 1.33 kilobase pairs long and includes 22 bp of 5`-untranslated sequence, an open reading frame of 1248 bp, and 55 bp of 3`-untranslated sequence including the stop codon (Fig. 1B). To see if additional related clones were present in the library, the 5` portion of clone 10.1, from the 5` EcoRI cloning site to the internal EcoRI site (Probe 2, Fig. 1A), was used as a probe to re-screen the library. Seven additional clones were isolated, three of which contained sequences related to, but distinct from the 10.1 clone. One of these related clones, clone 12.1, appears to be a partial but nearly full-length cDNA encoding human CKI. It contains an open reading frame identical to rat CKI for the first 380 amino acids, but lacks the first 20 bp (seven amino acids) of the CKI open reading frame. As discussed below, clones 10.1 and 12.1 appear to be derived from distinct genes.

The presumed start codon of the 1.33-kilobase pair clone 10.1 cDNA is at bases 23-25 (Fig. 1B). This ATG appears to be the authentic start site of the open reading frame because: 1) it lies within an optimal translational initiation consensus sequence of GCCATGG (Kozak, 1986, 1987); 2) it is immediately preceded by a stop codon in the same reading frame (bases 17-19); and 3) the predicted start site is consistent with those predicted for human and rat CKI as well as S. cerevisiae HRR25 (Hoekstra et al., 1991; Graves et al., 1993). Conceptual translation of the open reading frame predicts a polypeptide of 416 amino acids and a molecular mass of 47,314 daltons, with a predicted pI of 10.5.

The clone 10.1 cDNA shows a high degree of sequence similarity to members of the CKI family at both the nucleotide and amino acid levels, with the greatest similarity to the CKI isoform. The predicted kinase domain (residues 9 to 293) is 53-98% identical and 71-99% similar to the kinase domains of other human and yeast members of the CKI family. Although isolated in a screen for CKI, this cDNA appears to be the product of a distinct gene and we therefore propose the name of human casein kinase I epsilon (hCKI) for this new member of the casein kinase I gene family. Several lines of evidence indicate that hCKI is not allelic with CKI. Over a stretch of 179 amino acids where hCKI and hCKI are identical (Fig. 2), they share only 86% nucleic acid identity.()While CKI has 97.5% amino acid identity with hCKI over the kinase domain (residues 9-293, Fig. 2) it is only 53% identical in the carboxyl-terminal region (residues 294-416). Optimal alignment of the hCKI and hCKI carboxyl-terminal domains requires two insertions in the hCKI sequence. Overall, the hCKI cDNA is only 77% identical to the hCKI cDNA open reading frame at the nucleic acid level and the 5`- and 3`-untranslated regions of the two cDNAs are completely divergent. The close similarity between the coding sequences of the two genes suggests a fairly recent gene duplication event.

Like other members of the CKI family, hCKI is highly basic, with positively charged residues uniformly distributed over the length of the molecule. In common with other CKI family members, it lacks the salt-bridge forming APE motif seen in domain VIII of other protein kinases. The 123-residue carboxyl-terminal extension that follows the kinase domain begins with a 15-amino acid stretch (EDVDRERREHEREER) that contains 8 acidic and 6 basic residues. The homologous region of rat and human CKI contains 7 acidic and 7 basic residues in a 16-amino acid stretch. This motif is similar to the KEKE motif proposed to facilitate interactions with the intracellular multicatalytic protease (Realini et al., 1994). The last 100 amino acids include 18 arginine and 11 proline residues and only 6 acidic residues.

Northern Blot Analysis of hCKI Expression in Tissue Culture Cells

A Northern blot of poly(A)-selected RNA from log phase HeLa cells probed with hCKI demonstrated the presence of a major message of 2.7 kilobases (kb) and a lower abundance message of 1.6 kb (Fig. 3B, lanes 5 and 6). These mRNAs are unique to hCKI and not due to cross-hybridization with other known CKI isoforms, since in the same experiment a hCKI2 probe identified mRNAs of 4.0 and 2.2 kb, and a hCKI probe identified species of 3.5, 2.0, and 1.8 kb (Fig. 3B, lanes 1-4). The hCKI probe also identified species of 3.5 and 2.0 kb (Fig. 3B, lane 6); these may represent cross-hybridization with hCKI mRNAs.


Figure 3: Northern blot analysis of hCKI expression. A, hCKI is expressed in diverse tissue culture cell lines. Lanes 1-8 each contain 16 µg and lanes 9-12 each contain 50 µg of total cellular RNA isolated from the indicated human cell lines. In the upper panel, the filter was probed with P-labeled full-length hCKI cDNA, while in the lower panel the same filter was probed with P-labeled mouse actin cDNA. A star marks the 2.7- and 1.6-kb hCKI mRNAs, while the triangle () indicates a 3.5-kb mRNA species that may represent cross-reactivity with hCKI mRNA. HCT 15, colon adenocarcinoma; HCT 116, colon carcinoma; DLD1, colon carcinoma; HL60, promyelocytic cell line; HeLa, cervical carcinoma; 293, adenovirus-transformed embryonal kidney; MCF7, breast adenocarcinoma; NMB, neuroblastoma. B, hCKI mRNA is distinct from the mRNA of other members of the human CKI family. A Northern blot containing poly(A) selected RNA from log phase HeLa cells was probed with P-labeled hCKI2 (lanes 1 and 2), hCKI (lanes 3 and 4), or hCKI (lanes 5 and 6). Lanes 1, 3, and 5 each contain 2 µg, and lanes 2, 4, and 6 each contain 4 µg of RNA.



A Northern blot was performed on total RNA extracted from eight cultured human cell lines to determine the spectrum of hCKI expression. A major message of 2.9 kb was present in most cell lines, while a less abundant mRNA of 1.77 kb was also apparent (best seen in Fig. 3A, lanes 9, 11, and 12, and also detected upon longer exposure in other cell lines and in poly(A)- selected RNA, data not shown). Of note, hCKI mRNA was not detected in HL60 and MCF-7 cells (Fig. 3A, lanes 7 and 10). This broad distribution of hCKI expression is similar to that seen with rat and rabbit CKI, which was detected in virtually all tissues examined (Graves et al., 1993).

Genomic Localization of hCKI, hCKI, and hCKI

Mutations in human genes involved in DNA metabolism may have diverse clinical manifestations. It was of interest to determine whether: 1) hCKI and hCKI mapped to distinct loci, and 2) any of the known CKI genes were linked to any mapped human genetic disorders.

The genomic location of the CKI-related genomic clones A115C12, D76C5, E14G10, and cos3.3 (see ``Materials and Methods'') was determined by fluorescent in situ hybridization to metaphase chromosomes (Fig. 4). Hybridization efficiency for each genomic clone was greater than 90% for both metaphase and interphase chromosomes. A115C12 (hCKI) hybridized to a D group chromosome (chromosomes 13-15), D76C5 (hCKI) hybridized to an E group chromosome (chromosomes 16-18), and E14G10 and cos3.3 (both hCKI) hybridized to a G group chromosome (chromosomes 21, 22). Because E14G10 also hybridized to the Y chromosome, further localization of hCKI was performed only with cos3.3.


Figure 4: Fluorescent in situ hybridization of CKI-containing genomic clones to human metaphase chromosomes. Hybridized probes were detected with Cy3-avidin. A, hybridization of biotinylated A115C12 (hCKI) to a D group chromosome. B, hybridization of biotinylated D76C5 (hCKI) to an E group chromosome. C, hybridization of biotinylated cos3.3 (hCKI) to a G group chromosome. D, refined localization of CKI-containing genomic clones using FLpter%. The left, middle, and right panels show the FLpter% map positions (within 2 standard deviations) alongside chromosome ideograms (Francke, 1994) of chromosome 13 for A115C12 (39.7 ± 6.4%), chromosome 17 for D76C5 (93.8 ± 4.8%), and chromosome 22 for cos3.3 (73.7 ± 4.4%). Ideograms represent chromosome preparations at the 850 band level.



Chromosomal assignments were made by co-hybridizing a biotin-labeled CKI-containing genomic clone with a uniquely haptenated fiduciary marker originating from a candidate chromosome. Coincident hybridizations were achieved when fiduciary markers for chromosome 13 (D13S6), chromosome 17 (Yurov et al., 1987; Alexandrov et al., 1988), and chromosome 22 (D22S9) were co-hybridized with A115C12, D76C5, and cos3.3, respectively (data not shown).

Chromosomal localizations were refined by determining the chromosomal address in terms of FLpter%. As described by Lichter et al. (1990), the distance the hybridization signal was from the pterminus (pter) was expressed as a percentage of the actual contour length of the chromosome (pter to qter) and denoted as FLpter%. The results for each genomic clone are summarized in Fig. 4. A115C12 mapped to chromosome 13 with a FLpter% value of 39.7 ± 6.4%. D76C5 mapped to chromosome 17 with a FLpter% of 93.8 ± 4.8%. cos3.3 mapped to chromosome 22 with a FLpter% value of 73.7 ± 4.4%. When compared to banded chromosomes A115C12 originates from chromosome 13q13.1-14.1, confirming the recent report of Tapia and co-workers (Tapia et al., 1994). The fact that only a single genomic locus was found for hCKI supports the hypothesis that hCKI, hCKI2, and hCKIL (also called CKI3) are splice variants rather than the products of unique genes. D76C5 localizes to chromosome 17q25.2-25.3 in a region containing the thymidine kinase gene. The gene encoding hCKI is located on chromosome 22q12.3-13.1 telomeric to the NF2/MERLIN locus and lies in a region frequently deleted in familial and sporadic meningiomas (OMIM, 1994).

Expression of Recombinant Casein Kinase I in E. coli

The high degree of similarity between hCKI and hCKI strongly suggests that hCKI encodes an active kinase. To confirm this, and as an initial step in characterizing hCKI activity, the hCKI coding sequence was cloned into the pET16b bacterial expression vector (``Materials and Methods''). Cultures of E. coli strain BL21(DE3) pLysS transformed with empty pET16b vector or vector containing hCKI were grown as described under ``Materials and Methods.'' After induction with isopropyl-1-thio--D-galactoside, cells carrying the hCKI construct contained a 48-kDa protein that reacted with an anti-CKI/ antibody on immunoblot (Fig. 5A). As seen with CKI (Graves et al., 1993), expression of hCKI was at relatively low levels, since no significant bands of this size were evident on Coomassie-stained gels of total cellular extract (data not shown).


Figure 5: Expression of active hCKI in E. coli.A, hCKI encodes a 48-kDa polypeptide E. coli strain BL21(DE3) pLysS containing expression vector pET16b with or without hCKI cDNA insert were lysed before or after a 2-h induction at 37 °C with 0.4 mM isopropyl-1-thio--D-galactoside (IPTG). Expression of hCKI was detected by Western blot with a 1:1000 dilution of UT31 antiserum in the absence (lanes 1-4) or presence (lane 5) of 1 µg of the antigenic peptide. B, D4 peptide phosphorylation in crude extracts. Soluble extracts were prepared from isopropyl-1-thio--D-galactoside-induced and uninduced E. coli carrying pET16b or pET16b-hCKI. Kinase activity on the peptide substrate DDDDVASLPGLRRR was quantitated as described (``Materials and Methods''). C, substrate specificity of hCKI. Recombinant hCKI was immunoprecipitated from extracts with UT31 antiserum and incubated in the presence of [-P]ATP with 1 µg of phosvitin (lane 1), 10 µg of casein (lanes 2 and 5), 10 µg of histone H1 (lane 3), or no added substrate (lane 4). The kinase immunoprecipitation in lane 5 was performed in the presence of excess antigenic peptide. The arrow indicates the position of autophosphorylated hCKI (48 kDa). The 60-kDa band (marked with *) is a co-precipitating E. coli protein, since a band of identical size co-precipitates with a truncated 44-kDa hCKI (data not shown). D, hCKI kinase activity is inhibited by CKI-7. The activity of immunoprecipitated hCKI on the substrate peptide D4 was determined at increasing concentrations of the CKI inhibitor CKI-7.



Phosphorylation of Peptide by Crude Extracts

Casein kinase I family members are characterized by their ability to phosphorylate substrates with acidic or phosphoserine residues amino-terminal to the target serine or threonine. To determine if overexpressed hCKI had a similar sequence specificity, the D4 peptide (see ``Materials and Methods'') was incubated with soluble extracts from E. coli carrying the hCKI construct. CKI activity was present at low levels before induction and increased 5.5-fold after induction (Fig. 5B). Lower levels of D4 phosphorylation were found using extracts from induced cells carrying empty expression vector. These data indicate that hCKI can phosphorylate a well-defined casein kinase I substrate.

Phosphorylation of Protein Substrates by hCKI Immunoprecipitates

The activity of hCKI on a variety of protein substrates was tested, taking advantage of the finding that hCKI was active after immunoprecipitation from E. coli lysates with anti-hCKI antiserum. As expected for members of the casein kinase I family, immunoprecipitates from induced cells phosphorylated phosvitin (Fig. 5C, lane 1) and casein (lane 2), but not histone H1 (lane 3). Inclusion of the antigenic peptide in the immunoprecipitation reaction significantly reduced the kinase activity in the pellet (lane 5). Immunoprecipitated hCKI actively autophosphorylated (Fig. 5C, arrow), an activity well described for other CKI family members.

Inhibition of hCKI by the Casein Kinase I Inhibitor CKI-7

The compound N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7) has been reported to specifically inhibit most (Chijiwa et al., 1989), although not all (Vancura et al., 1993) isoforms of casein kinase I. To determine if CKI-7 (obtained from Seikagaku Corp.) inhibits hCKI activity, increasing concentrations of the compound were incubated with immunoprecipitated hCKI and the D4 peptide. CKI-7 inhibited phosphorylation with half-maximal inhibition of kinase activity at 18 µM CKI-7 (Fig. 5D). These results are consistent with those of Chijiwa et al.(1989), who found an K of 8.5 µM using rabbit skeletal muscle CKI, and Graves et al.(1993), who described an IC of 12 µM using recombinant rat CKI. Taken together these results support the hypothesis that hCKI is a bona fide member of the casein kinase I gene family.

Rescue of the hrr25 Slow Growth Phenotype by hCKI but Not hCKI2

Sequence analysis of the casein kinase I gene family members suggests that hCKI and hCKI are most closely related to S. cerevisiae HRR25 and S. pombe hhp1 and hhp2, the kinases involved in DNA repair. A prominent phenotype of yeast with a deletion of the HRR25 gene is very slow growth with a doubling time of greater than 12 h. To determine whether human CKI isoforms could complement this growth defect, cDNAs encoding hCKI and hCKI2 were cloned into a galactose-inducible high copy expression vector (see ``Materials and Methods''). Inducible expression of the cloned proteins in hrr25 cells was confirmed by immunoblot analysis of extracts from induced cultures (data not shown). We ascertained that active hCKI2 was produced by incubating extracts from uninduced and induced cells with CKI antiserum and assaying the immunoprecipitates for CKI activity.

As shown by the dilution spot assay (Fig. 6), under inducing conditions hCKI rescued the slow growth phenotype of the hrr25 cells almost as well as HRR25. The kinase activity of hCKI was required to rescue, since a kinase-deficient mutant did not complement. Hoekstra and co-workers have found that human CKI also complements the hrr25 cells,()consistent with the high degree of homology of the two proteins. Interestingly, the construct expressing hCKI2 grew almost as poorly as the deletion mutant bearing vector alone. Thus, hCKI2 and hCKI, while closely related, are functionally distinct.


Figure 6: Rescue of S. cerevisiae hrr25 slow growth phenotype by hCKI but not hCKI2. S. cerevisiae with a deletion of HRR25 were transformed with a high copy plasmid bearing the indicated cDNAs under the control of the Gal1-10 operator. Serial 10-fold dilutions of saturated cultures were spotted on selective medium containing glucose or galactose as the sole carbon source and incubated for 4 days at 30 °C.




DISCUSSION

Casein Kinase I Epsilon Is a New Member of the Casein Kinase I Gene Family

This report details the isolation and cDNA cloning of a new member of the casein kinase I gene family. This kinase, hCKI, contains a kinase domain highly related to other members of the family, and a carboxyl-terminal extension most closely related to that of CKI (Graves et al., 1993). Nucleic acid sequence and chromosomal localization indicate that hCKI is the product of a distinct gene, rather than an allele of CKI.

Many of the Casein Kinase I Genes Appear to be Redundant

A prominent feature of the casein kinase I gene family tree is the redundancy present in several of its branches. In fission yeast at least two pairs of CKI isoforms have been found. One pair, hhp1 and hhp2, play overlapping roles in DNA repair (Dhillon and Hoekstra, 1994). A second pair, cki1 and cki2, while homologous in the kinase domain, are distinct in the carboxyl-terminal tail and appear to play distinct roles in vivo (Wang et al., 1992, 1994). In budding yeast, YCK1 and YCK2 form an essential gene pair (Robinson et al., 1992, 1993), while HRR25 does not appear to have a partner. In mammals, CKI and CKI are most closely related, both containing only a short (24-35 amino acid) carboxyl-terminal extension. It is of note, however, that CKI has not yet been isolated from human cDNA libraries. Using bovine CKI as a probe, we have isolated several distinct human CKI cDNA clones, suggesting multiple related genes in this branch of the family (data not shown). The identification of hCKI, with its high degree of homology with CKI, demonstrates that this branch of the CKI tree has redundancy as well.

The sequence similarity of various CKI isoforms suggests they may fulfill similar but not necessarily the same functions in vivo. Thus, CKI and CKI may serve overlapping rather than identical functions in mammals. A functional overlap (as opposed to complete redundancy) may result from subtle or overt differences in enzyme regulation, substrate specificity, intracellular localization, or tissue specific expression. Thus, the redundancy suggested by the high degree of sequence similarity does not rule out clinical manifestations caused by mutations or disruptions of a single gene.

Several lines of evidence suggest that hCKI may play a role in DNA metabolism. First, the related CKI can inhibit the replication function of large T antigen in SV40 DNA replication (Cegielska and Virshup, 1993; Cegielska et al., 1994). Importantly, hCKI and hCKI but not hCKI2 rescues the growth phenotype of hrr25, a yeast kinase involved in DNA repair. This appears to confirm the suggestion of Kuret and co-workers (Kearney et al., 1994) who found that the CKI/ branch of the mammalian CKI family are most similar to HRR25, Hhp1 and Hhp2, the yeast kinases associated with DNA repair defects. Of note in this regard, hCKI shares with hCKI, hCKI, HRR25, Hhp1, and Hhp2 a potential nuclear localization signal (KRQK, residues 221-224) and purification of CKI activity from mammalian nuclei has yielded forms of CKI similar in size (50-55 kDa) to hCKI (Tuazon and Traugh, 1991). It was therefore of interest to determine whether any of the CKI genes were linked to any known DNA repair disorders. The hCKI gene indeed resides in a region (22q12.3-13.1) implicated in familial and sporadic meningiomas distinct from mutations in NF2 or the c-sis proto-oncogene (OMIM, 1994). Further studies will be required to determine if the hCKI gene is indeed mutant in this or other diseases.

Function of Carboxyl-terminal Extension

A prominent feature of hCKI is its carboxyl-terminal extension, a domain it shares with CKI. Three distinct functions of the carboxyl-terminal extension of other casein kinase I family members have been described to date. 1) Regulation of over-expressed rat CKI by heparin was abolished by removal of the tail (Graves et al., 1993). 2) Activity of S. pombe Cki1, using casein as a substrate, was increased severalfold by removal of the tail (Carmel et al., 1994). 3) Removal of the last 80 amino acids of YCK2, including a carboxyl-terminal prenylation signal, significantly impaired its ability to rescue a yck1/yck2 double mutant (Robinson et al., 1993). Thus, sequences in the carboxyl terminus can be involved in enzyme regulation, enzyme activity, and membrane localization. The ability of hCKI (with a 123-amino acid carboxyl-terminal extension) but not hCKI2 (with a 35-amino acid carboxyl-terminal extension) to rescue the growth defect of hrr25 yeast also suggests an important biological role for the carboxyl-terminal domain. How this domain functions in mammalian forms of CKI to regulate its in vivo function remains to be determined.


FOOTNOTES

*
This research was supported in part by National Institutes of Health Grant R01 AI31657 and the Primary Children's Medical Foundation (to D. M. V.). National Cancer Institute Grant 5 P30 CA42014 subsidized a portion of the costs of oligonucleotide and peptide synthesis. 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) L37043, (hCKI) and L37042 (hCKI2).

§
To whom correspondence should be addressed: Program in Human Molecular Biology and Genetics, University of Utah, Bldg. 533, Rm. 4420A, Salt Lake City, UT 84112. Internet: virshup@gene1.med.utah.edu.

The abbreviations used are: hCKI, human casein kinase I; bp, base pair; CKI, casein kinase I, PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair; FLpter%, fractional length from p terminus.

M. Hoekstra, personal communication.

K. J. Fish and D. M. Virshup, unpublished results.


ACKNOWLEDGEMENTS

We thank Erica Vielhaber for mutagenesis of hCKI, Mark Curran, Dick Lemons, Matt Rebentisch, and Darryl Gietzen for technical help, Andrew Thorburn for reviewing the manuscript, Joie Rowles and Melanie Cobb for bovine CKI clones, and Merl Hoekstra for shared reagents and productive discussions. Screening of the P1 library was facilitated by Mark Leppert and the Technical Access Section of the Utah Center for Human Genome Research (5P30 HG00199).


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