From the
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
Casein kinase I (CKI)
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,
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
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
Kinase reactions were
performed in buffer containing 20 µM ATP, 30 mM HEPES, pH 7.5, 7 mM MgCl
To ascertain whether the CKI
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
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
Like other members of the CKI family, hCKI
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
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
As shown by the dilution
spot assay (Fig. 6), under inducing conditions hCKI
The sequence similarity of various CKI
isoforms suggests they may fulfill similar but not necessarily the same
functions in vivo. Thus, CKI
Several lines of evidence suggest that hCKI
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Erica Vielhaber for mutagenesis of hCKI
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
,
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.
(
)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).
,
,
, 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).
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).
), 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.
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 hCKI
2. hCKI
2
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 hCKI
2
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 hCKI
2, 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 hCKI
2 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.
, 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
To generate antibodies against hCKI and
CKI
, 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
, hCKI
2, 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
To
generate an inactive form of hCKI and hCKI
2
, 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 hCKI
2 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 hCKI
2(pELV3) using the oligonucleotide CT CTT
CGT CTC TCA CCA TGG CGA GTA GC. hCKI
2
(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). hCKI
2 and hCKI
2
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.
2
construct indeed produced active kinase, duplicate transformants of
hCKI
2 and hCKI
2
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).
Isolation and Characterization of a Human Casein Kinase
I
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 cDNA Clone
. 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.
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.
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.
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
A Northern blot of poly(A)-selected RNA from log
phase HeLa cells probed with hCKI Expression in Tissue
Culture Cells
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 hCKI
2
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
hCKI
2 (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
Mutations in human genes involved in DNA metabolism
may have diverse clinical manifestations. It was of interest to
determine whether: 1) hCKI, hCKI
, and
hCKI
and hCKI
mapped to distinct loci,
and 2) any of the known CKI genes were linked to any mapped human
genetic disorders.
)
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).
supports the hypothesis that hCKI
, hCKI
2, and
hCKI
L (also called CKI
3) 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
The high degree of similarity between hCKI in E.
coli
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
The activity of hCKI
Immunoprecipitates
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
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 by the Casein Kinase I Inhibitor
CKI-7
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
Sequence analysis of the casein
kinase I gene family members suggests that hCKI Slow Growth Phenotype by
hCKI
but Not hCKI
2
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 hCKI
2 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 hCKI
2 was produced by incubating extracts
from uninduced and induced cells with CKI
antiserum and assaying
the immunoprecipitates for CKI activity.
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
hCKI
2 grew almost as poorly as the deletion mutant bearing vector
alone. Thus, hCKI
2 and hCKI
, while closely related, are
functionally distinct.
Figure 6:
Rescue
of S. cerevisiae hrr25 slow growth phenotype by hCKI
but not hCKI
2. 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.
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.
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.
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 hCKI
2 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 hCKI
2 (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.
/EMBL Data Bank with accession number(s) L37043,
(hCKI
) and L37042 (hCKI
2).
, human
casein kinase I
; bp, base pair; CKI
, casein kinase I
,
PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair; FLpter%,
fractional length from p terminus.
,
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).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.