From the
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
The term casein kinase I (CK1)
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
Enzymological studies of
mammalian CK1 had typically described a monomeric enzyme with
M
From the 1,582-bp cDNA of
CK1
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 CK1
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) .
The growth tests shown in
Fig. 10
illustrate the partial nature of complementation of the
growth defects of yck mutants by the rat CK1
In the present work, we define a novel subfamily of at least
three enzymes, CK1
The justification for classing the CK1
As judged by Northern analysis, the
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
There are notable similarities between the
CK1
The identities were computed over the kinase domain, residues
15-312 of casein kinase I
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
,
three full-length rat testis cDNAs were cloned that defined three
separate members of this subfamily. The isoforms, designated CK1
1,
CK1
2, and CK1
3, have predicted molecular masses of 43,000,
45,500, and 49,700. CK1
3 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
CK1
1 (2 kilobases (kb)), CK1
2 (1.5 and 2.4 kb), and CK1
3
(2.8 kb) were detected by Northern hybridization of testis RNA. Message
for CK1
3 was also observed in brain, heart, kidney, lung, liver,
and muscle whereas CK1
1 and CK1
2 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 CK1
s although less
effectively than the CK1
or CK1
isoforms. All three CK1
s
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 CK1
1 or CK1
3 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.
(
)
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.
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) .
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, CK1
1, CK1
2, and CK1
3,
that form a distinct subfamily within the CK1s. Although over 90%
homologous to each other, CK1
s are only 51-59% identical
within the protein kinase domain to other CK1 isoforms. CK1
1 and
CK1
3 were also found to complement defects in yeast cells
defective in the YCK genes.
Cloning of CKI
A
partial bovine cDNA of 900 bp which encodes for a CK1 isoform termed
CK11
2, and
3 cDNAs
(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 CK
1 and
CK
2 double-stranded sequencing was performed in pBluescript using
T7, T3, and sequence-specific primers. The CK1
3 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 CK1
1, a 361-bp EcoRI-StuI
fragment, position 1280-1641, composed of 50% coding and 50%
3`-untranslated sequences; for CK1
2, a 159-bp
EcoRI-PstI fragment, position 1430-1589, from
the 3`-untranslated region; for CK1
3, 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 CK1
1 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 pETCK1
1.
2, 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 CK1
2 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 pETCK1
2. Two complementary
oligonucleotides of 41 and 45 bp were synthesized corresponding to the
CK1
3 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 CK1
3 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 pETCK1
3.
1 and
CK1
3 coding sequences were excised from pBluescript plasmids and
inserted into the pYcDE2 plasmid. The resulting plasmids pMG1b and
pMG3b contain CK1
1 and CK1
3, respectively, in the correct
orientation. The plasmids pMG1a and pMG3a have CK1
1 and CK1
3
sequences in the antisense orientation.
Expression of CK1
The E. coli strain BL21/DE3, which
contains the T7 RNA polymerase gene under control of the lacUV5
promoter, was transformed with pETCK11,
2, and
3 in
Escherichia coli
1, pETCK1
2, or
pETCK1
3. 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.
CK1
1,
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.
(
)
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) .
Cloning of cDNAs Encoding CKI
From screening a rat testis library using the bovine
partial CK11, CKI
2, and
CKI
3
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 CK1
1, CK1
2, and CK1
3 in the order in which
the corresponding cDNAs were characterized. The partial bovine CK1
sequence was most similar, 97% identical, to that of CK1
3. Of the
11 positives, two corresponded to CK1
1, seven to CK1
2, and
two to CK1
3. CK1
1, CK1
2, and CK1
3 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 CK1
3 (designated CK1
3L) 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 CK1
3 sequence beginning at Cys-424.
Figure 1:
Restriction maps of cDNA clones
encoding CK11, CK1
2, and CK1
3. The coding sequences are
shown as filled boxes; the inserted sequence in one CK1
3
clone is labeled CK1
3L. The regions of probes used for Northern
hybridization are indicated as A-C.
Figure 2:
Alignment of predicted protein sequences
of CK1, CK1
1, CK1
2, and
CK1
3.
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). CK1
1 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 CK1
2 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 CK1
3
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 CK1
3 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 CK1
3 probe as
described under ``Experimental Procedures.'' An autoradiogram
is shown.
Expression and Characterization of CK1
The three CK1 isoforms could be expressed as
active proteins in E. coli. and were partially purified.
Incubation with ATP and Mg1, CK1
2,
and CK1
3
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 CK1
2 and CK1
3 was
stimulated over 10-fold, compared with about 6-fold activation for
CK1
. The CK1
1 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 CK1
2 and CK1
3, as had been observed for
CK1
(14) . The CK1
1 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 CK1
s 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 CK1
1 and CK1
2
were 200 and 60 µM, respectively. CK1
3 was the least
affected by CK1-7, with only 30% inhibition at 300
µM inhibitor, the highest concentration tested.
Figure 5:
Autophosphorylation of CK11,
CK1
2, and CK1
3. Purified protein kinase was
autophosphorylated as described under ``Experimental
Procedures,'' analyzed by SDS-polyacrylamide gel electrophoresis,
and an autoradiogram prepared. Track 1, CK1
1; track
2, CK1
2; track 3,
CK1
3.
Figure 6:
Effects of heparin on CK11,
CK1
2, and CK1
3 activity. Protein kinase activity of CK1
1
(squares), CK1
2 (triangles), or CK1
3
(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, CK1
2, and
CK1
3 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
Little is known regarding the biological
functions of any of the known CK1 enzymes. To determine whether the
CK1 Proteins
proteins possess functional similarity with the S.
cerevisiae Yck proteins, we first expressed the CK1
1 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 CK1
1 and CK1
3 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 CK1
1 and CK1
3 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).
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.
1, CK1
2, and CK1
3, within the CK1
family of protein kinases. One cDNA for CK1
3L 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
CK1
s, identified by cDNA cloning, be considered CK1s rests on
several factors. First, the CK1
s 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 CK1
s carry several signature sequences
that are characteristic of other CK1s, such as LLGPSLEDLF, HIPXR,
EQSRRDD, and LPWQGLKA. Secondly, biochemical analysis of the three
CK1
s 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 CK1
s 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.
s 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
CK1
s have a 2-amino-acid insertion (after residue 146 of
CK1
), a feature in common with the yeast Yck1 and Yck2 proteins.
CK1
2 and CK1
3 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 CK1
3L 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) .
3 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 CK1
1 and CK1
2. Thus, testis is
the only tissue expressing mRNAs for all the known CK1s and three
isoforms, CK1
1, CK1
2, 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
.
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.
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
.
/EMBL Data Bank with accession number(s) U22296, U22297,
and U22321.
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 CK1
1,
CK1
2, and CK1
3, respectively, in relation to the protein
sequences reported in this paper. Subsequently, we have also expressed
full-length CK1
3 and found no major differences in properties as
compared with the shorter form described herein (L. Zhai and P. J.
Roach, unpublished results).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.