(Received for publication, January 31, 1997, and in revised form, February 24, 1997)
From the Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037
The Schizosaccharomyces pombe gene
pch1+ (ombe cyclin
omology) was isolated in a two-hybrid screen
for proteins that interact with Cdc2. The cyclin box region of Pch1
protein shares greatest sequence identity with mammalian and
Drosophila C-type cyclins (~33% identity). Pch1 is
significantly less similar to Mcs2 (19% identity), a second member of
the C-type cyclin family in S. pombe. Cdc2 co-precipitates
with Pch1 in S. pombe cell lysates, although Cdc2 may not
be the major catalytic partner of a Pch1 kinase in vivo.
Purified Pch1-associated kinase phosphorylated myelin basic protein,
histone H1, and a peptide corresponding to the carboxyl-terminal domain
repeat of RNA polymerase II. The amount of pch1 mRNA
does not oscillate during the cell cycle, as is the case for mRNA
transcripts of other C-type cyclin genes. pch1 cells are
inviable, therefore S. pombe has two essential genes that
encode members of the C-type cyclin family,
pch1+ and mcs2+. The
pch1 mutation causes pleiotropic morphological defects and an associated growth deficiency, but loss of Pch1 activity does not
result in a cdc cell cycle-arrest phenotype.
In Schizosaccharomyces pombe, cell cycle progression is
determined by the activation state of a single
cyclin-dependent kinase (CDK),1
Cdc2 (1, 2). Activation of Cdc2 requires the formation of a complex
between itself and a cyclin. In S. pombe, Cdc2 has been
shown to form a complex with a B-type cyclin, Cdc13 (3, 4). This
cyclin-kinase complex accumulates during interphase, and the cyclin
component is rapidly degraded upon exit from mitosis (M-phase) (4).
Although Cdc13-Cdc2 complex is required for the onset of M-phase, Cdc2
functions at both the G1-S-phase and G2-M-phase
transitions (5). Thus, there must be an additional cyclin-Cdc2 complex
that promotes the onset of S-phase. Recent studies have shown that
fission yeast Cig2 B-type cyclin is most abundant and has the highest
associated kinase activity during S-phase (6, 7). In cig2
cells, S-phase is delayed, and Cdc13 becomes essential for DNA
replication, suggesting that Cig2-Cdc2 normally promotes the onset of
S-phase, but in a
cig2 strain this function can be
performed by Cdc13-Cdc2 (7, 8). The role of B-type cyclins at both the
G1-S-phase and G2-M-phase transitions seems to
be a common feature between the budding yeast Saccharomyces cerevisiae and S. pombe (9-11). Three additional
S. pombe cyclin genes have been previously identified,
although their function in the cell cycle is unclear. The first of the
genes, cig1+, encodes a B-type cyclin that is
most abundant and has the highest associated kinase activity during
M-phase (12). Disruption of cig1+ causes no
obvious phenotype (13-16). The second gene,
puc1+, which encodes an unusual type of cyclin,
was initially proposed to function as a G1 cyclin, although
more recent studies have suggested that it might have a role in
delaying G1 arrest in nitrogen-starved cells (16, 17). The
third cyclin gene, mcs2+, encodes an essential
C-type cyclin that shares some of the characteristics of
pch1+, the cyclin gene described in this study
(18). Recent studies have shown that Mcs2 is the cyclin partner of Mcs6
kinase, which is also known as Crk1 or Mop1 (19, 20). The Mcs2-Mcs6
kinase can carry out the activating threonine 167 phosphorylation of Cdc2 in vitro, although it is unknown whether it performs
this function in vivo.
Human and Drosophila cyclin C cDNA clones were initially identified by virtue of their ability to rescue the cell cycle-arrest defect of a S. cerevisiae strain lacking G1 cyclins CLN1-3 (21-23). Human G1 cyclins, cyclin D1 and cyclin E, were also obtained in the same screen (21). Unlike the G1 functions of cyclin D1 and cyclin E, which are well characterized, the function of cyclin C is unknown. Cyclins with significant homology to the cyclin box region of cyclin C seem to be involved in the initiation of RNA polymerase II transcription and also in the activation of other CDKs through phosphorylation. Cyclin H, a human C-type cyclin, is the regulatory subunit of a mammalian kinase known as CDK-activating kinase that carries out the activating phosphorylation of Cdc2 and Cdk2 kinase in vitro (24). The cyclin H complex is also part of the general transcription factor IIK, a subunit of transcription factor IIH, which is essential for initiation of transcription (25-27). In addition to activating CDKs, the cyclin H complexes can phosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II, which consists of heptapeptide repeats (YSPTSPS) (28). Phosphorylation of the CTD seems to be important for the regulation of transcription (29, 30). In S. cerevisiae, the SRB10 and SRB11 genes, which encode a kinase and C-type cyclin, respectively, were isolated as high copy suppressors of a CTD truncation mutant (31). Independently, SRB10 (SSN3) and SRB11 (SSN8) were isolated as suppressors of a snf1 mutation (32, 33). Snf1, a serine/threonine kinase, is involved in the regulation of glucose-repressed genes (34). SRB10 and SRB11 were shown to be involved in transcriptional repression and activation of a variety of genes in S. cerevisiae (33). CDK8, the associated kinase of cyclin C, shares significant homology with Srb10, although the functional homology between the two complexes is unknown (35, 36). Another S. cerevisiae C-type cyclin and kinase pair, Ccl1 and Kin28, form a complex that phosphorylates CTD but not the mitotic CDK Cdc28 in vitro. Loss of Kin28 activity results in a decrease in the abundance of all mRNA species, suggesting that Kin28 kinase activity is essential for all transcription catalyzed by RNA polymerase II (37). Recently, a gene expressing the C-type cyclin subunit of a CTDK1 (CTD kinase) activity, CTK2, was cloned in S. cerevisiae (38). This cyclin-kinase pair, Ctk2-Ctk1, co-purifies with CTDK1 activity, although the in vivo role of this kinase in transcription is unknown.
We report the isolation of pch1+, a S. pombe gene that encodes a cyclin that is most closely related to the C-type cyclin genes of higher eukaryotes. We demonstrate that pch1+ is an essential gene, showing that there are at least two essential cyclin C genes in fission yeast.
Methods and media used for general genetics and biochemical procedures with fission yeast have been described (39, 40).
Cloning and Sequencing of the pch1+ GeneA 1,147-bp pch1 cDNA in pACT2 was obtained in a two-hybrid screen of 2 × 107 S. pombe cDNA clones using pAS1-Cdc2 as the bait (41). The cDNA was sequenced in both orientations and found to encode the entire Pch1 protein. The pch1 cDNA was used to probe filters containing an ordered array of cosmid and P1 genomic clones of S. pombe to determine the chromosomal location of pch1+ (42, 43). A 4.5-kb genomic clone of pch1+ was obtained by colony hybridization of XhoI/XbaI fragments from P1 clone 7B10p inserted between the XhoI and XbaI sites in pBluescript II (pBF108). Sequence analysis of the genomic clone was performed in both orientations upstream and downstream of the pch1+ coding region.
Construction of theA plasmid carrying the pch1+
genomic clone, pBF108, was digested with SalI and
SmaI to remove a 900-bp fragment containing the majority of
the pch1+ open reading frame. This DNA fragment
was replaced with the 1.9-kb SalI/SmaI fragment
containing his7+ derived from pEA2 (44) to
create pBF110. The 3.5-kb EcoRI fragment of pBF110 was used
to replace pch1+ with the disrupted allele
(pch1::his7+) by transformation into a
diploid strain generated by mating CH428 and CH429 (44). The
pch1::his7+/pch1+
diploid strain was induced to sporulate by inoculating malt extract medium with log-phase cultures grown in yeast extract with supplements medium. After 2 days at 22 °C, asci were collected, washed twice with water, and resuspended in 0.4% glusulase. After an overnight incubation at 30 °C, spores were washed twice with water and stored at 4 °C. Spores at 0.1 A600 were germinated
in Edinburgh minimal medium 2 plus leucine, uracil, and adenine at
30 °C. At the indicated times, samples containing 10 A600 of cells were taken for microscopic observation, Northern analysis, and kinase assays. For both Northern mRNA analysis and kinase assays, cells were harvested by
filtration, washed once with ice-cold water, frozen in liquid nitrogen,
and stored at 70 °C. For microscopic observation, cells were
harvested by centrifugation, resuspended in ice-cold water, and mixed
with 100% ethanol to adjust to a final concentration of 70% ethanol, and samples were stored at 4 °C overnight. After fixation, 1 µg/ml 4
, 6-diamidino-2-phenylindole or 50 ng/ml Calcofluor was added to
samples that were resuspended in PBS.
Total yeast RNA from a 10 A600 cell pellet was extracted as described (39). Ten µg of the total RNA preparation was resolved on a 1% formaldehyde agarose gel and transferred to nitrocellulose. The filters were hybridized with random-primed 32P-labeled probes (Prime-It II; Stratagene) in 50% formamide, 1× Denhardt's solution, 0.15% SDS, 5× SSC, 10% dextran sulfate, and 100 µg/ml denatured salmon sperm. After an overnight incubation at 42 °C, the filters were washed at 42 °C twice with 2× SSC/0.1% SDS and once with 0.2× SSC/0.1% SDS and then subjected to autoradiography or detection using a Molecular Dynamics PhosphorImager.
Expression of GST-Pch1 and Antibody ProductionThe 1,147-bp
XhoI fragment from pACT-Pch1 was cloned into the
SalI site of pGEX-KG (45) in frame with GST to create
pBF103. Expression of the GST-Pch1 fusion protein was performed as
described (46). Fresh overnight cultures of Escherichia coli
BL21(DE3)pLysS transformed with pBF103 were diluted 1:20 in LB medium
containing both chloramphenicol (34 µg/ml) and ampicillin (50 µg/ml). After 2 h in a 37 °C shaker,
isopropyl-1-thio--D-galactopyranoside was added to a
final concentration of 0.1 mM, and the incubation was
continued for an additional 2 h. The culture was harvested by
centrifugation at 2,500 × g for 10 min at 4 °C,
resuspended in ice-cold 1× PBS, and stored at
70 °C. The sample
was lysed by rapid thawing at 37 °C and mild sonication. After
centrifugation at 10,000 × g for 15 min at 4 °C,
GST-Pch1 was recovered from the supernatant by incubation with
glutathione-Sepharose 4B beads (Pharmacia) for 15 min at 4 °C. The
glutathione-Sepharose beads complexed with the fusion protein were
washed three times with ice-cold PBS. GST-Pch1 was eluted from the
beads at room temperature for 15 min in 20 mM glutathione,
100 mM Tris (pH 8.0), and 120 mM NaCl.
Purified GST-Pch1 protein was used to immunize rabbit #0645. The polyclonal anti-Pch1 antibodies from rabbit serum were purified first by subtraction against GST on nitrocellulose blocked with 2.5% fetal calf serum, 0.1% Tween 20, and TBS and then absorbed to GST-Pch1 on nitrocellulose blocked with 2.5% fetal calf serum. After a 1-h incubation at 4 °C, the nitrocellulose with anti-Pch1 antibodies was washed twice with 0.1% Tween 20 in TBS at 4 °C. Antibodies were eluted in 1 ml of 100 mM glycine (pH 2.2) for 10 min at 4 °C followed by neutralization with 0.1 ml of 1 M Tris (pH 8.0).
GST-Pch1 was expressed in S. pombe from the nmt1
promoter in the pREPGST construct (41). pBF123 was constructed by
polymerase chain reaction (PCR) amplification of the coding region of
pch1 from pBF108 with the following primers:
5Pch-BamHI,
5
-CGCAATGAGTGAAGTAATAAAATCTGTACC-3
; and
3
Pch-BamHI, 5
-CATTTATGAAGCTTCCGTCTC-3
.
Both the 5
and the 3
primers contain a BamHI site
(underlined) adjacent to a initiation codon or the stop codon,
respectively. PCR reactions and PCR product purification were performed
as described (47). The isolated PCR-amplified DNA was digested with
BamHI for 2 h and subjected to gel electrophoresis
through a 1% agarose gel. The PCR fragments were isolated from agarose
by using the QIAquick gel extraction kit (Qiagen) as specified by the
manufacturer and ligated into the BamHI site of pREPGST,
which had been treated with calf intestinal phosphatase (Promega). The
resulting construct, pBF123, was transformed into a
pch1::his7+/pch1+
diploid strain and selected on Edinburgh minimal medium 2. A transformed diploid strain was induced to sporulate on malt extract medium, and spores were collected and processed as indicated above. A
pch1::his7+ haploid strain isolated on
Edinburgh minimal medium 2 was rescued by pBF123.
Frozen cell pellets containing 10 A600 of cells in 1.5-ml microcentrifuge tubes were thawed and resuspended in 0.5 ml of lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, 50 mM NaF, 0.1 mM sodium orthovanadate supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 µM microcystin, and 5 µg/ml aprotinin, leupeptin, and pepstatin. Cells were lysed by vortexing with glass beads for 5 min at 4 °C. Debris was removed by centrifugation at 14,000 × g for 15 min at 4 °C, and cell extracts were normalized by the addition of lysis buffer based on protein concentrations determined by a Bradford assay (Bio-Rad).
Cyclin-kinase complexes were isolated from cell extracts by glutathione-Sepharose (Pharmacia), p13suc1-Sepharose (48), or protein A-Sepharose (Pharmacia) prebound to GST-Pch1 antibodies or preimmune sera from the same rabbit. Precipitation of cyclin complexes was carried out on a rocker at 4 °C for 1-2 h. Isolated complexes were washed three times with lysis buffer.
For Western blot analysis, a fraction of each co-precipitation was boiled in Laemmli sample buffer and loaded onto SDS-polyacrylamide gels. After electrophoresis, the separated proteins were transferred to nitrocellulose as described (49). The blots were blocked for at least 1 h in 5% (w/v) nonfat dry milk dissolved in TBS/0.1% Tween 20. The blots were then incubated with a 1:1,000 dilution of either purified polyclonal anti-Pch1 (#0645) or polyclonal anti-Cdc2 antibodies (#9808) for 1 h at room temperature. After washing with TBS/0.1% Tween 20, the blots were incubated with a 1:3,000 dilution of horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Promega), washed, and developed by enhanced chemiluminescence (Amersham) according to the manufacturer's specifications.
Kinase AssaysProtein A- and glutathione-Sepharose
precipitates were washed three times with lysis buffer as described
above followed by three washes with ice-cold kinase assay buffer (50 mM Tris (pH 7.4) and 10 mM MgCl2).
The bound complexes were resuspended in 50 µl of kinase assay buffer
containing 50 µCi of [-32P]ATP, 100 µM
ATP, and either 50 µg of histone H1 (Boehringer Mannheim), 6.25 µg
of myelin basic protein (MBP) (Boehringer Mannheim), or 2 µg of CTD
peptide (a gift from Geoffrey Laff and Mark Solomon, New Haven, CT).
The assays containing MBP and CTD were incubated at room temperature
for 5 and 30 min, respectively; the samples containing histone H1 were
incubated at 30 °C for 15 min. After the incubation, 50 µl of
Laemmli sample buffer was added, and the samples were boiled for 2 min.
Half of each reaction was resolved on either a SDS-12% polyacrylamide
gel for histone H1 or a SDS-15% polyacrylamide gel for MBP and CTD.
Dried gels were subjected to quantitation (Molecular Dynamics
PhosphorImager) and autoradiography.
The GenBankTM accession number for pch1+ is U92879[GenBank].
A
yeast two-hybrid screen was used to identify proteins that interact
with Cdc2 (50). A hybrid protein containing the Gal4 DNA-binding domain
(Gal41-147) fused to Cdc2 was co-expressed with the hybrid
proteins from the library containing the Gal4 activation domain fused
to S. pombe cDNA clones in the S. cerevisiae strain Y190. Protein-protein interactions were detected by
transcriptional activation of both HIS3 and lacZ
reporter genes. In this screen, 2 × 107 transformants
were screened and yielded 112 positive cDNA clones representing 7 genes. Two of these genes have been described previously, orp2+ (41) and suc1+
(51). One gene for which 10 independent isolates were obtained is
represented by the positive clone pACT-JL21, which interacted specifically with the Gal4(1-147)Cdc2 fusion protein in the presence of 25 mM 3-aminotriazole (Fig.
1).
The nucleotide sequence of the longest (1,147 bp) cDNA insert from
the clones that cross-hybridized with pACT-JL21 was found to encode a
cyclin-like protein. The gene was named pch1+
for ombe cyclin
omology. The chromosomal location of
pch1+, determined by hybridization of the
cDNA insert to an ordered array of cosmid and P1 clones (42, 43),
is on chromosome II between top1+ and
cdc10+. A genomic clone was obtained by the
cloning of a 4.5-kb XbaI/XhoI fragment from P1
clone 7B10p between the XbaI and XhoI sites of pBluescript II. Sequence analysis of the genomic clone confirmed that
the cDNA was full-length, and the open reading frame is encoded by
a single exon (Fig. 2A). The encoded Pch1
protein contains 342 amino acids and has a predicted
Mr of 38,000.
Pch1 is a Member of the Cyclin C Family
The cyclin box region of Pch1 was used in a data base homology search. Drosophila and human cyclin C showed the highest identity to the cyclin box region of Pch1 (35 and 33%, respectively), especially within regions 1, 2, and 4 (Fig. 2B). Pch1 is more distantly related to human cyclin H (52), S. cerevisiae Srb11 (31, 33), S. pombe Mcs2 (18), S. cerevisiae Ccl1 (53), and S. cerevisiae Ctk2 (38) in which the sequence identity ranges from 17-24% (Table I). These cyclin C family members have been implicated in the regulation of RNA polymerase II transcription and in the activating phosphorylation of cyclin-dependent kinases. Pch1 is only very weakly related to A-type and B-type cyclins, for example S. pombe Cdc13, a B-type cyclin that is required for mitosis, shares only 13% identity with Pch1 in the cyclin box domain.
|
Experiments were carried out to characterize the strength
of the in vivo interactions involving Cdc2 and Pch1. These
experiments used a plasmid construct that expressed GST-Pch1 from the
nmt1 promoter in S. pombe. This construct fully
complemented the pch1 mutation (see below), indicating
that the GST-Pch1 protein was functional. The rescued strain was used
to isolate either GST-Pch1-associated proteins using
glutathione-Sepharose beads or Cdc2-associated proteins using
p13Suc1-Sepharose. A wild-type strain expressing unfused
GST served as a control. Western blot analysis was performed in
duplicate with an affinity-purified polyclonal
-Pch1 antibody and
with a polyclonal
-Cdc2 antibody (Fig. 3). A Cdc2
signal was detected in the lane containing proteins that associate with
GST-Pch1. This signal was absent in the lane containing proteins that
associate with GST, indicating that the association specifically
involved Cdc2 and Pch1. The Cdc2 signal in the GST-Pch1 sample was much
less than that detected with p13Suc1-Sepharose A,
suggesting that the Pch1 co-precipitates with only a very small
fraction of Cdc2. This finding was underscored by the failure to detect
GST-Pch1 in the mixture of proteins that precipitate with
p13Suc1-Sepharose A (Fig. 3), although it may be that Suc1
and Pch1 associations with Cdc2 are mutually exclusive.
Pch1-associated Kinase Phosphorylates Various Substrates
To
assay for Pch1 associated-kinase activity, Pch1 and associating
proteins were immunoprecipitated from a wild-type cell extract with
-Pch1 sera or glutathione-Sepharose beads were used to purify
GST-Pch1 from S. pombe cells transformed with a plasmid that
expressed GST-Pch1 from the nmt1 promoter. Pch1
associated-kinase activity was detected using the following exogenously
supplied substrates: MBP, a peptide of the CTD from RNA polymerase II, and histone H1 (Fig. 4). The phosphorylation of the
three substrates was rather poor, with myelin basic protein seeming to
be the best substrate (Fig. 4, lane 4). To show that the
kinase activity was specific to Pch1, protein A-Sepharose beads either
alone (lane 2) or with preimmune sera (lane 3)
were used in the assay. Kinase activity was minimal in both controls.
Glutathione-Sepharose beads precipitated a Pch1-associated kinase
activity that was detected in cell extracts from cells expressing
GST-Pch1 (lane 7), and as expected this kinase activity was
absent in extracts from cells that did not express GST-Pch1 (lane
8). Cdc2 kinase precipitated with p13suc1-Sepharose
was used as a positive control for all three substrates (lane
9). The results of preliminary experiments suggest that the MBP
kinase activity of Pch1 kinase does not oscillate during the cell
cycle,2 although definitive studies await
the identification of a better substrate.
pch1 mRNA Remains Constant During the Cell Cycle
To
determine if pch1 mRNA levels are regulated in a cell
cycle manner, Northern analysis was performed on total RNA collected at
20-min intervals from a culture of cells undergoing a synchronous cell
cycle. These cells carried the cdc25-22 mutation; they were arrested in late G2 by incubating at the restrictive
temperature of 36 °C and then induced to enter M-phase synchronously
by adjusting the culture to the permissive temperature of 26 °C.
pch1 mRNA was detected at all time points (Fig.
5). There was an approximate 2-fold difference between
some samples, but there seemed to be no periodic change in the Pch1
mRNA signal during the cell cycle (Fig. 5). In contrast, the
abundance of cdc22 mRNA underwent a highly periodic
change during the two cell cycles, in agreement with previous studies
(54).
pch1+ is an Essential Gene
The function of Pch1
protein was investigated using a pch1 null mutation. This
mutation was constructed by replacing the
SmaI/SalI fragment (codons 11-321) with the
S. pombe his7+ gene (44) (Fig.
6A). The EcoRI fragment containing
the disrupted gene was used to replace one copy of
pch1+ in a diploid strain. Two independent gene
disruption transformants were confirmed by Southern hybridization
analysis (data not shown). These
pch1+/pch1::his7+
diploids were sporulated and analyzed by tetrad dissection. All 36 tetrads gave rise to 2 viable and 2 inviable segregants (Fig. 6B). The inviable segregants formed microcolonies consisting
of approximately 100 cells; their phenotypes are described in more detail below. All viable segregants were his7,
thus the inviable segregants carried the
pch1::his7+ marker. The lethal phenotype of
pch1 segregants was rescued by plasmids carrying the
pch1+ genomic clone or the
pch1+ cDNA expressed from the
nmt1 promoter in pREP1 (55). Thus, the lethality of the
pch1::his7+ haploid strain was a
direct result of the loss of pch1+.
To more accurately characterize the pch1 phenotype, a
pch1/pch1::his7+ diploid
was induced to sporulate, and then spores were inoculated into liquid
minimal medium. This medium lacked histidine, thus only the cells that
contained the pch1::his7+ gene
germinated. Every 4 h, aliquots of cells were taken for cell
number determination via Coulter counter and for ethanol fixation for
microscopic analysis. Fixed cells were stained with DNA dye
diamidinophenylindole (4
, 6-diamidino-2-phenylindole) to observe the
nuclear structure and with Calcofluor to visualize septum formation
(Fig. 7). Both wild-type and
pch1 spores
germinated approximately 8 h after inoculation into media.
However,
pch1 cells arrested after 4-5 cell divisions
with a variety of morphological defects, whereas the wild-type cells
continued to divide normally. At 12 h after inoculation, the
pch1 cells seem fairly normal, however, although a small
population of cells had off-centered nuclei. The 24-h time point
revealed a large amount of septal material in a portion of the cells as
well as a population of anucleate cells. A combination
4
,6-diamidino-2-phenylindole and Calcofluor staining revealed improper
nuclear segregation in some
pch1 cells, resulting in one
daughter cell with two nuclei and the other with none (data not shown).
A similar pattern was also observed in
pch1 cells fixed
36 h after inoculation.
In this report we have described the cloning and characterization of the pch1+ gene of S. pombe. pch1+ was isolated based on the ability of Pch1 protein to interact with a hybrid protein composed of the Gal4 DNA-binding domain and Cdc2. This protein-protein interaction activates the transcription of both lacZ and HIS3 in the S. cerevisiae strain Y190. Sequence analysis indicates that Pch1 protein shares significant homology with members of the cyclin C family. Pch1 is 35% identical and 60% similar to Drosophila cyclin C in the cyclin box region. The Drosophila and human cyclin C genes share 72% identity and were cloned by complementation of a S. cerevisiae strain lacking G1 cyclins CLN1-3 (21, 23). Although expression of pch1+ in S. cerevisiae does not complement the triple CLN disruption (data not shown), we do not believe this reflects a fundamental difference between Pch1 and other cyclin C proteins. At this point we cannot speculate as to whether the Pch1 is a functional homolog of C-type cyclins in higher eukaryotes. Cyclin C complementation of the triple CLN mutant requires high level overexpression and might result from cross-reactivity between the cdc28 kinase and a class of cyclins normally dedicated to transcription. We propose that a similar cross-reaction between highly overexpressed proteins accounts for our recovery of pch1 in the screen for cdc2 interacting factors.
Like mcs2+, another C-type cyclin gene in
S. pombe, pch1+ is an essential gene
(18). Spores that carry a pch1 null allele germinate
normally and proceed to form microcolonies of inviable cells arrested
with heterogeneous phenotypes. pch1 cells seem swollen
and display pleiotropic morphological defects such as abnormal septal
formation, apparent aggregation of mitochondria, and misplacement or
absence of a nucleus.
pch1 cells do not display a
cdc phenotype, indicating that Pch1 is not required for a
progression through a specific phase of the cell cycle. Although an
association involving Pch1 and Cdc2 was detected by both two-hybrid and
co-precipitation assays, we think it unlikely that Cdc2 is the major
catalytic partner of Pch1 in vivo. The protein-protein
interactions detected between Cdc2 and Pch1 in the two-hybrid system
could be accounted for by significant similarity between Cdc2 and the
true catalytic partner of Pch1. Cyclin-dependent kinases
are similar in overall structure, but some CDKs contain amino acid
differences within the region responsible for cyclin binding. The
cyclin binding region in Cdc2 and its homologs contains the PSTAIRE
motif, whereas the C-type cyclin-associated kinases do not have the
PSTAIRE motif, but instead, many embody the sequence SACRE (31, 33,
36). Western analysis of GST-Pch1 affinity-purified proteins with
-PSTAIRE antibody indicates that the Pch1-associated kinase does not
contain a PSTAIRE motif.3 Furthermore,
overproduction of Pch1 at an intermediate temperature (32 °C) in
strains having temperature-sensitive alleles of cdc2 (cdc2-33 and cdc2-L7) had very little effect,
although it did cause the cells to be slightly more elongated at
division.3 This finding suggests that Pch1 is not a major
partner of Cdc2 in vivo.
Another possibility to account for the Cdc2 and Pch1 interaction is
that Cdc2 may be a substrate of the Pch1-associated kinase. Cyclin H is
a C-type cyclin that is the regulatory subunit of a human kinase that
is able to carry out the activating phosphorylation of
cyclin-dependent kinases in vitro (24). In
S. pombe, high expression of a mutant form of Cdc2 that has
a nonphosphorylatable alanine residue substituted for threonine 167 results in a typical cdc arrested phenotype, in which
cells continue to grow but are unable to divide (56). Spore germination
of
pch1 cells results in a multitude of morphological
defects instead of a cdc
arrested phenotype, suggesting
that Pch1 function is not primarily involved in the activation of Cdc2.
Recent studies have shown that Mcs2-Mcs6 kinase exhibits a
CDK-activating kinase-like activity in vitro, although it is
unknown whether Mcs2-Mcs6 kinase contributes to the activation of Cdc2
in vivo (19, 20).
mcs2,
mcs6, and
pch1 mutations cause somewhat similar phenotypes in
that all three types of mutant spores germinate to form small
microcolonies of inviable cells that are not highly elongated, but the
majority contain septa (18-20). It is possible that Mcs2- and
Pch1-associated kinases share the responsibility for carrying out the
activating phosphorylation of Cdc2, which could account for the fact
that the loss of either cyclin does not cause a cell cycle-arrest
phenotype similar to that of cdc mutants. However, it is
clear that Mcs2 and Pch1 have distinct essential functions that do not
seem to be directly involved in promoting cell cycle progression. As
such, the in vivo functions of both Mcs2 and Pch1 remain
obscure, as is the case with most members of the C-type cyclin family.
Additional genetic and biochemical studies of fission yeast have the
potential to provide important insights into this enigmatic class of
cyclins.
We thank Steve Elledge for providing the S. pombe two-hybrid library and plasmids, Elmar Maier for S. pombe cosmid and P1 filters for chromosomal mapping of pch1+, Charles Hoffman for pEA2, and Geoffrey Laff and Mark Solomon for the CTD peptide. We thank Clare McGowan, Kazuhiro Shiozaki, and other members of the Russell lab for helpful suggestions.