(Received for publication, July 6, 1995)
From the
The catalytic subunit of Saccharomyces cerevisiae casein kinase II (Sc CKII) is encoded by the CKA1 and CKA2 genes, which together are essential for viability. Five
independent temperature-sensitive alleles of the CKA2 gene
were isolated and used to analyze the function of CKII during the cell
cycle. Following a shift to the nonpermissive temperature, cka2 strains arrested within a single cell cycle
and exhibited a dual arrest phenotype consisting of 50% unbudded and
50% large-budded cells. The unbudded half of the arrested population
contained a single nucleus and a single focus of microtubule staining,
consistent with arrest in G
. Most of the large-budded
fraction contained segregated chromatin and an extended spindle,
indicative of arrest in anaphase, though a fraction contained an
undivided nucleus with a short thick intranuclear spindle, indicative
of arrest in G
and/or metaphase. Flow cytometry of
pheromone-synchronized cells confirmed that CKII is required in
G
, at a point which must lie at or beyond Start but prior
to DNA synthesis. Similar analysis of hydroxyurea-synchronized cells
indicated that CKII is not required for completion of previously
initiated DNA replication but confirmed that the enzyme is again
required for cell cycle progression in G
and/or mitosis.
These results establish a role for CKII in regulation and/or execution
of the eukaryotic cell cycle.
Casein kinase II (CKII) ()is a serine/threonine
protein kinase which is ubiquitous among eukaryotic organisms (for
review, see Issinger, 1993; Pinna, 1990; Tuazon and Traugh, 1991). The
enzyme is composed of a catalytic
and regulatory
subunit
that combine to form a native
holoenzyme which is constitutively active in vitro. How
(and indeed whether) the enzyme is regulated in vivo is
unknown, though regulation via allosteric effectors (e.g. polyamines), covalent modification, cellular redistribution, and
substrate-directed effects have all been proposed. CKII recognizes a
Ser of Thr residue followed by a series of acidic residues and
phosphorylates a broad and intriguing spectrum of both nuclear and
cytoplasmic substrates.
Although the physiological role of CKII is
not known, several lines of evidence suggest a role for the enzyme in
cell proliferation. First, CKII activity is elevated in rapidly
dividing cells, both normal and transformed (reviewed in Issinger,
1993). CKII activity has also been reported to increase in response to
stimulation of cells by mitogenic stimuli, though these effects have
been difficult to reproduce (see Litchfield et al., 1994, for
discussion). Second, CKII phosphorylates a number of proteins known to
play crucial roles in cell proliferation (Meisner and Czech, 1991),
including the nuclear oncogene proteins c-Myc, c-Myb, and c-Jun, the
tumor suppressor protein p53, and the cyclin-dependent protein kinase
p34 (Russo et al., 1992).
Cyclin-dependent protein kinases (CDKs) constitute the engine of the
eukaryotic cell cycle and are essential for Start as well as the
G
/S and G
/M transitions (Morgan, 1995). Third,
CKII activity has been found to fluctuate in a cell cycle-dependent
manner in mammalian cells (Carroll and Marshak, 1989; DeBenedette and
Snow, 1991). Fourth and most importantly, experimental manipulation of
CKII activity in vivo can have profound effects on cell
proliferation. Disruption of CKII is lethal in Saccharomyces
cerevisiae (Padmanabha et al., 1990) and probably also in Dictyostelium discoideum (Kikkawa et al., 1992) and Schizosaccharomyces pombe (Snell and Nurse, 1994). In
mammalian cells, inhibition of CKII activity by microinjection of
antibodies directed against the
subunit into
G
-synchronized human fibroblasts has been shown to inhibit
cell cycle progression in response to serum stimulation (Pepperkok et al., 1994). Microinjection of nanomolar quantities of
active CKII into Xenopus oocytes accelerates meiotic maturation induced
by mitosis promoting actor (Mulner-Lorillon et al., 1988).
Perhaps most remarkably, the catalytic subunit of CKII functions as an
oncogene when expressed as a transgene in lymphocytes of transgenic
mice, leading to the stochastic production of lymphomas when expressed
alone and to the production of acute lymphocytic leukemia when
co-expressed with a c-Myc transgene (Seldin and Leder, 1995).
We
have exploited molecular and genetic methods available in the budding
yeast S. cerevisiae (Sc) to explore the physiological
role of CKII in this organism. Sc CKII consists of two catalytic
subunits, and
`, and two regulatory subunits,
and
`, which are encoded by the CKA1, CKA2, CKB1, and CKB2 genes, respectively (for review, see
Glover et al., 1994). Deletion of either catalytic subunit
gene alone has few if any phenotypic consequences, but simultaneous
disruption of both CKA1 and 2 is lethal (Padmanabha et al., 1990). Cells arrested by depletion of CKII arrest as a
population of unbudded and budded cells, with a significant proportion
of the latter exhibiting an elongated bud characteristic of several
cell division cycle mutants (Hartwell et al., 1973). Arrested
cells continue to grow, suggesting that metabolism continues in the
absence of cell division.
To further explore the role of CKII during
growth and division, S. cerevisiae mutant strains
temperature-sensitive for CKII have been constructed. In contrast to
previously constructed strains that deplete kinase activity by
attrition (Padmanabha et al., 1990), these mutants allow CKII
activity to be inactivated within a time short with respect to the
length of the cell cycle. Analysis of these temperature-sensitive
strains reveals that CKII is required for cell cycle progression at two
points in the cell cycle, one in G and in the other in
G
and/or mitosis. Furthermore, the data indicate that CKII
activity is not required for ongoing DNA replication during S phase.
For stationary phase G synchronization, cultures were
inoculated into YPD at 8
10
cells/ml and grown at
25 °C until stationary phase (approximately 60 h; 90-95%
unbudded cells). Cells were released from stationary phase arrest by
dilution to 8
10
cells/ml in fresh YPD at 25 °C
or the appropriate nonpermissive temperature. For arrest in G
in response to mating pheromone or in early S phase in response
to hydroxyurea, cells were inoculated into YPD at 2-8
10
cells/ml, grown at 25 °C for 6 h (log phase), and
then treated either with
-factor at 2.5 µg/ml for 4.5 h or
with hydroxyurea (added directly to the culture as a powder) at 0.1 M for 5 h. Synchronized cultures were released by filtering
through a 2.5-cm HA 0.45-µm filter (Millipore) mounted in a Swinnex
25 filter apparatus, followed by washing and resuspending the cells at
the original density in fresh YPD at 25 °C or the appropriate
nonpermissive temperature.
The nucleotide substitution(s) and corresponding
amino acid replacement(s) present in each temperature-sensitive cka2 allele were determined by sequencing the protein coding
region. As expected for hydroxylamine, which deaminates cytosines, all
mutations could be explained by C T transitions (see Table 2). One allele (cka2-13) contained a single
substitution while the remaining four (cka2-7, -8, -11, and -12) contained two, and every
substitution resulted in an amino acid replacement. The temperature
sensitivity of the single mutant (cka2-13) is presumably
explained by the sole amino acid replacement in this mutant (D225N).
The affected residue, which corresponds to D220 of cAMPdPK (Table 2), is invariant in the protein kinase family (Hanks and
Quinn, 1991) and is postulated to function in stabilizing the catalytic
loop (Knighton et al., 1991). The D225N replacement also
occurs as one of the two mutations present in the cka2-11 allele. The A190T and A190V replacements of cka2-7 and cka2-12, respectively, affect a residue which
is moderately variable among protein kinases generally but is invariant
among known CKIIs. Replacements in the other two alleles do not affect
strongly conserved residues either in protein kinases generally or in
CKII (Hanks and Quinn, 1991).
The maximum permissive and minimum restrictive temperatures of the five mutant strains are shown in Table 3. The lower temperature transition of YDH11 relative to YDH13 implies that the E299K replacement in the cka2-11 allele is also a destabilizing mutation, at least in the context of the D225N mutation.
All five strains exhibited some phenotypic defect at permissive temperature. As shown in Table 3, all five strains exhibited some increase in flocculation. This effect was severe in those strains showing the greatest temperature sensitivity, most notably YDH12, which grew essentially as a pellet at 25 °C in liquid medium. Strains displaying the greatest temperature sensitivity also exhibited a modest slow growth phenotype at permissive temperature, as assessed by colony size on plates (data not shown). Because flocculation and slow growth are both characteristic of CKII depletion (Padmanabha et al., 1990), these results imply a reduction in CKII activity in these mutants at permissive temperature. Based on the above results, YDH8 was selected for the bulk of the studies described below because it has the lowest transition temperature consistent with a near normal growth rate (Fig. 1A) and a tractable level of flocculation (Table 3) at permissive temperature.
Figure 1:
Growth curves of asynchronously growing CKA2 and cka2-8 strains at 25 and 37 °C.
Strains were inoculated into YPD, grown for 6 h at 25 °C, and then
either maintained at 25 °C (A) or shifted to 37 °C (B). Cell density was determined as described under
``Experimental Procedures.'' , YDH6 (CKA2);
, YDH8 (cka2-8).
Budding profiles for the experiment shown in Fig. 1B are presented in Table 4. Both YDH6 and YDH8 exhibited a transient, heat shock-induced increase in the proportion of unbudded cells during the first 1-2 h following the shift to restrictive temperature. YDH8 cells reached a stable state (terminal phenotype) by 4-6 h after the shift. The arrested population consisted of approximately equal numbers of unbudded and large-budded cells, with small-to-medium-budded cells being present at very low levels. In contrast, YDH6 gradually returned to the roughly equal mixture of unbudded and small-to-medium-budded cells characteristic of log phase growth. Flow cytometry of YDH8 cells 4-6 h after the shift indicated a mixture of cells containing 1 and 2 N DNA complement (data not shown). These results imply the existence of at least two distinct arrest points in the mutant. Because YDH8 cells do not arrest with a single terminal morphology, cka2-8 does not qualify as a classical cell division cycle mutation (Hartwell et al., 1973).
To determine whether the dual arrest phenotype was specific to YDH8, the budding profile of two other temperature-sensitive strains was examined at the restrictive temperature. Strains YDH11 and 13, whose cka2 alleles bear replacement(s) distinct from those of cka2-8 (Table 2), also arrested as a 50:50 mixture of unbudded and large-budded cells (data not shown). This outcome indicated that the dual arrest phenotype is not an allele-specific response.
To better
define the points of arrest, YDH8 cells incubated at the restrictive
temperature for 5 h were double-stained with the DNA-binding dye DAPI
(to visualize nuclear morphology) and an antitubulin monoclonal
antibody (to visualize the tubulin cytoskelton, including the spindle).
Stained cells were analyzed by immunofluorescence microscopy (Fig. 2B). The unbudded half of the arrested population
uniformly displayed a single mass of DAPI-stainable material,
indicative of an undivided nucleus. The majority of these cells
contained an array of cytoplasmic microtubules radiating from a single
focus, a morphology typical of cells arrested in G. The
large-budded half of the arrested population was heterogeneous. The
majority of these cells (approximately two-thirds) contained two lobes
of DAPI-stainable material and an elongated spindle. Cells in which the
two lobes remained joined through the bud aperture and cells in which
the two nuclei appeared to be fully separated were both observed, at
approximately equal frequency. Both phenotypes are indicative of arrest
in anaphase (Surana et al., 1993). The remaining third of
budded cells contained a single, round nucleus traversed by a short,
thick intranuclear spindle (not shown). The latter phenotype is
characteristic of arrest in G
or metaphase (Irniger et
al., 1995; Surana et al., 1991). At the level of
resolution achieved by immunocytochemistry, the morphology of the
spindle did not appear to be abnormal in any of these arrested cells.
The mutant grown at 25 °C (Fig. 2A) and the
wild-type strain grown at either 25 or 37 °C (not shown) exhibited
the expected array of nuclear and cytoskeletal morphologies typical of
logarithmically growing cells.
Figure 2: Nuclear morphology and microtubule cytoskeleton of YDH8 (cka2-8) at 25 and 37 °C. YDH8 was inoculated into YPD as in Fig. 1, grown for 6 h at 25 °C, and then either maintained at 25 °C for an additional 5 h (A) or shifted to 37 °C for 5 h (B). Nuclear morphology was visualized with DAPI, and the microtubule cytoskeleton was visualized by staining with a monoclonal anti-tubulin antibody, as described under ``Experimental Procedures.'' The identical field of cells is shown in the left and right panels.
The results obtained with
asynchronous cultures suggested that CKII is required for cell cycle
progression in G as well as in G
and/or M. In
order to probe these arrest points independently, we analyzed cell
cycle progression of synchronized mutant and wild-type cultures. Cells
were synchronized in G
by two different protocols, nutrient
limitation and exposure to mating pheromone, and at the G
/S
boundary by treatment with hydroxyurea.
Figure 3:
Growth curves and budding profiles of CKA2 and cka2-8 strains following release from
stationary phase arrest. Cultures were grown in YPD at 25 °C until
stationary phase (90-95% unbudded cells) and then inoculated at 8
10
cells/ml in YPD prewarmed to 37 °C. Cell
density (A) and the percentage of budded cells (B)
were determined as described under ``Experimental
Procedures.''
, YDH6 (CKA2);
, YDH8 (cka2-8).
The failure to obtain a quantitative G arrest could be explained by the finite time required to
inactivate the cka2-8 allele (or to dephosphorylate the
relevant substrates) or by residual activity of the enzyme at the
restrictive temperature. Preincubation of YDH8 at 37 °C for either
1 or 2 h prior to release had little if any effect on the proportion of
cells able to pass the G
block. In contrast, preincubation
for 1 h at 38.5 °C (the maximum permissive temperature of the
control strain; Table 3) and subsequent release at that
temperature resulted in a complete G
block (data not
shown). This result suggests some leakiness of the cka2-8 allele at 37 °C.
Wild-type cells exposed to the mating
pheromone -factor arrest cell division, but not growth, at a later
stage in G
than cells in stationary phase. To determine
whether the point of G
arrest in response to CKII depletion
lies before or after the point of
-factor arrest, YDH6 and YDH8
were grown to log phase, synchronized in G
with
-factor and then released at 25, 37, or 38.5 °C. As shown in Fig. 4A, YDH8 responded to and recovered from
-factor with kinetics identical to those of the wild-type at 25
°C. At 37 °C (without preincubation) YDH8 exhibited only a
slight G
defect (data not shown). The strain budded with
near normal efficiency, albeit approximately 1 h slower than wild-type,
and did not arrest until the G
and/or M block (75-80%
budded cells, the vast majority large-budded). In contrast, at 38.5
°C (and with a 1-h preincubation) the mutant exhibited a complete
G
arrest (Fig. 4, B and C). Again,
cell number did not increase (Fig. 4B), and in this
case virtually all cells remained unbudded (Fig. 4C).
At 38.5 °C the control strain, YDH6, exhibited an approximately 50%
reduction in growth rate and also reached stationary phase at a
slightly lower cell density (Fig. 4B). Nevertheless,
this strain was clearly able to exit G
and complete
multiple rounds of cell division at this temperature.
Figure 4:
Growth curves and budding profiles of CKA2 and cka2-8 strains following release from
mating pheromone-induced G arrest. Cultures were inoculated
into YPD, grown for 6 h at 25 °C, and then synchronized in G
by exposure to
-factor. Cells were released from
-factor arrest at either 25 or 38.5 °C (in the latter case,
cultures were shifted to the nonpermissive temperature 1 h prior to the
removal of
-factor). A, release at 25 °C. B,
release at 38.5 °C.
, YDH6 (CKA2);
, YDH8 (cka2-8). C, budding profile from an experiment
identical to that in panels A and B, except that an
additional YDH8 culture, released at 38.5 °C, was returned to 25
°C 4 h after release.
, YDH8 released at 25 °C;
,
YDH6 released at 38.5 °C;
, YDH8 released at 38.5 °C;
, YDH8 released at 38.5 °C and then returned to 25 °C
after 4 h.
To confirm
that arrest was indeed in G, the DNA content of mating
pheromone-synchronized mutant and wild-type cells was analyzed by flow
cytometry (Fig. 5). At the time of
-factor release, all
cells were arrested with a 1 N DNA content. After release, the
mutant at 38.5 °C remained arrested with a 1 N complement
of DNA for up to 11 h, while both the mutant at 25 °C and wild-type
at 38.5 °C proceeded through S and into G
/M, eventually
losing synchrony after multiple cell divisions. The G
peak
of the arrested mutant exhibited a rightward drift upon prolonged
incubation at 38.5 °C. Such drifts have been noted before and are
due to increased autofluorescence from cell enlargement (Reed and
Wittenberg, 1990). This was consistent with the observed increase in
the average size of the arrested cells throughout the course of the
experiment. These experiments with pheromone-synchronized cells confirm
that CKII is required for cell cycle progression in G
, at a
point which must lie between the point of
-factor arrest and the
onset of S phase.
Figure 5:
Flow cytometry of CKA2 and cka2-8 strains following release from mating
pheromone-induced G arrest. Data shown are from the
experiment described in the legend of Fig. 4C. Cellular
DNA content was determined by flow cytometry of propidium
iodide-stained cells as described under ``Experimental
Procedures.'' The left peak in each profile reflects
cells with 1 N DNA content, the right peak, cells
with 2N DNA content. Times shown refer to time after release
from
-factor.
In order to determine whether arrest at the
G block is reversible, G
-arrested YDH8 cells
were shifted back to 25 °C after 4 h at 38.5 °C. Approximately
3-4 h after being returned to 25 °C these cells
simultaneously initiated bud formation (Fig. 4C) and
entered S phase (Fig. 5). By 5 h of recovery approximately 80%
of the cells were budded and contained a 2 N DNA complement.
However, few if any cells appeared to be competent to divide at this
point, and nearly all of these recovered cells formed an aberrant,
elongated bud. Cells with the latter morphology have been observed
previously following gradual depletion of CKII activity in a null
background (Padmanabha et al., 1990) and are also prominent in cka2
strains incubated at a semipermissive
temperature. (
)We speculate that this phenotype is
associated with intermediate levels of CKII activity during
G
/M, such that some CKII-dependent functions are completed
but not others.
Figure 6:
Growth curves of CKA2 and cka2-8 strains following release from
hydroxyurea-induced S phase arrest. Cultures were inoculated into YPD,
grown for 6 h at 25 °C, and then synchronized in early S phase by
exposure to hydroxyurea. Cells were released from hydroxyurea arrest at
either 25 or 38 °C (in the latter case, cultures were shifted to
the nonpermissive temperature 1 h prior to the removal of hydroxyurea). A, release at 25 °C. B, release at 38 °C.
, YDH6 (CKA2);
, YDH8 (cka2-8).
In
order to define the position of the second arrest point, cells were
analyzed by flow cytometry (Fig. 7). Prior to release from
hydroxyurea, the mutant at 25 and 38 °C and the wild-type at 38
°C all exhibited a DNA content between 1 and 2 N,
indicative of S phase arrest. Within the first 2 h after release, all
three cultures resumed DNA synthesis, completed S, and acquired a 2 N DNA content. This result established that the mutant is able
to complete previously initiated DNA synthesis at the restrictive
temperature. At later time points, the two control cultures continued
to cycle and ultimately lost synchrony, whereas the mutant at 38 °C
remained arrested with a 2 N DNA complement for up to 9 h. The
flow cytometry data thus positioned the second cell cycle block in
G and/or M. The G
/M-arrested cells remained
large-budded and, when examined for their nuclear and spindle
morphologies, displayed the same characteristic range of large-budded
phenotypes seen in asynchronous cultures at 37 °C (data not shown).
This suggested that the G
/M block defined using hydroxyurea
synchronization is the same as that observed in the asynchronous
cultures.
Figure 7: Flow cytometry of CKA2 and cka2-8 strains following release from hydroxyurea-induced S phase arrest. Data shown are from an experiment identical to that in Fig. 6, except that cells were exposed to hydroxyurea for 5.5 rather than 5 h. Cellular DNA content was determined by flow cytometry of propidium iodide-stained cells as described under ``Experimental Procedures.'' The left peak in each profile reflects cells with 1 N DNA content, the right peak, cells with 2 N DNA content. Times shown refer to time after release from hydroxyurea.
The reversibility of the G/M block was
assessed by returning a culture to 25 °C after 4 h at 38 °C.
YDH8 remained large-budded after such a shift and did not resume normal
cell cycle progression (data not shown), consistent with the results
obtained with G
-arrested cells allowed to recover at 25
°C (see above).
Because of potential artifacts associated with
hydroxyurea treatment, we confirmed the G/M block using a
second synchronization protocol. Wild-type and mutant cells were
synchronized in G
with
-factor and then released into
fresh medium at 25 °C. By 100 min after release approximately 80%
of the mutant cells had initiated bud formation and DNA synthesis,
indicating that they had passed the CKII G
block. When
shifted to 38 °C 100 min after release, the vast majority of these
cells failed to complete division but arrested as large-budded cells
with a 2 N DNA complement. These arrested cells displayed the
same range of nuclear morphologies seen with hydroxyurea
synchronization (data not shown). We conclude that the G
/M
arrest is not an artifact of hydroxyurea treatment.
Figure 8:
Viability of asynchronously growing CKA2 and cka2-8 strains following a shift to
nonpermissive temperature. Strains were inoculated into YPD at a
starting density of 2 10
cells/ml, grown for 8 h at
25 °C, and then shifted to 38.5 °C. Aliquots were removed at
the indicated times after the shift, and percent viability was
determined as described under ``Experimental Procedures.''
, YDH6 (CKA2);
, YDH8 (cka2-8).
Figure 9:
Rates of RNA and protein synthesis in CKA2 and cka2-8 strains at 25 and 38 °C.
Cells were grown in SMM to mid-log phase at 25 °C and then either
maintained at 25 °C or shifted to 38 °C. Aliquots were removed
at the indicated times after the shift and pulse-labeled for 10 min
with either [C]uracil (A) or
[
S]methionine (B). Incorportation into
acid-precipitable material was measured as described under
``Experimental Procedures.'' The data are expressed as
counts/min incorporated/cell.
, YDH6 (CKA2) at 25 °C;
, YDH8 (cka2-8) at 25 °C;
, YDH6 at 38
°C;
, YDH8 at 38 °C;
, YDH6 at 25 °C in the
presence of 10 µg/ml cycloheximide (added immediately after the
zero time point).
The reduction in
the rate of total RNA synthesis was not reflected in a comparable
reduction in the rate of total protein synthesis (Fig. 9B). While uptake of
[S]methionine in the mutant was consistently
lower than that of the wild-type, the magnitude of this effect
(approximately 2-fold) was no greater at 38 °C than at 25 °C.
By contrast, treatment with the protein synthesis inhibitor,
cycloheximide, inhibited [
S]methionine
incorporation more than 20-fold for up to 4 h (Fig. 9B). The arrest of growth in the mutant strain,
therefore, does not appear to be due to an overall inhibition of
protein synthesis, although an effect on the synthesis of specific
messages limiting for cell cycle progression cannot be ruled out by
these experiments. Also, because of the energetic expense of protein
synthesis, cell cycle arrest is unlikely to be due to a general decline
in metabolic activity.
We have used these temperature-sensitive
alleles to define the requirement for CKII activity during the cell
cycle in S. cerevisiae. As in any experiment employing a
temperature-sensitive mutation, an important caveat is that the
observed effects may be specific to heat-shocked cells. This concern is
exacerbated in this case because of the higher temperatures required to
obtain a tight arrest. While we cannot eliminate this caveat, we note
that temperatures identical to those employed here have been
effectively used to analyze cell cycle mutants in S.
cerevisiae. For example, a temperature of 38 °C was required
to identify the G/M function of Cdc28 (Reed and Wittenberg,
1990). Similarly, Tang and Reed(1993) used 38.5 °C to obtain a
tight G
arrest of a cks1 allele.
The targets of CKII
which are essential for G progression remain to be
identified. In S. cerevisiae, progression through Start is
regulated by a series of complexes between the Cdc28 protein kinase and
five different cyclins, Cln1, Cln2, Cln3, Clb5, and Clb6 (Schwob and
Nasmyth, 1993). Russo et al. (1992) have shown that
p34
is phosphorylated in vitro by purified CKII
at Ser
and that this site is phosphorylated in vivo during the G
phase of the cell cycle. Whether Cdc28 is
subject to the same modification is not known, but the relevant CKII
phosphorylation site is conserved in Cdc28. Other proteins required for
Start also represent potential targets. In an effort to identify genes
which interact genetically with CKII, we have carried out a screen for
multicopy suppressors of the cka2-13 allele. (
)Among the genes identified in this screen is CDC37, a previously characterized gene required for Start
(Reed, 1980). We have found that Cdc37 is a physiological substrate of
CKII and that mutation of the CKII recognition site impairs CDC37 function in vivo. (
)Although the biochemical
function of Cdc37 is not known, failure to phosphorylate Cdc37 could
explain the G
arrest of cka2 mutants.
Our
analysis of cells released from hydroxyurea arrest suggests that CKII
activity is not required for completion of S phase, consistent with the
low level of CKII activity detected during S phase in mammalian systems
(Carroll and Marshak, 1989; DeBenedette and Snow, 1991). However, we
emphasize that this conclusion rests upon the dual assumption that the
enzyme is fully inactivated by a 1-h preincubation at 38 °C and
that all relevant substrates become dephosphorylated. While the former
appears likely in view of our studies of G arrest, there is
no information concerning the latter. At least one well characterized
substrate of CKII is involved in DNA replication (DNA ligase I), and
the activity of this protein is increased in response to CKII
phosphorylation (Prigent et al., 1992). We also emphasize that
our results do not preclude a requirement for CKII at the
G
/S transition, as hydroxyurea arrest occurs after this
point in the cycle.
Although the
targets of CKII relevant to arrest in G/M remain to be
identified, a promising candidate is the well characterized CKII
substrate, topoisomerase II. This enzyme is a major structural
component of the metaphase chromosome scaffold and is essential both
for chromosome condensation in metaphase and for sister chromatid
segregation during anaphase (for review, see Cardenas and Gasser,
1993). S. cerevisiae topoisomerase II is an excellent
substrate of CKII in vitro, and phosphorylation strongly
stimulates enzyme activity (Cardenas et al., 1993). The
protein is phosphorylated in vivo and becomes
hyperphosphorylated during mitosis (Cardenas et al., 1992).
The excellent correlation between the in vivo and in vitro sites suggests that topoisomerase II is a physiological target of
CKII in yeast. Consistent with this, phosphorylation of the protein is
temperature sensitive in the YDH8 strain (Cardenas et al.,
1992). Temperature-sensitive topoisomerase II (top2
) mutants arrest in mitosis with an
elongated spindle and a nucleus which is stretched through the bud
aperture (Holm et al., 1985), a phenotype similar to that of
anaphase-arrested cka2
cells. Moreover, like cka2
mutants, top2
mutants
become inviable at the nonpermissive temperature, apparently during a
failed attempt to segregate daughter chromosomes (Holm et al.,
1985). Collectively, these data suggest that the anaphase arrest of cka2
strains may result from failure to
phosphorylate and activate topoisomerase II. It appears unlikely that
failure to activate topoisomerase II can account for those cka2
cells which arrest with a
G
/metaphase phenotype, suggesting that additional
substrates must be involved.
The properties of the CKII mutations described here
differ in several respects from those reported recently for the orb5 allele of the S. pombe cka1 gene (Snell and Nurse, 1994). Following a shift to the
nonpermissive temperature, orb5 cells undergo several cell
divisions and ultimately die as small spherical cells. No cell cycle
defects were noted in this mutant, which was interpreted as a
morphological mutant defective in reinitialization of polarized growth
following cytokinesis (Snell and Nurse, 1994). At face value, the
different behavior of temperature-sensitive CKII mutations in the two
organisms suggests significant differences in the physiological role of
CKII in S. pombe and S. cerevisiae. However, we have
recently isolated two temperature-sensitive alleles of the Sc CKA1 gene (encoding the CKII
subunit) and find that these exhibit
a behavior strikingly similar to that of the orb5 mutation,
including the absence of first cycle arrest and adoption of a highly
spherical morphology. (
)This result implies some functional
specialization of CKA1 and CKA2 in S. cerevisiae and argues that the function of CKII in the two yeasts may be
similar. Additional studies in both organisms will be required to
clarify and correlate the physiological role of CKII in the two
species.