Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan
* Author for correspondence (e-mail: tmiyaka{at}hiroshima-u.ac.jp)
Accepted 9 June 2005
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Summary |
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Key words: Pkc1p, Mpk1p, Cln2p, Saccharomyces cerevisiae, Calcium
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Introduction |
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As yeast cells begin the G1/S transition, Cln1p and Cln2p G1 cyclins accumulate, activate the cyclin-dependent kinase Cdc28p, and induce clustering of actin at the presumptive bud site, thus focusing secretion of new cell-wall components onto the nascent bud (Pruyne and Bretscher, 2000). Continued Cdc28p-Cln1/2p activity maintains this clustered distribution of actin in the bud tip and polarized growth (Lew and Reed, 1993
). Cells over-expressing Cln1p or Cln2p form a highly elongated bud because of sustained polarized growth (Lew and Reed, 1995
). Polarized growth continues in G2, as the mitotic cyclin Clb1/2p-Cdc28p activity remains sequestered via inhibitory phosphorylation by Swe1p, a protein kinase that inhibits Cdc28p (Booher et al., 1993
; Lew and Reed, 1995
). Recently, we reported that Ca2+-signaling pathways induced the formation of highly polarized bud and delayed the cell-cycle progression in G2 (Mizunuma et al., 1998
; Mizunuma et al., 2001
). The effect of Ca2+ on cell-cycle regulation and polarized bud growth are pronounced on a zds1
background lacking the negative regulator for SWE1 and CLN2 transcription (Bi and Pringle, 1996
; Ma et al., 1996
; Yu et al., 1996
; Mizunuma et al., 1998
; Mizunuma et al., 2001
; Mizunuma et al., 2004
). These phenotypes are peculiar to calcium, because a similar effect was not observed with sorbitol (300 mM) or MgCl2 (100 mM; our unpublished data).
In S. cerevisiae, a unique protein kinase C, Pkc1p, functions in a multiplicity of pathways, including those related to cell wall integrity, bud emergence, stretching of the plasma membrane (Mazzoni et al., 1993; Levin and Errede, 1995
; Gustin et al., 1998
; Heinisch et al., 1999
) and organization of the actin cytoskeleton (Heinisch et al., 1999
). One well-known pathway for Pkc1p involves the sequentially activated protein kinases Bck1p, the redundant Mkk1p/Mkk2p and Mpk1p (Slt2p), which ultimately activates by phosphorylation of transcription factors, including the Rlm1p and SBF (Swi4p-Swi6p) complex (Dodou and Treisman, 1997
; Madden et al., 1997
; Baetz et al., 2001
). This pathway is essential for cell wall integrity, bud emergence, responses to hypotonic shock, stretching of the plasma membrane, and repolarization of actin upon cell-wall stress (Levin et al., 1990
; Levin and Bartlett-Heubusch, 1992
; Paravicini et al., 1992
; Yoshida et al., 1992
; Lew and Reed, 1995
; Davenport et al., 1995
; Kamada et al., 1995
; Igual et al., 1996
; Marini et al., 1996
; Zarzov et al., 1996
; Gray et al., 1997
; Gustin et al., 1998
; Helliwell et al., 1998
; Delley and Hall, 1999
). In addition, Pkc1p is involved in controlling the depolarization of actin during adaptation to growth at higher temperatures in a manner independent of the Mpk1p MAP kinase pathway (Delley and Hall, 1999
). However, little is known about this Pkc1p effecter branch.
Much of our understanding of Pkc1p functions has come from the phenotypes caused by MPK1 deletion and/or severe temperature-sensitive alleles of PKC1, all of which involve a defect in the cell wall. The identification of a Mpk1p-independent role of Pkc1p will be facilitated by the isolation and characterization of a PKC1 allele specifically defective in the novel function.
Here, we describe the isolation and characterization of a pkc1 mutant allele (pkc1-834) defective in a previously unknown pathway. The pkc1-834 allele was defective in the maintenance of Ca2+-induced F-actin polarization in a manner independent of Mpk1p activation. This phenotype appeared to be caused by decreased expression of Cln2p. Pkc1p was required for posttranscriptional upregulation of Cln2p. The Rho1 small G protein molecular switch was suggested to be involved in the novel Pkc1p function.
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Materials and Methods |
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DNA sequence
Sequencing of double strand DNA was done by the cycle sequencing method, using a ALFred DNA sequencer (Amersham Biosciences). For PCR amplification of the genomic DNA, a Thermo Sequenase fluorescently labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Biosciences) was used. M13 universal and reverse primers from Cy5TM AutoRead Sequencing Kit (Amersham Biosciences) were also used. The entire open reading frame of PKC1 was divided into four regions. PCR products were synthesized using primers, region I: 5'-AAAGAGCTCATGAGTTTTTCACAAT-3' (sense strand, the SacI site is underlined) and 5'-TGCACCTGCAGATTCTTTGCTGCTGAAGTAGAC-3' (antisense strand, the PstI site is underlined). Region II: 5'-AAACTGCAGGTGCAAGGCGAAAATA-3' (sense strand, the PstI site is underlined), and 5'-AACTTCTAGATCCTCTTGGATTTCC-3' (antisense strand, the XbaI site is underlined). Region III: 5'-AAATCTAGAAGTTGATCACATTGAT-3' (sense strand, the XbaI site is underlined), and 5'-GATAGGTCGACTCTTTGGTTTTGAA-3' (antisense strand, the SalI site is underlined). Region IV: 5'-AAAGTCGACCTATCTGTAAGAAGGG-3' (sense strand, the SalI site is underlined), and 5'-TTTCTAAGCTTTCATAAATCCAAATCATCT-3' (antisense strand, the HindIII site is underlined).
Site-directed mutagenesis
To construct plasmids containing the PKC1 gene with single mutations, we employed site-directed mutagenesis. The mutagenic primers for PKC1 (P1102S) were 5'-CCACCCTACATCTCAGAAATTAAATCTCCG-3' and 5'-CGGAGATTTAATTTCTGAGATGTAGGGTGG-3'; and those for the PKC1 (N834D), 5'-GTTCTTGGTAAAGGTGATTTTGGTAAAG-3' and 5'-CTTTACCAAAATCACCTTTACCAAGAAC-3'. PCR was performed with PfuTurbo DNA polymerase (Stratagene) and pUC119-PKC1 as a PCR template. Successful mutagenesis was confirmed by DNA sequencing. The low-copy plasmid YCp50-PKC1 (P1102S) or YCp50-PKC1 (N834D) construct was generated as follows. The SalI-PvuII fragment of pUC119-PKC1 containing PKC1 (P1102S) or PKC1 (N834D) was ligated into the SalI-NruI site of YCp50. YCp50-PKC1 (P1102S) or YCp50-PKC1 (N834D) was used for complementation analysis for pkc1 or zds1
pkc1
.
Gene disruption and Strain construction
The pkc1 strain in the W303 background was constructed by gene replacement. Genomic DNA was isolated from the pkc1
::LEU2 (DL376) strain (Levin et al., 1990
). The PKC1 locus was amplified by PCR using primers 5'-ATGTTTGTCCCACTCCAGGTTGCAC-3' and 5'-TTTGAGACGTCATGAACTCTCGCGG-3'. The amplified fragment was used to transform a diploid W303 strain [ZDS1/ZDS1 (W303-1D) or zds1
::TRP1/ZDS1(YMM235)]. Replacement was confirmed by PCR. The diploid was sporulated and the tetrads were dissected and analyzed. When the spores were grown at room temperature, all of the tetrads produced two colonies that grew at the wild-type rate and two colonies that grew very poorly even in the presence of 1 M sorbitol. The fast growing colonies all had a Leu phenotype, indicating that the very poor growth results from the integration of the knockout construct. Deletion of the PKC1 gene in the Leu+ strains was further confirmed by complementation of the very poor growth by a centromere plasmid carrying the PKC1 gene.
The pkc1 zds1
(pkc1
) strain bearing the N834D, P1102S or wild-type PKC1 allele was constructed as follows. The YCp50-PKC1 (P1102S), YCp50-PKC1 (N834D) or YCp50-PKC1 plasmid was transformed into a diploid strain (YMM236 or YMM235). The diploid was sporulated and the tetrads were dissected and analyzed. The spores that were grown minus uracil (Ura), tryptophan (Trp) and leucine (Leu) (which selects for the zds1
pkc1
strain bearing the N834D, P1102S or wild-type PKC1 allele) or minus Ura and Leu (which selects for the pkc1
strain bearing N834D, P1102S or the wild-type PKC1 allele) were isolated and their phenotypes examined.
The zds1 strain of the YPH500 background was constructed by gene replacement. Disruption of the ZDS1 gene was performed using a zds1 disruption plasmid pUC119-zds1::URA3. The zds1::URA3 fragment was used to transform the YPH500 background.
Actin staining
Actin was visualized in formaldehyde-fixed cells by using Rhodamine-conjugated phalloidin as described previously (Adams and Pringle, 1991).
Assessment of cell lysis
Qualitative assessment of cell lysis in colonies was done using the alkaline phosphatase assay described previously (Saka et al., 2001). Colonies were formed on YPD agar plates at 25°C for 2 days, and the plates were then shifted to 35°C and overlaid with an alkaline phosphatase assay solution. Colonies that contain lysed cells stain blue, whereas intact colonies remain white.
Cell culture synchronization, RNA isolation and northern blot analysis
Cell culture synchronization was done by the procedure described previously (Mizunuma et al., 1998). Total RNA was isolated by the hot phenol method. The isolated RNA was separated on a 1% agarose gel, transferred to a nylon membrane, and then subjected to northern blot analysis. The SWE1, CLN2, and ACT1 probes were generated by random-primed labeling of a 0.7 kb BglII fragment of SWE1, a 1.3 kb NcoI-XhoI fragment of CLN2 and a 1.1 kb XhoI-KpnI fragment of ACT1, respectively, with [
-32P]dCTP by use of a multiprime DNA labeling kit (Amersham Biosciences).
Western blot analysis
Preparation of cell extracts and immunoprecipitations were performed basically as described previously (Mizunuma et al., 2001; Mizunuma et al., 2004
). For detection of the HA-tagged proteins, Myc-tagged proteins, Pkc1p, phosphorylated Mpk1, Mpk1p and Cdc28p, monoclonal antibodies 12CA5 (BAbCO) against the HA epitope, 9E10 (BAbCO) against the Myc epitope, polyclonal anti-PKC1 (Santa Cruz Biotech.), anti-phospho-p44/42 MAP kinase (New England Biolabs Inc.), anti-MAPK antibody (Santa Cruz Biotech.), and anti-PSTAIRE (Santa Cruz Biotech.), respectively, were used.
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Results |
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We next investigated the effect of exogenous calcium on Swe1p and Cln2p levels by western blot analysis of synchronized cell cultures of strains with chromosomally integrated constructs for both Myc-tagged Swe1p and HA-tagged Cln2p (Fig. 3A). The Swe1p and Cln2p levels in the cells released from G1 arrest were analyzed by western blotting. As previously reported, Swe1p and Cln2p levels in wild-type cells oscillated during the cell cycle, peaking at the time of bud emergence and declining before nuclear division (McMillan et al., 1998; Sia et al., 1998
). Exogenous calcium caused a 20-minute lag in the increase in Swe1p and Cln2p levels, reflecting the delays in the elevation of the respective mRNA (Fig. 2); and the increased protein levels were sustained longer by calcium (Fig. 3A). There was no significant difference among the strains when cultivated in YPD or YPD plus CaCl2 medium with regard to the periodic patterns of the Swe1p level. In the presence of exogenous calcium, Swe1p in the pkc1-834 and zds1
pkc1-834 cells accumulated, consistent with the observation that the scz6 mutation failed to suppress the G2 delay caused by calcium (Fig. 1C and Fig. 3A). The G2 delay was partially suppressed by the deletion of the SWE1 gene, suggesting that the delay was indeed mediated at least partially by the activation of Swe1p (data not shown). However, Cln2p levels in the pkc1-834 cells were significantly lower than those in the wild-type strain. Moreover, Cln2p levels in the pkc1-834 strain, in contrast to those in the wild-type strain, were not elevated by exogenous calcium in either ZDS1 or zds1
backgrounds, suggesting that Pkc1p is important for the elevation and maintenance of Cln2p levels in response to exogenous calcium (Fig. 3A). The defect in Cln2p accumulation in the pkc1-834 cells could be due to a decreased rate of Cln2p synthesis, the destabilization of Cln2p, or both.
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Using the same synchronous cell cultures as used in the experiment for Fig. 3A, we determined the cellular DNA content (Fig. 3C). As previously reported (Mizunuma et al., 1998; Mizunuma et al., 2001
), a delay in G1, as well as in G2, was induced by calcium in all the strains examined (compare YPD and YPD + Ca2+ of Fig. 3C). The G1 delay seemed to be due to the downregulation of CLN2 mRNA (Fig. 2). The pkc1-834 cells showed a G1 delay after release from G1 arrest, even in the absence of exogenous calcium, indicating that Pkc1p is required for the G1-S transition (Fig. 3C).
Pkc1p is required for the maintenance of calcium-induced F-actin polarization
Both actin polarization and hyper-polarized bud growth is dependent on the G1 cyclins (Lew and Reed, 1993). Cell polarization can be estimated by the distribution of F-actin. We therefore examined the ability of the pkc1-834 cells to polarize their F-actin cytoskeleton in response to calcium. Using the same synchronous cell cultures described in the above section, we examined the effects of exogenous calcium on bud emergence and F-actin polarization at the bud site (Fig. 4). After a 2-hour treatment of the cells with
-factor, about 80% of wild-type cells yielded a mating projection, whereas in the pkc1-834 cells it was about 30%, although the treatment resulted in a G1 block in the mutant cells (data not shown and Fig. 3C). However, pkc1-834 cells on the zds1
background formed a mating projection at a ratio comparable to that of the wild-type cells. Therefore, we used the zds1
mutant background in this study (Fig. 4). Upon release of the
-factor-treated cells to YPD medium, bud emergence of the zds1
pkc1-834 cells was delayed by 20 minutes compared with that of the zds1
cells. Bud emergence of both zds1
pkc1-834 and pkc1-834 cells was delayed by exogenous calcium similarly (Fig. 4B). These results suggested that the suppression of the calcium-induced polarized bud growth by the pkc1-834 mutation is not due to the defect in bud emergence.
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Activation of Mpk1p and cell wall integrity are not impaired in the pkc1-834 mutant
Yeast bearing the stt1-1 mutation, a previously characterized temperature-sensitive PKC1 allele, exhibits a defect in cell wall maintenance and in G2-M transition (Yoshida et al., 1992). So we examined whether the stt1-1 mutation could suppress the calcium sensitivity phenotypes of the zds1
strain. However, the stt1-1 allele only partially suppressed the phenotype, suggesting that the defect of the pkc1-834 allele may be functionally distinct from that of the stt1-1 allele (Fig. 1A,B).
In characterization of the pkc1-834 and stt1-1 mutations, the former mutation was found to involve an asparagine to aspartic acid substitution at amino acid position 834, and the latter, a proline to serine substitution at amino acid position 1102 of Pkc1p (Fig. 5A). The stt1-1 mutation (P1102S) was within the kinase domain in a highly conserved proline residue, whereas the pkc1-834 mutation (N834D) was within the predicted ATP-binding site of Pkc1p in an evolutionarily non-conserved residue. We constructed N834D and P1102S mutant versions of the PKC1 gene on a centromeric plasmid. The N834D and P1102S alleles could suppress any of the phenotypes of the pkc1-834 or stt1-1 mutation, respectively (data not shown). Furthermore, the pkc1 zds1
strain bearing the N834D or P1102S allele exhibited calcium phenotypes similar to those of the pkc1-834 zds1
or stt1-1 zds1
strain, respectively, confirming that each mutant allele is responsible for the observed phenotype (Fig. 5B).
It was previously shown that the growth of the pkc1-1 strain (on EG123 background) was dependent on the presence of exogenous calcium in the medium (Levin and Bartlett-Heubusch, 1992). Interestingly, the mutation site of pkc1-834 (N834D) coincided with that of this pkc1-1 mutation (N834K). The pkc1-834 mutant was able to grow on a YPD plate (without added CaCl2) at a rate slower than that of the wild-type strain, and the growth rate could be restored to an apparently normal level by the addition of CaCl2, but not MgCl2 (Fig. 1A and Fig. 5B; data not shown). Thus, the N834 residue seems to be involved somehow in the regulation of Pkc1p by calcium. Western blot analysis of Pkc1p in cell extracts of wild-type, pkc1-834 and stt1-1 strains, using an anti-Pkc1p antibody, demonstrated that the expression level of Pkc1-834p and of Stt1-1p was comparable to that of the wild-type Pkc1p (data not shown).
We further genetically characterized the defects of the two PKC1 mutant alleles. Homo- and hetero-allelic diploids were constructed and examined for their temperature sensitivities and resistance to calcium on the zds1 background. In the presence of 100 mM CaCl2 at 37°C, the homo-allelic diploids pkc1-834/pkc1-834 and stt1-1/stt1-1 grew poorly, whereas the hetero-allelic diploid (pkc1-834/stt1-1) was able to grow (all on the zds1
background) showing an intragenic complementation (Fig. 6A). This result indicated that the defects of the stt1-1 and pkc1-834 mutations were functionally different. In addition, various diploid strains, with respect to the PKC1 allele, exhibited a range of calcium resistance: (in decreasing order) pkc1-834/pkc1-834, pkc1-834/stt1-1, stt1-1/stt1-1 and PKC1/PKC1 (all on the zds1
background), suggesting that the wild-type Pkc1p inhibits growth in the presence of calcium and that the mutation pkc1-834 reduces the inhibitory effect more than stt1-1 (Fig. 6A).
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The degradation of Hsl1p, a negative regulator of Swe1p, via the ubiquitin-mediated 26S proteasome pathway is triggered by the activation of calcineurin and the Mpk1p-Mck1p pathway (Mizunuma et al., 2001). Pkc1p is known as an upstream regulator of the Mpk1p MAPK pathway in cell wall construction. We expected that calcium-induced Hsl1p degradation, which is dependent on the activation of Mpk1p occurs normally in the pkc1-834 strain, but not in the stt1-1 strain. However, the level of Hsl1p in these strains, in contrast to that in the mpk1
strain, decreased more rapidly, in a similar manner as in wild-type strain (Fig. 7). Consistent with this observation, both pkc1-834 and stt1-1 alleles failed to suppress the Ca2+-induced G2 delay of the zds1
strain (Fig. 1C), suggesting that this effect is due to the activation of Swe1p. Thus, it was suggested that the Pkc1-834p and Stt1-1p mutant proteins are functional in regulating the Hsl1p degradation. Alternatively, it was also possible that lower activity of Mpk1p in the stt1-1 cells is still sufficient to trigger Hsl1p degradation.
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Rho1p GTPase is involved in the Ca2+-induced polarized bud growth
In the activation of the Mpk1p MAPK pathway, Pkc1p is a down-stream component of Rho1p, a small G protein molecular switch (Nonaka et al., 1995; Kamada et al., 1996
; Helliwell et al., 1998
; Delley and Hall, 1999
). Because Rho1p is an essential protein, several conditional, lethal (high temperature-sensitive) mutations in the RHO1 gene have been isolated and characterized (Helliwell et al., 1998
; Saka et al., 2001
). Of these, rho1-2 and rho1-5 strains are defective in the activation of Mpk1p, whereas rho1-3 and rho1-4 mutants are not (Saka et al., 2001
). We examined allele specificity of various rho1 mutations on the Ca2+-induced polarized bud growth of the zds1
strain. Consistent with the distinct role of Pkc1p in Ca2+-induced polarized bud growth, the rho1-3 and rho1-4 mutations, but not the rho1-2 and rho1-5 mutations, suppressed the calcium phenotypes of the zds1
strain, suggesting that the defect caused by the rho1-3 and rho1-4 alleles, rather than the rho1-2 and rho1-5 alleles, are related in having the novel Pkc1p function (Fig. 8). Consistent with the notion that the functional defects of the rho1-3 and rho1-4 alleles are related with that of the pkc1-834 allele (Fig. 3A), the levels of Cln2p in rho1-3 and rho1-4 strains and the pkc1-834 strain were similarly low compared with those of wild-type and rho1-2 and rho1-5 strains (data not shown). The typical effects of calcium, observed in zds1
mutants of W303 strain background, namely Ca2+-induced growth arrest, hyper-polarized bud growth and G2 delay, were also observed in the YPH500 strain background at calcium concentrations higher than those for the W303 strain. The pkc1-834 mutation, but not that of stt1-1, suppressed the Ca2+-induced hyper-polarized bud growth on this strain background (data not shown). Taken together, these data implicated the Mpk1p-independent novel pathway of Pkc1p in Ca2+-induced polarized bud growth in a manner dependent on Rho1p.
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Discussion |
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The polarized bud growth and defect in cell proliferation, but not the G2 delay, caused by high calcium in the zds1 strain were abolished by the pkc1-834 mutation (Fig. 1). This suggested the possibility that the growth inhibition of zds1
cells by high calcium is due to the hyperpolarization of bud growth induced by high Cln2p levels. However, we consider this possibility unlikely for the following reasons. The growth of the cln2
zds1
strains was more sensitive to calcium than growth of the zds1
strain, although cln2
zds1
cells failed to show hyperpolarized bud growth in response to calcium (data not shown). Moreover, the rho1-3 and rho1-4 mutant strains (like the pkc1-834 strain) that expressed lower levels of Cln2p than the wild-type strain could not suppress calcium sensitivity of zds1
strain (data not shown). Furthermore, double mutant strains having the pkc1-834 mutation in combination with either of the rho1-2, -3, -4 or -5 mutations, exhibited more severe growth defect on YPD medium at a permissive temperature (25°C) than either of the single mutants, suggesting that rho1-2, 3, 4 and 5 mutations may have additional defect besides the defect in the activation of the Pkc1p (data not shown).
We have shown that the regulation of Cln2p expression (and probably Cln1p, as well) is mediated by Pkc1p at the posttranscriptional level (Fig. 3A,B). However, its molecular mechanism still remains to be elucidated. Thus, the level of Cln2p in the pkc1-834 strain, defective in the regulatory mechanism, was lower that in the wild-type strain, and Cln2p was more stable in the pkc1-834 strain (Fig. 3A,B). Since the phosphorylation of G1 cyclin, which is reported to be due to the Cdc28-dependent autophosphorylation of the Cln subunit, is required for their rapid degradation (Lanker et al., 1996), the stable nature of Cln2p in the pkc1-834 strain may be the result of the lower activity of Cln2p-Cdc28p in this strain. Alternatively, it is also possible that Pkc1p may regulate Cln2p at the posttranscriptional level.
An earlier study suggested that the activation of Cdc28p-Cln1/2p in G2 leads to hyperpolarization of F-actin and formation of elongated buds (Lew and Reed, 1993). Thus, the effect of the pkc1-834 mutation on the calcium-induced hyperpolarized bud growth of the zds1
strain can be explained by the reduced activity of the Cdc28p-Cln1/2p kinase, which is insufficient to fully promote F-actin polarization. During adaptation to higher temperature conditions, Pkc1p is required for both de- and repolarization of the actin cytoskeleton. The former is independent of the Mpk1p MAP kinase pathway, while the latter is dependent on it (Delley and Hall, 1999
). By contrast, in the response to calcium, Pkc1p was not required for the de- and repolarization of F-actin (Fig. 4). Instead, Pkc1p was required for the maintenance of F-actin polarization (Fig. 4). We also found that Rho1p Rho GTPase is involved in Ca2+-induced polarized bud growth in a manner independent of Mpk1p. Pkc1p interacts with Rho1p in vivo and in vitro (Nonaka et al., 1995
; Kamada et al., 1996
). Pkc1p mutant protein (Pkc1-834p) of the pkc1-834 strain can physically interact with Rho1p, as revealed by the two-hybrid assay using LexA-Rho1p (Q68L, GTP-bound form) with GAD-Pkc1-834p (data not shown). Co-immunoprecipitation of Pkc1-834p and Rho1p in vivo was also observed (data not shown). The interaction of Rho1p and Pkc1-834p is consistent with the ability of Pkc1-834p to activate the Mpk1p pathway, leading to cell wall construction.
How does Pkc1p regulate the establishment and maintenance of polarized bud growth in zds1 strain? Earlier studies showed that the Pkc1p-Mpk1p pathway is activated at the G1/S transition, concomitant with bud emergence, and that its activation is partly dependent on the activation of the Cdc28p-Cln1/2p complex (Marini et al., 1996
; Zarzov et al., 1996
; Gray et al., 1997
). The pkc1-834 zds1
cells were able to initiate bud formation, but were unable to sustain polarized bud growth by exogenous calcium (Fig. 4). Even the reduced levels of Cln1p and Cln2p in pkc1-834 zds1
cells appeared to be sufficient to establish polarized bud growth, but insufficient to support Ca2+-induced hyper-polarized bud growth. Our data indicated that Pkc1p is important for maintaining the Cln2p levels, placing the Pkc1p function upstream of Cln1p and Cln2p. Our results and those of others indicate that the Pkc1p-Mpk1p pathway is required for the establishment of polarized bud growth, while the Mpk1p-independent Pkc1p pathway is required for its maintenance.
How does Ca2+ signaling induce polarized bud growth and G2 cell-cycle delay? These may be achieved by maintaining a high level of the G1 cyclins and by triggering the Swe1p-mediated inhibition of Cdc28p-Clb1/2p. Of these, the former is dependent on the novel Pkc1p function, whereas the latter is mediated by a coordinated action of the calcineurin and Mpk1p-Mck1p pathways to activate Swe1p, which is achieved by elevating its abundance and downregulating Hsl1p (a Swe1p negative regulator) leading to the accumulation of the hyperphosphorylated, active form of Swe1p (Mizunuma et al., 2001). However, the possibility that Pkc1p is involved in the downregulation of Hsl1p could not be excluded, because the calcium-induced Hsl1p degradation in pkc1
strain could not be determined because the growth of this strain was extremely poor even in the presence of an osmo-stabilizer. Significantly, the swe1
pkc1-834 double mutant exhibits a severe growth defect even at the temperatures permissive for the pkc1-834 strain (data not shown), suggesting that Ca2+-mediated coordinated regulation of polar bud growth and cell-cycle is important for normal cell growth. It will be of considerable interest to study whether a similar mechanism operates in higher eukaryotes. A model summarizing our results is shown in Fig. 9
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Acknowledgments |
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References |
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