Orthologues of the anaphase-promoting complex/cyclosome coactivators Cdc20p and Cdh1p are important for mitotic progression and morphogenesis in Candida albicans

The conserved anaphase-promoting complex/cyclosome (APC/C) system mediates protein degradation during mitotic progression. Conserved coactivators Cdc20p and Cdh1p regulate the APC/C during early to late mitosis and G(1) phase. Candida albicans is an important fungal pathogen of humans, and it forms highly polarized cells when mitosis is blocked through depletion of the polo-like kinase Cdc5p or other treatments. However, the mechanisms governing mitotic progression and associated polarized growth in the pathogen are poorly understood. In order to gain insights into these processes, we characterized C. albicans orthologues of Cdc20p and Cdh1p. Cdc20p-depleted cells were blocked in early or late mitosis with elevated levels of Cdc5p and the mitotic cyclin Clb2p, suggesting that Cdc20p is essential and has some conserved functions during mitosis. However, the yeast cells formed highly polarized buds in contrast to the large doublets of S. cerevisiae cdc20 mutants, implying a distinct role in morphogenesis. In comparison, cdh1Δ/cdh1Δ cells were viable but showed enrichment of Clb2p and Cdc5p, suggesting that Cdh1p may influence mitotic exit. The cdh1Δ/cdh1Δ phenotype was pleiotropic, consisting of normal or enlarged yeast, pseudohyphae, and some elongated buds, whereas S. cerevisiae cdh1Δ yeast cells were reduced in size. Thus, C. albicans Cdh1p may have some distinct functions. Finally, absence of Cdh1p or Cdc20p had a minor or no effect on hyphal development, respectively. Overall, the results suggest that Cdc20p and Cdh1p may be APC/C activators that are important for mitosis but also morphogenesis in C. albicans. Their novel features imply additional variations in function and underscore rewiring in the emerging mitotic regulatory networks of the pathogen.     

Two different modes of cyclin clb2 proteolysis during mitosis in Saccharomyces cerevisiae

Sister chromatid separation and mitotic exit are triggered by the anaphase-promoting complex (APC/C) which is a multi-subunit ubiquitin ligase required for proteolytic degradation of various target proteins. Cdc20 and Cdh1 are substrate-specific activators of the APC/C. It was previously proposed that Cdh1 is essential for proteolysis of the yeast mitotic cyclin Clb2. We show that Clb2 proteolysis is triggered by two different modes during mitosis. A fraction of Clb2 is degraded during anaphase in the absence of Cdh1. However, a second fraction of Clb2 remains stable during anaphase and is degraded in a Cdh1-dependent manner as cells exit from mitosis. Most of cyclin Clb3 is degraded independently of Cdh1. Our data imply that degradation of mitotic cyclins is initiated by a Cdh1-independent mechanism.     

Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20

Cdc20, an activator of the anaphase-promoting complex (APC), is also required for the exit from mitosis in Saccharomyces cerevisiae. Here we show that during mitosis, both the inactivation of Cdc28-Clb2 kinase and the degradation of mitotic cyclin Clb2 occur in two steps. The first phase of Clb2 proteolysis, which commences at the metaphase-to-anaphase transition when Clb2 abundance is high, is dependent on Cdc20. The second wave of Clb2 destruction in telophase requires activation of the Cdc20 homolog, Hct1/Cdh1. The first phase of Clb2 destruction, which lowers the Cdc28-Clb2 kinase activity, is a prerequisite for the second. Thus, Clb2 proteolysis is not solely mediated by Hct1 as generally believed; instead, it requires a sequential action of both Cdc20 and Hct1.     

Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC

Ubiquitin-mediated proteolysis has emerged as a key mechanism of regulation in eukaryotic cells. During cell division, a multi-subunit ubiquitin ligase termed the anaphase promoting complex (APC) targets critical regulatory proteins such as securin and mitotic cyclins, and thereby triggers chromosome separation and exit from mitosis. Previous studies in the yeast Saccharomyces cerevisiae identified the conserved WD40 proteins Cdc20 and Hct1 (Cdh1) as substrate-specific activators of the APC, but their precise mechanism of action has remained unclear. This study provides evidence that Hct1 functions as a substrate receptor that recognizes target proteins and recruits them to the APC for ubiquitylation and subsequent proteolysis. By co-immunoprecipitation, we found that Hct1 interacted with the mitotic cyclins Clb2 and Clb3 and the polo-related kinase Cdc5, whereas Cdc20 interacted with the securin Pds1. Failure to interact with Hct1 resulted in stabilization of Clb2. Analysis of Hct1 derivatives identified the C-box, a motif required for APC association of Hct1 and conserved among Cdc20-related proteins. We propose that proteins of the Cdc20 family are substrate recognition subunits of the ubiquitin ligase APC.     

Cell cycle-dependent nuclear export of Cdh1p may contribute to the inactivation of APC/C(Cdh1)

Cdh1p is a substrate-specific subunit of the anaphase-promoting complex (APC/C), which functions as an E3 ubiquitin ligase to degrade the mitotic cyclin Clb2p and other substrates during the G(1) phase of the cell cycle. Cdh1p is phosphorylated and thereby inactivated at the G(1)/S transition predominantly by Cdc28p-Clb5p. Here we show that Cdh1p is nuclear during the G(1) phase of the cell cycle, but redistributes to the cytoplasm between S phase and the end of mitosis. Nuclear export of Cdh1p is regulated by phosphorylation and requires active Cdc28p kinase. Cdh1p binds to the importin Pse1p and the exportin Msn5p, which is necessary and sufficient to promote efficient export of Cdh1p in vivo. Although msn5delta cells are viable, they are sensitive to Cdh1p overexpression. Likewise, a mutant form of Cdh1p, which is constitutively nuclear, prevents accumulation of Clb2p and leads to cell cycle arrest when overexpressed in wild-type cells. Taken together, these results suggest that phosphorylation-dependent nuclear export of Cdh1p by Msn5p contributes to efficient inactivation of APC/C(Cdh1).     

Dephosphorylation of threonine 169 of Cdc28 is not required for exit from mitosis but may be necessary for start in Saccharomyces cerevisiae.

Entry into mitosis requires activation of cdc2 kinase brought on by its association with cyclin B, phosphorylation of the conserved threonine (Thr-167 in Schizosaccharomyces pombe) in the T loop, and dephosphorylation of the tyrosine residue at position 15. Exit from mitosis, on the other hand, is induced by inactivation of cdc2 activity via cyclin destruction. It has been suggested that in addition to cyclin degradation, dephosphorylation of Thr-167 may also be required for exit from the M phase. Here we show that Saccharomyces cerevisiae cells expressing cdc28-E169 (a CDC28 allele in which the equivalent threonine, Thr-169, has been replaced by glutamic acid) are able to degrade mitotic cyclin Clb2, inactivate the Cdc28/Clb2 kinase, and disassemble the anaphase spindles, suggesting that they exit mitosis normally. The cdc28-E169 allele is active with respect to its mitotic functions, since it complements the mitosis-defective cdc28-1N allele. Whereas replacement of Thr-169 with serine affects neither Start nor the mitotic activity of Cdc28, replacement with glutamic acid or alanine renders Cdc28 inactive for Start-related functions. Coimmunoprecipitation experiments show that although Cdc28-E169 associates with mitotic cyclin Clb2, it fails to associate with the G1 cyclin Cln2. Thus, an unmodified threonine at position 169 in Cdc28 is important for interaction with G1 cyclins. We propose that in S. cerevisiae, dephosphorylation of Thr-169 is not required for exit from mitosis but may be necessary for commitment to the subsequent division cycle.     

Genetic and biochemical evaluation of the importance of Cdc6 in regulating mitotic exit

We evaluated the hypothesis that the N-terminal region of the replication control protein Cdc6 acts as an inhibitor of cyclin-dependent kinase (Cdk) activity, promoting mitotic exit. Cdc6 accumulation is restricted to the period from mid-cell cycle until the succeeding G1, due to proteolytic control that requires the Cdc6 N-terminal region. During late mitosis, Cdc6 is present at levels comparable with Sic1 and binds specifically to the mitotic cyclin Clb2. Moderate overexpression of Cdc6 promotes viability of CLB2Deltadb strains, which otherwise arrest at mitotic exit, and rescue is dependent on the N-terminal putative Cdk-inhibitory domain. These observations support the potential for Cdc6 to inhibit Clb2-Cdk, thus promoting mitotic exit. Consistent with this idea, we observed a cytokinesis defect in cdh1Delta sic1Delta cdc6Delta2-49 triple mutants. However, we were able to construct viable strains, in three different backgrounds, containing neither SIC1 nor the Cdc6 Cdk-inhibitory domain, in contradiction to previous work. We conclude, therefore, that although both Cdc6 and Sic1 have the potential to facilitate mitotic exit by inhibiting Clb2-Cdk, mitotic exit nevertheless does not require any identified stoichiometric inhibitor of Cdk activity.     

Cdk and APC activities limit the spindle-stabilizing function of Fin1 to anaphase

The fidelity of chromosome segregation depends on proper regulation of mitotic spindle behaviour. In anaphase, spindle stability is promoted by the dephosphorylation of cyclin-dependent kinase (Cdk) substrates, which results from Cdk inactivation and phosphatase activation. Few of the critical Cdk targets have been identified. Here, we identify the budding-yeast protein Fin1 (ref. 7) as a spindle-stabilizing protein whose activity is strictly limited to anaphase by changes in its phosphorylation state and rate of degradation. Phosphorylation of Fin1 from S phase to metaphase, by the cyclin-dependent kinase Clb5-Cdk1, inhibits Fin1 association with the spindle. In anaphase, when Clb5-Cdk1 is inactivated, Fin1 is dephosphorylated by the phosphatase Cdc14. Fin1 dephosphorylation targets it to the poles and microtubules of the elongating spindle, where it contributes to spindle integrity. A non-phosphorylatable Fin1 mutant localizes to the spindle before anaphase and impairs efficient chromosome segregation. As cells complete mitosis and disassemble the spindle, the ubiqutin ligase APC(Cdh1) targets Fin1 for destruction. Our studies illustrate how phosphorylation-dependent changes in the behaviour of Cdk1 substrates influence complex mitotic processes.     

The Bfa1/Bub2 GAP complex comprises a universal checkpoint required to prevent mitotic exit

At the end of the cell cycle, cyclin-dependent kinase (CDK) activity is inactivated to allow mitotic exit [1]. A protein phosphatase, Cdc14, plays a key role during mitotic exit in budding yeast by activating the Cdh1 component of the anaphase-promoting complex to degrade cyclin B (Clb) and inducing the CDK inhibitor Sic1 to inactivate Cdk1 [2]. To prevent mitotic exit when the cell cycle is arrested at G2/M, cells must prevent CDK inactivation. In the spindle checkpoint pathway, this is accomplished through Bfa1/Bub2, a heteromeric GTPase-activating protein (GAP) that inhibits Clb degradation by keeping the G protein Tem1 inactive [3-5]. Tem1 is required for Cdc14 activation. Here we show that in budding yeast, BUB2 and BFA1 are also required for the maintenance of G2/M arrest in response to DNA damage and to spindle misorientation. cdc13-1 bub2 and cdc13-1 bfa1 but not cdc13-1 mad2 double mutants rebud and reduplicate their DNA at the restrictive temperature. We also found that the delay in mitotic exit in mutants with misoriented spindles depended on BUB2 and BFA1, but not on MAD2. We propose that Bfa1/Bub2 checkpoint pathway functions as a universal checkpoint in G2/M that prevents CDK inactivation in response to cell-cycle delay in G2/M.     

KEN-box-dependent degradation of the Bub1 spindle checkpoint kinase by the anaphase-promoting complex/cyclosome

The spindle checkpoint is a cell cycle surveillance mechanism that ensures the fidelity of chromosome segregation during mitosis and meiosis. Bub1 is a protein serine-threonine kinase that plays multiple roles in chromosome segregation and the spindle checkpoint. In response to misaligned chromosomes, Bub1 directly inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) by phosphorylating its activator Cdc20. The protein level and the kinase activity of Bub1 are regulated during the cell cycle; they peak in mitosis and are low in G1/S phase. Here we show that Bub1 is degraded during mitotic exit and that degradation of Bub1 is mediated by APC/C in complex with its activator Cdh1 (APC/C(Cdh1)). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Bub1, whereas depletion of Cdh1 by RNA interference increases the level of the endogenous Bub1 protein. Bub1 is ubiquitinated by immunopurified APC/C(Cdh1) in vitro. We further identify two KEN-box motifs on Bub1 that are required for its degradation in vivo and ubiquitination in vitro. A Bub1 mutant protein with both KEN-boxes mutated is stable in cells but fails to elicit a cell cycle phenotype, indicating that degradation of Bub1 by APC/C(Cdh1) is not required for mitotic exit. Nevertheless, our study clearly demonstrates that Bub1, an APC/C inhibitor, is also an APC/C substrate. The antagonistic relationship between Bub1 and APC/C may help to prevent the premature accumulation of Bub1 during G1.     

Adaptation to the spindle checkpoint is regulated by the interplay between Cdc28/Clbs and PP2ACdc55

The spindle checkpoint arrests cells in metaphase until all chromosomes are properly attached to the chromosome segregation machinery. Thereafter, the anaphase promoting complex (APC/C) is activated and chromosome segregation can take place. Cells remain arrested in mitosis for hours in response to checkpoint activation, but not indefinitely. Eventually, they adapt to the checkpoint and proceed along the cell cycle. In yeast, adaptation requires the phosphorylation of APC/C. Here, we show that the protein phosphatase PP2A^Cdc55 dephosphorylates APC/C, thereby counteracting the activity of the mitotic kinase Cdc28. We also observe that the key regulator of Cdc28, the mitotic cyclin Clb2, increases before cells adapt and is then abruptly degraded at adaptation. Adaptation is highly asynchronous and takes place over a range of several hours. Our data suggest the presence of a double negative loop between PP2A^Cdc55 and APC/C^Cdc20 (i.e., a positive feedback loop) that controls APC/C^Cdc20 activity. The circuit could guarantee sustained APC/C^Cdc20 activity after Clb2 starts to be degraded.     

The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae.

Proteolysis mediated by the anaphase promoting complex (APC) has a crucial role in regulating the passage of cells through anaphase. Destruction of the anaphase inhibitor Pds1p is necessary for separation of sister chromatids, whereas destruction of the mitotic cyclin Clb2p is important for disassembly of the mitotic spindle, cytokinesis and re-replication of the genome. Pds1p proteolysis precedes that of Clb2p by at least 15 min, which helps to ensure that cells never re-replicate their genome before they have separated sister chromatids at the previous mitosis. What triggers Pds1p proteolysis and why does it not also trigger that of Clb2p? Apart from sharing a dependence on the APC, these two proteolytic events differ in their dependence on other cofactors. Pds1p proteolysis depends on a WD-repeat protein called Cdc20p, whereas Clb2p proteolysis depends on another, related WD protein called Hct1/Cdh1p. On the other hand, destruction of Clb2p, but not that of Pds1p, depends on the Polo-like kinase, Cdc5p. Cdc20p is essential for separation of sister chromatids, whereas Cdc5p is not. We show that both Cdc5p and Cdc20p are unstable proteins whose proteolysis is regulated by the APC. Both proteins accumulate during late G2/M phase and disappear at a late stage of anaphase. Accumulation of Cdc20p contributes to activation of Pds1p proteolysis in metaphase, whereas accumulation of Cdc5p facilitates the activation of Clb2p proteolysis.     

Budding yeast RSI1/APC2, a novel gene necessary for initiation of anaphase, encodes an APC subunit.

SIC1 is a non-essential gene encoding a CDK inhibitor of Cdc28-Clb kinase activity. Sic1p is involved in both mitotic exit and the timing of DNA synthesis. To identify other genes involved in controlling Clb-kinase activity, we have undertaken a genetic screen for mutations which render SIC1 essential. Here we describe a gene we have identified by this means, RSI1/APC2. Temperature-sensitive rsi1/apc2 mutants arrest in metaphase and are unable to degrade Clb2p, suggesting that Rsi1p/Apc2p is associated with the anaphase promoting complex (APC). This is an E3 ubiquitin-ligase that controls anaphase initiation through degradation of Pds1p and mitotic exit via degradation of Clb cyclins. Indeed, the anaphase block in rsi1/apc2 temperature-sensitive mutants is overcome by removal of PDS1, consistent with Rsi1p/Apc2p being part of the APC. In addition, like our rsi1/apc2 mutations, cdc23-1, encoding a known APC subunit, is also lethal with sic1Delta. Thus SIC1 clearly becomes essential when APC function is compromised. Finally, we find that Rsi1p/Apc2p co-immunoprecipitates with Cdc23p. Taken together, our results suggest that RSI1/APC2 is a subunit of APC.     

New insights into the regulation of anaphase by mitotic cyclins in budding yeast

Mitotic cyclins drive initiation and progression through mitosis. However, their role during progression remains poorly understood due to their essential function in initiation of mitosis and redundant activities. The function of the principal mitotic cyclin, Clb2, in S. cerevisiae, was investigated during progression through anaphase in diploid cells after DNA damage and during normal growth using fixed and live cell fluorescence techniques. I find that during anaphase, absence of Clb2 affects chromosome movement and plays an important role in inhibiting kinetochore microtubules regrowth. In addition, absence of Clb2 leads to defects and the collapse of spindle pole body separation. Most unexpectedly, new bipolar spindle forms and spindle re-forms. The intensity of the defects appears to correlate with strength of checkpoint activation, and during adaptation to DNA damage, these defects lead to important chromosome missegregation, during normal growth, defects are resolved rapidly. During recovery, intermediate phenotypes are observed. Altogether, data reveal new and unexpected roles for mitotic cyclins during progression through mitosis; results indicate that mitotic cyclins play key role in growth suppression of kinetochore microtubules and suggest that new bipolar spindle formation might be actively inhibited by mitotic cyclins during anaphase.     

NAP1 acts with Clb1 to perform mitotic functions and to suppress polar bud growth in budding yeast

NAP1 is a 60-kD protein that interacts specifically with mitotic cyclins in budding yeast and frogs. We have examined the ability of the yeast mitotic cyclin Clb2 to function in cells that lack NAP1. Our results demonstrate that Clb2 is unable to carry out its full range of functions without NAP1, even though Clb2/p34CDC28-associated kinase activity rises to normal levels. In the absence of NAP1, Clb2 is unable to efficiently induce mitotic events, and cells undergo a prolonged delay at the short spindle stage with normal levels of Clb2/p34CDC28 kinase activity. NAP1 is also required for the ability of Clb2 to induce the switch from polar to isotropic bud growth. As a result, polar bud growth continues during mitosis, giving rise to highly elongated cells. Our experiments also suggest that NAP1 is required for the ability of the Clb2/p34CDC28 kinase complex to amplify its own production, and that NAP1 plays a role in regulation of microtubule dynamics during mitosis. Together, these results demonstrate that NAP1 is required for the normal function of the activated Clb2/p34CDC28 kinase complex, and provide a step towards understanding how cyclin- dependent kinase complexes induce specific events during the cell cycle.     

Human T-lymphotropic virus type 1 oncoprotein tax promotes unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1 and causes chromosomal instability

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. The HTLV-1 transactivator, Tax, is implicated as the viral oncoprotein. Na?ve cells expressing Tax for the first time develop severe cell cycle abnormalities that include increased DNA synthesis, mitotic arrest, appearance of convoluted nuclei with decondensed DNA, and formation of multinucleated cells. Here we report that Tax causes a drastic reduction in Pds1p/securin and Clb2p/cyclin B levels in yeast, rodent, and human cells and a loss of cell viability. With a temperature-sensitive mutant of the CDC23 subunit of the anaphase-promoting complex (APC), cdc23(ts); a temperature-sensitive mutant of cdc20; and a cdh1-null mutant, we show that the diminution of Pds1p and Clb2p brought on by Tax is mediated via the Cdc20p-associated anaphase-promoting complex, APC(Cdc20p). This loss of Pds1p/securin and Clb2p/cyclin B1 occurred before cellular entry into mitosis, caused a G(2)/M cell cycle block, and was accompanied by severe chromosome aneuploidy in both Saccharomyces cerevisiae cells and human diploid fibroblasts. Our results support the notion that Tax aberrantly targets and activates APC(Cdc20p), leading to unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1, a delay or failure in mitotic entry and progression, and faulty chromosome transmission. The chromosomal instability resulting from a Tax-induced deficiency in securin and cyclin B1 provides an explanation for the highly aneuploid nature of adult T-cell leukemia cells.     

Phosphorylation and dephosphorylation regulate APC/CCdh1 substrate degradation

The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C^Cdh1 mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1^m11 mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/C^Cdh1 substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation.     

Enhanced Cell Polarity in Mutants of the Budding Yeast Cyclin-dependent Kinase Cdc28p

The yeast cyclin-dependent kinase Cdc28p regulates bud morphogenesis and cell cycle progression via the antagonistic activities of Cln and Clb cyclins. Cln G1 cyclins direct polarized growth and bud emergence, whereas Clb G2 cyclins promote isotropic growth of the bud and chromosome segregation. Using colony morphology as a screen to dissect regulation of polarity by Cdc28p, we identified nine point mutations that block the apical-isotropic switch while maintaining other functions. Like a clb2 Delta mutation, each confers tubular bud shape, apically polarized actin distribution, unipolar budding, and delayed anaphase. The mutations are all suppressed by CLB2 overexpression and are synthetically lethal with a CLB2 deletion. However, defects in multiple independent pathways may underlie their common phenotype, because the mutations are scattered throughout the CDC28 sequence, complement each other, and confer diverse biochemical properties. Glu12Gly, a mutation that alters a residue involved in Swe1p inhibition of Cdc28p, was unique in being suppressed by deficiency of SWE1 or CLN1. With wild-type CDC28, filament formation induced by CLN1 overexpression was markedly decreased in a SWE1 deletion. These results suggest that Swe1p, via inhibition of Clb2p/Cdc28p, may mediate much of the effect of Cln1p on filamentous morphogenesis.     

Regulation of the anaphase-promoting complex by the dual specificity phosphatase human Cdc14a

Two forms of the anaphase-promoting complex (APC) mediate the degradation of critical cell cycle regulators. APC(Cdc20) promotes sister-chromatid separation by ubiquitinating securin, whereas APC(Cdh1) ubiquitinates mitotic cyclins, allowing the exit from mitosis. Here we show that phosphorylation of human Cdh1 (hCdh1) by cyclin B-Cdc2 alters the conformation of hCdh1 and prevents it from activating APC. A human homologue of yeast Cdc14, human Cdc14a (hCdc14a), dephosphorylates hCdh1 and activates APC(Cdh1). In contrast, hCdc14a does not affect the activity of APC(Cdc20). hCdc14a is a major phosphatase for hCdh1 and localizes to centrosomes in HeLa cells. Therefore, hCdc14a may promote the activation of APC(Cdh1) and exit from mitosis in mammalian cells.     

Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins

Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.