CDK-dependent phosphorylation of Mob2 is essential for hyphal development in Candida albicans

Nuclear Dbf2-related (NDR) protein kinases are essential components of regulatory pathways involved in cell morphogenesis, cell cycle control, and viability in eukaryotic cells. For their activity and function, these kinases require interaction with Mob proteins. However, little is known about how the Mob proteins are regulated. In Candida albicans, the cyclin-dependent kinase (CDK) Cdc28 and the NDR kinase Cbk1 are required for hyphal growth. Here we demonstrate that Mob2, the Cbk1 activator, undergoes a Cdc28-dependent differential phosphorylation on hyphal induction. Mutations in the four CDK consensus sites in Mob2 to Ala significantly impaired hyphal development. The mutant cells produced short hyphae with enlarged tips that displayed an illicit activation of cell separation. We also show that Cdc28 phosphorylation of Mob2 is essential for the maintenance of polarisome components at hyphal tips but not at bud tips during yeast growth. Thus we have found a novel signaling pathway by which Cdc28 controls Cbk1 through the regulatory phosphorylation of Mob2, which is crucial for normal hyphal development.     

CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth

Polarisome is a protein complex that plays an important role in polarized growth in fungi by assembling actin cables towards the site of cell growth. For proper morphogenesis, the polarisome must localize to the right place at the right time. However, the mechanisms that control polarisome localization remain poorly understood. In this study, using the polymorphic fungus Candida albicans as a model, we have discovered that the cyclin‐dependent kinase (CDK) Cdc28 phosphorylates the polarisome scaffold protein Spa2 to govern polarisome localization during both yeast and hyphal growth. In a yeast cell cycle, Cdc28‐Clb2 phosphorylates Spa2 and controls the timing of polarisome translocation from the bud tip to the bud neck. And during hyphal development, Cdc28‐Clb2 and the hyphal‐specific Cdc28‐Hgc1 cooperate to enhance Spa2 phosphorylation to maintain the polarisome at the hyphal tip. Blocking the CDK phosphorylation causes premature tip‐to‐neck translocation of Spa2 during yeast growth and inappropriate septal localization of Spa2 in hyphae and abnormal hyphal morphology under certain inducing conditions. Together, our results generate new insights into the mechanisms by which fungi regulate polarisome localization in the control of polarized growth.     

Sep7 is essential to modify septin ring dynamics and inhibit cell separation during Candida albicans hyphal growth

When Candida albicans yeast cells receive the appropriate stimulus, they switch to hyphal growth, characterized by continuous apical elongation and the inhibition of cell separation. The molecular basis of this inhibition is poorly known, despite its crucial importance for hyphal development. In C. albicans, septins are important for hypha formation and virulence. Here, we used fluorescence recovery after photobleaching analysis to characterize the dynamics of septin rings during yeast and hyphal growth. On hyphal induction, septin rings are converted to a hyphal-specific state, characterized by the presence of a frozen core formed by Sep7/Shs1, Cdc3 and Cdc12, whereas Cdc10 is highly dynamic and oscillates between the ring and the cytoplasm. Conversion of septin rings to the hyphal-specific state inhibits the translocation of Cdc14 phosphatase, which controls cell separation, to the hyphal septum. Modification of septin ring dynamics during hyphal growth is dependent on Sep7 and the hyphal-specific cyclin Hgc1, which partially controls Sep7 phosphorylation status and protein levels. Our results reveal a link between the cell cycle machinery and septin cytoskeleton dynamics, which inhibits cell separation in the filaments and is essential for hyphal morphogenesis.     

Cell Cycle Regulated Interaction of a Yeast Hippo Kinase and Its Activator MO25/Hym1

Hippo pathways are ancient signaling systems that contribute to cell growth and proliferation in a wide diversity of eukaryotes, and have emerged as a conserved regulator of organ size control in metazoans. In budding yeast, a Hippo signaling pathway called the Regulation of Ace2 and Morphogenesis (RAM) network promotes polarized cell growth and the final event in the separation of mother and daughter cells. A crucial regulatory input for RAM network control of cell separation is phosphorylation of a conserved hydrophobic motif (HM) site on the NDR/LATS family kinase Cbk1. Here we provide the first direct evidence that the Hippo-like kinase Kic1 in fact phosphorylates the HM site of Cbk1, and show that Kic1 is allosterically activated by Hym1, a highly conserved protein related to mammalian MO25. Using the structure of mammalian MO25 in complex with the Kic1-related pseudokinase STRAD, we identified conserved residues on Kic1 that are required for interaction with Hym1. We find that Kic1 and Hym1 protein levels remain constant throughout the cell cycle but the proteins’ association is regulated, with maximal interaction coinciding with peak Cbk1 HM site phosphorylation. We show that this association is necessary but not sufficient for this phosphorylation, suggesting another level of regulation is required to promote the complex to act upon its substrates. This work presents a previously undiscovered cell cycle regulated interaction between a Hippo kinase and a broadly conserved allosteric activator. Because of the conserved nature of this pathway in higher eukaryotes, this work may also provide insight into the modularity of Hippo signaling pathways.     

Cyclin-dependent kinases control septin phosphorylation in Candida albicans hyphal development

Cyclin-dependent kinases (Cdks) control cytoskeleton polarization in yeast morphogenesis. However, the target and mechanism remain unclear. Here, we show that the Candida albicans Cdk Cdc28, through temporally controlled association with two cyclins Ccn1 and Hgc1, rapidly establishes and persistently maintains phosphorylation of the septin cytoskeleton protein Cdc11 for hyphal development. Upon hyphal induction, Cdc28-Ccn1 binds to septin complexes and phosphorylates Cdc11 on Ser394, a nonconsensus Cdk target. This phosphorylation requires prior phosphorylation on Ser395 by the septin-associated kinase Gin4. Mutating Ser394 or Ser395 blocked Cdc11 phosphorylation on Ser394 and impaired hyphal morphogenesis. Reconstitution experiments using purified Cdc28-Ccn1, Gin4, and septins reproduced phosphorylations on the same residues. Transient septin-Cdc28 associations were also detected prior to bud and mating-projection emergence in S. cerevisiae. Our study uncovers a direct link between the cell-cycle engine and the septin cytoskeleton that may be part of a conserved mechanism underlying polarized morphogenesis.     

Phosphorylation of Rga2, a Cdc42 GAP, by CDK/Hgc1 is crucial for Candida albicans hyphal growth

Cyclin-dependent kinases (CDKs) control yeast morphogenesis, although how they regulate the polarity machinery remains unclear. The dimorphic fungus Candida albicans uses Cdc28/Hgc1, a CDK/cyclin complex, to promote persistent actin polarization for hyphal growth. Here, we report that Rga2, a GTPase-activating protein (GAP) of the central polarity regulator Cdc42, undergoes Hgc1-dependent hyperphosphorylation. Using the analog-sensitive Cdc28as mutant, we confirmed that Cdc28 controls Rga2 phosphorylation in vitro and in vivo. Deleting RGA2 produced elongated yeast cells without apparent effect on hyphal morphogenesis. However, deleting it or inactivating its GAP activity restored hyphal growth in hgc1Delta mutants, suggesting that Rga2 represses hyphal development and Cdc28/Hgc1 inactivates it upon hyphal induction. We provide evidence that Cdc28/Hgc1 may act to prevent Rga2 from localizing to hyphal tips, leading to localized Cdc42 activation for hyphal extension. Rga2 also undergoes transient Cdc28-dependent hyperphosphorylation at bud emergence, suggesting that regulating a GAP(s) of Cdc42 by CDKs may play an important role in governing different forms of polarized morphogenesis in yeast. This study reveals a direct molecular link between CDKs and the polarity machinery.     

Depletion of a polo-like kinase in Candida albicans activates cyclase-dependent hyphal-like growth

Morphogenesis in the fungal pathogen Candida albicans is an important virulence-determining factor, as a dimorphic switch between yeast and hyphal growth forms can increase pathogenesis. We identified CaCDC5, a cell cycle regulatory polo-like kinase (PLK) in C. albicans and demonstrate that shutting off its expression induced cell cycle defects and dramatic changes in morphology. Cells lacking CaCdc5p were blocked early in nuclear division with very short spindles and unseparated chromatin. GFP-tagged CaCdc5p localized to unseparated spindle pole bodies, the spindle, and chromatin, consistent with a role in spindle elongation at an earlier point in the cell cycle than that described for the homologue Cdc5p in yeast. Strikingly, the cell cycle defects were accompanied by the formation of hyphal-like filaments under yeast growth conditions. Filament growth was determinate, as the filaments started to die after 24 h. The filaments resembled serum-induced hyphae with respect to morphology, organization of cytoplasmic microtubules, localization of nuclei, and expression of hyphal-specific components. Filament formation required CaCDC35, but not EFG1 or CPH1. Similar defects in spindle elongation and a corresponding induction of filaments occurred when yeast cells were exposed to hydroxyurea. Because CaCdc5p does not appear to act as a direct repressor of hyphal growth, the data suggest that a target of CaCdc5p function is associated with hyphal-like development. Thus, an internal, cell cycle-related cue can activate hyphal regulatory networks in Candida.     

A cell cycle checkpoint monitors cell morphogenesis in budding yeast

Checkpoint controls are regulatory pathways that inhibit cell cycle progression in cells that have not faithfully completed a prior step in the cell cycle. In the budding yeast Saccharomyces cerevisiae, DNA replication and spindle assembly are monitored by checkpoint controls that prevent nuclear division in cells that have failed to complete these processes. During the normal cell cycle, bud formation is temporally coincident with DNA replication and spindle assembly, and the nucleus divides along the mother-bud axis in mitosis. In this report, we show that inhibition of bud formation also causes a dramatic delay in nuclear division. This allows cells to recover from a transient disruption of cell polarity without becoming binucleate. The delay occurs after DNA replication and spindle assembly, and results from delayed activation of the master cell cycle regulatory kinase, Cdc28. Cdc28 activation is inhibited by phosphorylation of Cdc28 on tyrosine 19, and by delayed accumulation of the B-type cyclins Clb1 and Clb2. These results suggest the existence of a novel checkpoint that monitors cell morphogenesis in budding yeast.     

Hyphal growth in Candida albicans requires the phosphorylation of Sec2 by the Cdc28-Ccn1/Hgc1 kinase

Polarized growth is a fundamental property of cell growth and development. It requires the delivery of post‐Golgi secretory vesicles to the site of polarized growth. This process is mediated by Rab GTPases activated by their guanine exchange factors (GEFs). The human fungal pathogen, Candida albicans, can grow in a budded yeast form or in a highly polarized hyphal form, and thus provides a model to study this phenomenon. During hyphal, but not yeast growth, secretory vesicles accumulate in an apical body called a Spitzenkörper, which acts to focus delivery of the vesicles to the tip. Post‐Golgi transport of secretory vesicles is mediated by the Rab GTPase Sec4, activated by its GEF Sec2. Using a combination of deletion mapping, in vitro mutagenesis, an analogue‐sensitive allele of Cdc28 and an in vitro kinase assay, we show that localization of Sec2 to the Spitzenkörper and normal hyphal development requires phosphorylation of Serine 584 by the cyclin‐dependent kinase Cdc28. Thus, as well as controlling passage through the cell cycle, Cdc28 has an important function in controlling polarized secretion.     

Hyphal Elongation Is Regulated Independently of Cell Cycle in Candida albicans

The mechanism for apical growth during hyphal morphogenesis in Candida albicans is unknown. Studies from Saccharomyces cerevisiae indicate that cell morphogenesis may involve cell cycle regulation by cyclin-dependent kinase. To examine whether this is the mechanism for hyphal morphogenesis, the temporal appearance of different spindle pole body and spindle structures, the cell cycle-regulated rearrangements of the actin cytoskeleton, and the phosphorylation state of the conserved Tyr19 of Cdc28 during the cell cycle were compared and found to be similar between yeast and serum-induced hyphal apical cells. These data suggest that hyphal elongation is not mediated by altering cell cycle progression or through phosphorylation of Tyr19 of Cdc28. We have also shown that germ tubes can evaginate before spindle pole body duplication, chitin ring formation, and DNA replication. Similarly, tip-associated actin polarization in each hypha occurs before the events of the G(1)/S transition and persists throughout the cell cycle, whereas cell cycle-regulated actin assemblies come and go. We have also shown that cells in phases other than G(1) can be induced to form hyphae. Hyphae induced from G(1) cells have no constrictions, and the first chitin ring is positioned in the germ tube at various distances from the base. Hyphae induced from budded cells have a constriction and a chitin ring at the bud neck, beyond which the hyphae continue to elongate with no further constrictions. Our data suggest that hyphal elongation and cell cycle morphogenesis programs are uncoupled, and each contributes to different aspects of cell morphogenesis.     

Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p

In budding yeast cells, the cytoskeletal polarization and depolarization events that shape the bud are triggered at specific times during the cell cycle by the cyclin-dependent kinase Cdc28p. Polarity establishment also requires the small GTPase Cdc42p and its exchange factor, Cdc24p, but the mechanism whereby Cdc28p induces Cdc42p-dependent polarization is unknown. Here we show that Cdc24p becomes phosphorylated in a cell cycle-dependent manner, triggered by Cdc28p. However, the role of Cdc28p is indirect, and the phosphorylation appears to be catalyzed by the p21-activated kinase family member Cla4p and also depends on Cdc42p and the scaffold protein Bem1p. Expression of GTP-Cdc42p, the product of Cdc24p-mediated GDP/GTP exchange, stimulated Cdc24p phosphorylation independent of cell cycle cues, raising the possibility that the phosphorylation is part of a feedback regulatory pathway. Bem1p binds directly to Cdc24p, to Cla4p, and to GTP-bound Cdc42p and can mediate complex formation between these proteins in vitro. We suggest that Bem1p acts to concentrate polarity establishment proteins at a discrete site, facilitating polarization and promoting Cdc24p phosphorylation at specific times during the cell cycle.     

Cdc28 and Cdc14 control stability of the anaphase-promoting complex inhibitor Acm1

The anaphase-promoting complex (APC) regulates the eukaryotic cell cycle by targeting specific proteins for proteasomal degradation. Its activity must be strictly controlled to ensure proper cell cycle progression. The co-activator proteins Cdc20 and Cdh1 are required for APC activity and are important regulatory targets. Recently, budding yeast Acm1 was identified as a Cdh1 binding partner and APC(Cdh1) inhibitor. Acm1 disappears in late mitosis when APC(Cdh1) becomes active and contains conserved degron-like sequences common to APC substrates, suggesting it could be both an inhibitor and substrate. Surprisingly, we found that Acm1 proteolysis is independent of APC. A major determinant of Acm1 stability is phosphorylation at consensus cyclin-dependent kinase sites. Acm1 is a substrate of Cdc28 cyclin-dependent kinase and Cdc14 phosphatase both in vivo and in vitro. Mutation of Cdc28 phosphorylation sites or conditional inactivation of Cdc28 destabilizes Acm1. In contrast, inactivation of Cdc14 prevents Acm1 dephosphorylation and proteolysis. Cdc28 stabilizes Acm1 in part by promoting binding of the 14-3-3 proteins Bmh1 and Bmh2. We conclude that the opposing actions of Cdc28 and Cdc14 are primary factors limiting Acm1 to the interval from G(1)/S to late mitosis and are capable of establishing APC-independent expression patterns similar to APC substrates.