Cla4-PAK Signaling in Budding Yeast
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Cla4-PAK Signaling in Budding Yeast

Simple eukaryotes such as yeasts and molds encode multiple PAKs (p21-Activated Kinases) that, like their orthologs in other systems, act downstream of Rho-family GTPases to regulate cytoskeletal structure and gene transcription. All PAKs contain an N-terminal PBD (p21 GTPase-Binding-Domain), which confers binding to small GTPases such as CDC42 (Cell Division Cycle-42) or Rac, and a C-terminal protein kinase domain (Ref.1). Saccharomyces cerevisiae cells contain three members of this family, Ste20, Cla4, and Skm1, each of which performs a distinct set of cellular roles.

S. cerevisiae Cla4 contains a CDC42-binding domain, a PH (Pleckstrin Homology) domain, and a serine/threonine kinase domain and is required for viability in the absence of the G1-Cyclins Cln1 and Cln2. Like Ste20, Cla4 is part of a multicomponent complex and can activate MAPK pathways by phosphorylating Ste11. Cla4 has unique functions in mitotic exit and in cytokinesis. Cla4 promotes normal Septin function and the subsequent regulation of polarized growth. Activation of CDC42 by its exchange factor CDC24 is required to organize the actin cytoskeleton towards the incipient bud site in S. cerevisiae or towards the pheromone-secreting partner during mating. Cytoskeletal polarization also requires Bem1, which functions as an adaptor for CDC42 and CDC24 (Ref.2). Cla4 is also required for the phosphorylation of GEF (Guanine-Nucleotide-Exchange Factor) LTE1. Ras2 is also essential for LTE1 localization, and active Ras2-GTP anchors LTE1 at the bud cortex via the direct interaction through the CHD (CDC25 Homology Domain) of LTE1. LTE1 is an activator of the GTPase Tem1 which, following inactivation of the CDC28-Clb (Cyclin-B) complex, promotes the release of cell cycle phosphatase CDC14 from the nucleolus. The budding yeast MEN (Mitotic Exit Network) control the exit from mitosis by facilitating the release of CDC14. CDC42, its GEF CDC24 and its effector Cla4 control the initial binding and activation of LTE1 to the bud cortex. Moreover, CDC24, CDC42 and Ste20, another PAK function parallel to LTE1 in facilitating mitotic exit. Tem1, CDC15, and other MEN proteins asymmetrically localize to the spindle poles. After spindle elongation into the bud, Tem1 is activated by the putative GEF-LTE1 at the bud cell cortex to promote CDC15 localization to the SPB (Spindle Pole Body), beginning the MEN signal cascade. In the absence of Bub2, Tem1 does not localize to the SPB until late anaphase and CDC15 localizes before anaphase resulting in inappropriate mitotic exit if spindle abnormalities exist (Ref.3&4).

During mitosis, S. cerevisiae expresses five septins: CDC3, CDC10, CDC11, CDC12, and Shs1/Sep7. Early in the cell cycle, Septins assemble into a patch at the incipient bud site. Among the proteins recruited to the daughter side are a protein-arginine methyltransferase, Hsl7, and protein kinases, Hsl1 and Swe1. Swe1 blocks mitotic entry by inhibiting Clb-bound CDC28 protein kinase. Swe1, in turn, is inactivated via concerted action of Hsl1 and Hsl7. Hsl1 and Hsl7 activity depends on their localization to properly assembled septins at the bud neck. If the collar is perturbed, Swe1 remains active, inhibits CDC28-Clb complexes, and prevents passage through G2/M, providing a mechanism to monitor the status of septin assembly and to retard cell cycle progression if there is a defect (Ref.5). Cln1, Cln2/CDC28 plays a direct role in initiating bud emergence, while CDC28-Clb2 requires the function of Bud2 to promote bud emergence. After bud emergence, Cla4 becomes activated by binding to GTP-CDC42, which, along with activated Ste20 and Gin4, may lead to cytokinesis. Myosins such as Myo3 and Myo5 are also are required for budding and are phosphorylated by Ste20 and Cla4. Furthermore, Cla4 also function late in the cell cycle because its kinase activity peaks near mitosis.