Regulation of G2M transition in budding yeast

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Entry into mitosis in eukaryotic organisms is regulated by an intricate network of kinases and phosphatases that coordinately bring about reorganization of various subcellular structures. A key regulatory component for this event is the conserved cyclin B-bound Cdc2. In fission yeast and higher eukaryotes, Cdc2 is phosphorylated at Tyr15 and negatively regulated by Wee1, an event that is reversed by the activity of Cdc25C phosphatase. These critical steps at mitotic entry appear to be largely conserved throughout evolution. Studies on the G2/M regulation in genetically-amenable organisms such as budding yeast have provided valuable insights into how eukaryotic organisms bring about timely activation of cyclin B-Cdc2 activity prior to mitotic entry. In budding yeast, Swe1 (Wee1 ortholog) negatively regulates mitotic Clb (collectively for the B-type cyclins - Clb1, Clb2, Clb3, and Clb4) associated-Cdc28 (Cdc2 homolog) by phosphorylating the equivalent Tyr19 residue, a modification that is reversed by Mih1 (Cdc25 ortholog). We previously showed that Cla4 (PAK homolog) and Cdc5 (Polo homolog) phosphorylate Swe1 in a step-wise manner. Our recent study found that Hsl1 (Nim1 ortholog) and Hsl7, which are critical for Swe1 localization to the bud-neck, are also required for proper localization of Cdc5 to the bud-neck and the Cdc5-dependent Swe1 phosphorylation. Mitotic Clb-bound Cdc28, but not G1 or S cyclin-bound Cdc28, directly phosphorylated Swe1 and this phosphorylation step appears to be important to prime Swe1 for the subsequent Cdc5-dependent Swe1 phosphorylation. We would like to further investigate the mechanism of how Hsl1 and Hsl7 cooperate with multi-kinases (Cla4, Cdc28, and Cdc5) to facilitate Swe1 hyperphosphorylation and subsequent degradation prior to mitotic entry. Our current model is that Swe1 functions as a nodal point to integrate multi-kinase-dependent signals that license passage into mitosis.In a separate study, we have been interested in understanding the regulation of one of the budding yeast Nim1-related kinases Gin4. Gin4 plays an important role in proper organization of septin ring at the mother-bud neck, a filamentous structure that is critical for diverse cellular processes including the regulation of mitotic entry and cytokinesis. Here we showed that a neck-associated Ser/Thr kinase Elm1, which is important for septin assembly, is critical for proper modification of Gin4 and its physiological substrate Shs1. Using purified recombinant proteins, we demonstrated that Elm1 directly phosphorylates and activates Gin4, which in turn phosphorylates Shs1. A Gin4 mutant lacking the Elm1-dependent phosphorylation sites appeared to be impaired in localization with a diminished kinase activity. Consistent with these observations, this mutant exhibited mild growth defect with frequently elongated bud morphology. Thus, we propose that Elm1 contributes to proper septin organization by directly regulating the Gin4-dependent Shs1 pathway. How the Elm1-Gin4-Shs1 pathway cooperates with other pathways leading to the regulation of septins and ultimately G2/M transition will be an intriguing question that requires further investigation.