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Contents
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Integration of Cell Cycle Signals by Swe1/Wee1 in Budding Yeast
Recent studies in budding yeast revealed that these cells use the level of Swe1 (the Wee1 ortholog in this organism) to keep track of cell cycle progression and determine the timing of entry into mitosis. Swe1 is negatively regulated by a complex signaling network that relays upstream signals during assembly of the filamentous septin collar at the bud-neck, suggesting that Swe1 functions as a nodal point for integrating multiple signals that license passage into mitosis. During an unperturbed cell cycle, Swe1 localizes to the interphase nucleus and to the bud-neck at the late stages of the cell cycle. Swe1 begins to accumulate in S phase and becomes cumulatively phosphorylated as cells proceed through the cell cycle. These phosphorylation events tag Swe1 for ubiquitin-dependent degradation, which in turn obliterates Swe1’s inhibitory phosphorylation of the cyclin-dependent Cdc28 (Cdk1 homolog). Interestingly, disruption of the septin collar at the bud-neck, by using temperature-sensitive septin mutants, or loss of the septin collar–associated kinase Hsl1 (Nim1 homolog) or its adaptor Hsl7 delocalizes Swe1 from the bud-neck. The resulting hypo-phosphorylated and stabilized Swe1 imposes a G2 delay. These findings suggest that Swe1 degradation requires components associated with septin filaments and that regulation of Swe1 is critical for coordinating morphogenetic events with the commencement of mitosis. An initial search for the kinases responsible for Swe1 phosphorylation identified two bud-neck–associated kinases, Cla4 (PAK homolog) and Cdc5 (Polo kinase homolog), which appear to phosphorylate Swe1 in a temporally regulated manner (Sakchaisri K et al. Proc Natl Acad Sci U S A 101: 4124–9, 2004). In an early stage of the cell cycle, Cla4 phosphorylates Swe1 at the bud-neck (Figure 1), by associating tightly with septins, and promotes septin filament assembly. Cdc5 localizes to the bud-neck and hyper-phosphorylates Swe1 late in the cell cycle (Figure 1). Subsequent studies showed that mitotic cyclin (Clb2)–associated Cdc28 also functions in a positive feedback loop to downregulate Swe1. Further biochemical analyses revealed that Clb2-Cdc28 promotes Cdc5-dependent Swe1 phosphorylation and degradation by generating a phospho-recognition motif that enhances the interaction between Swe1 and the non-catalytic polo-box domain of Cdc5 (Asano S et al. EMBO J 24: 2194–204, 2005) (Figure 1). Septin filament assembly is dependent on Cla4 function and is required for localization of Hsl1 and Hsl7, which are, in turn, required to ensure proximity between Cdc5 and Swe1. Therefore, downregulation of Swe1 is achieved through the coordination of both septin-collar formation and stepwise phosphorylation of Swe1 by multiple kinases. Analogous interactions between the polo-box domain of mammalian polo-like kinase Plk1 and its phosphorylated substrates by Cdk1 have been previously reported (Lowery DM et al. Oncogene 24: 248–59, 2005). Thus, the concerted action of Cdc28/Cdk1 and Cdc5/Polo on their common substrates appears to be an evolutionarily conserved mechanism that is designed to effectively bring about various mitotic events. Figure 1. Model illustrating multi-kinase–dependent Swe1 phosphorylation and degradation. In an early stage of the cell cycle, Cla4 phosphorylates Swe1 at the bud-neck, but this phosphorylation is not sufficient to trigger Swe1 degradation. Later in the cell cycle, such as in G2, Swe1 is moderately phosphorylated by nascent Clb2-Cdc28 activity, as the level of Clb2 rises in the nucleus, and this promotes Cdc5-dependent Swe1 hyper-phosphorylation and, subsequently, Swe1 degradation. Once unleashed from negative regulation by Swe1, the Clb2-Cdc28 complex becomes fully active and induces mitosis. Multi-kinase phosphorylation of substrates has recently been recognized as a means of regulating diverse cellular functions. The Cla4/Cdc28/Cdc5–dependent Swe1 regulation described above highlights how yeast cells orchestrate various cellular and biochemical events to ensure completion of earlier events prior to mitotic entry. Moreover, stepwise Swe1 phosphorylation that impinges on ordered assemblies of septin filaments and the Hsl1-Hsl7 platform at the bud-neck provides a mechanism of integrating temporally distinct signals from preceding cell cycle events into one protein that plays a critical role in modulating the timing of mitosis. Whether the mechanism of Swe1 regulation is uniquely evolved in budding yeast to accurately coordinate morphogenesis throughout the budding process or if analogous mechanisms exist in other eukaryotic organisms are intriguing questions that remain to be answered.
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itosis comprises a series of biochemical steps and coordinated cellular events that ensure faithful partitioning of genetic and cytoplasmic components. Failure in these processes leads to genomic instability and cell death. In higher eukaryotes and fission yeast, entry into mitosis is induced by the activation of cyclin B–bound Cdk1 (Harper JW and Adams PD. Chem Rev 101: 2511–26, 2001), which is held in check by the protein kinase Wee1 (Russell P and Nurse P. Cell 49: 559–67, 1987). Wee1 is, in turn, negatively regulated via phosphorylation by Nim1/Cdr1 (Russell P and Nurse P. Cell 49: 569–76, 1987). These regulatory steps appear to have been conserved throughout evolution.