ControL of the G2 Phase by SAPKs

G2 transition depends on the activity of CDK associated with mitotic cyclins. Wee kinases are negative regulators of such activity and control the length of G2 phase under specific conditions. In S. pombe, the Wee1 kinase allows entry into mitosis when cells reach a minimum threshold size and, in S. cerevisiae, Swe1 activates the morphogenetic checkpoint. The physiological role of this later checkpoint is under discussion (Keaton and Lew, 2006; Kellogg, 2003; McNulty and Lew, 2005) and is not clear whether it responds to cytoskeletal perturbations or monitors the size of the bud like in S. pombe cells. The molecular basis of the morphogenetic checkpoint seems firmly established: the septin ring formed on the neck between mother and daughter cell recruits Hsl1 (a septin-dependent protein kinase), which interacts with Hsl7 (a protein that serves as a holder to recruit Swe1); this complex is necessary to localize Swe1 to the neck and target it for destruction, as well as to activate Cdc28/Clb2. When bud formation (or growth) is impaired, Swe1 remains active and cells do not progress into mitosis (McMillan et al., 1999).

Activation of Hog1 upon osmostress induces a delay in G2 by the decrease of Clb2/Cdc28 activity and by the downregulation of CLB2 transcription (Alexander et al., 2001; Clotet et al., 2006). The mechanisms that Hog1 uses to downregulate Clb2 transcription are not known, but this could be a secondary effect caused by the decrease on Clb2/Cdc28 activity. Instead, the mechanism required to decrease Clb2 activity seems clear: Hog1 is acting over the machinery of the morphogenetic checkpoint that controls Swe1 levels. Activated Hog1 interacts and directly phosphorylates Hsl1 in a residue within the Hsl7-docking site, which promotes the delo-calization of Hsl7 from the neck that results in Swe1 accumulation (Clotet et al., 2006). Upon Hog1 activation, cells containing a nonphosphorylatable Hsl1 are unable to promote Hsl7 delocalization, fail to accumulate Swe1, and arrest at G2. Thus they are sensitive to osmotic stress.

It is worth noting that the same machinery used by the cells to monitor morphogenetic defects can integrate other signals. Interestingly, whereas the morphogenetic checkpoint monitors the absence of a septin ring, which produces the absence of Hsll, Hsl7, and Swel at the bud neck, the osmocheckpoint specifically delocalizes Hsl7 without affecting Hsll or septins (Clotet et al., 2006).

In S. pombe, the Skbl protein has been described as the homologue of Hsl7 in S. cerevisiae. In response to osmostress, Skbl delocalizes from the cell ends and nuclei; in addition, skb1 deletion strains are osmosensitive (Bao et al., 200l). However, there is not a perfect homologue of Hsll, and the exact mechanism by which Skbl is delocalized and confers osmoresistence has not been described. Another important difference is the fact that Skbl has been described as a factor that regulates Weel positively. This is the opposite of what has been described in S. cerevisiae and mammalian cells in which it has been demonstrated that SkblHs plays a role in destabilizing Weel (Lew, 2003).

Another interesting mechanism to slow down G2 progression in response to osmotic stress consists of regulation of the Cdc25 phosphatase by Srkl (Lopez-Aviles et al., 2005). Cdc25 is the key factor governing the initiation of mitosis that reverses inhibition of the Cdc2—mitotic complex because of phosphorylation by the Weel kinase (Gould and Nurse, l989; Millar et al., l99l). Srkl is related to the mammalian MAPKAP kinases and its overexpression causes cell cycle arrest in late G2 phase. Srkl interacts with and phosphorylates Cdc25. After exposure of cells to KCl, Styl phosphorylates Srkl and the catalytic activity of Srkl increases more than fivefold. Phosphorylation of Cdc25 by Srkl causes Cdc25 to bind to Rad24, a l4-3-3 protein family member, which helps retain Cdc25 in the cytoplasm and protect it from degradation. These results suggest that Styl SAP kinase is the effector of a checkpoint pathway that controls Cdc25 activity in response to osmotic stress.

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