The protein kinase swe1: new players in its regulatory pathway and analysis of its involvement in mitotic spindle dynamics

Swe1 is the effector kinase of the morphogenesis checkpoint that, in budding yeast, provides a link between cell morphology and entry into mitosis. Although there are some differences due to the particular kind of cell division established by the budding, Swe1 functions and regulators are evolutionarily conserved, indicating that this is an ancient cell cycle control strategy that has been adapted to respond to cytoskeletal signals in vertebrate as well as in S. cerevisiae cells. Swe1 blocks entry into mitosis through inhibitory phosphorylation of the catalytic subunit of the cyclin-dependent kinase Cdk1, Cdc28, and this modification is reversed by the protein phosphatase Mih1. Cdc28 activity is required both for entry into mitosis and for the switch from polar to isotropic bud growth, so when Cdc28 is phosphorylated on Tyr19 (Y19) both these events are inhibited. Timely degradation of Swe1 is important for cell survival in case of DNA replication stress, while it is inhibited by the morphogenesis checkpoint in response to alterations in actin cytoskeleton or septins’ structure. We show here that the lack of the Dma1 and Dma2 ubiquitin ligases, which moderately affects Swe1 localization and degradation during an unperturbed cell cycle with no apparent phenotypic effects, is toxic for cells that are partially defective in Swe1 down-regulation. Interestingly Swe1 is stabilized, but differently from morphogenesis checkpoint activation, restrained at the bud neck and hyperphosphorylated in dma1∆ dma2∆ cells subjected to DNA replication stress, indicating that the mechanism stabilizing Swe1 under these conditions is different from the one triggered by the morphogenesis checkpoint. Finally, the Dma proteins are required for proper Swe1 ubiquitylation. Altogether, our data highlight a previously unknown role of these proteins in the complex regulation of Swe1 degradation and suggest that they might contribute to control, directly or indirectly, Swe1 ubiquitylation. As already said, Swe1 stabilization prevents mitotic entry in response to different problems. In addition, elevated Swe1 levels inhibits mitotic spindle formation and elongation and several data indicate that, apart Cdc28, other Swe1 targets are likely involved in this process. In fact, the expression of Cdc28 alleles that could escape from Swe1 inhibition is not sufficient neither to restore proper spindle elongation nor progression through mitosis of cells that overexpress Swe1. We tried to identify new Swe1 targets acting in mitotic spindle dynamics and progression through mitosis by performing a genetic screen and by analyzing putative candidates among factors known to be involved in these processes. About the genetic screen, we found twelve recessive spontaneous suppressors that are able to restore spindle elongation and viability of SWE1 overespressing cells that lack the APC regulatory subunit CDH1. Interestingly, the suppression phenotype is due to inactivation of the same gene in all the suppressors. We are now trying to identify this gene and to characterize the suppression mechanism. In parallel, we pursued the identification of Swe1 targets by analyzing spindle associated factors and proteins involved in spindle dynamycs. In particular, the MAP Bik1 was found in a proteome chip array as a protein phosphorylated by Swe1. We found that, differently from what is published, high Swe1 levels reduce Bik1 phosphorylation independently of Swe1 inhibitory activity on Cdc28. Further analysis will be required to better understand the molecular details of the indirect Swe1 action on Bik1 phosphorylation. In addition, we found that high levels of the Polo kinase CDC5 partially restore spindle elongation and viability of SWE1 overexpressing cells, but only in the presence of functional Mih1. In particular, high Swe1 levels cause the accumulation of fully phosphorylated Mih1, that is not functional, and CDC5 overexpression in these cells restores the proper balance between Mih1 phosphorylation forms, and so its functionality. Further analysis will be required to better understand the interaction between Swe1, Cdc5 and the protein phosphatase Mih1. Altogether our data shed new light on Swe1 regulation mechanism and put the basis for the identification its new target(s) .