Clb2 and the APC/CCdh1 regulate Swe1 stability

Abstract

Swe1/Wee1 regulates mitotic entry by inhibiting Clb2-Cdk1 and its accumulation is involved in stress induced G2 arrest. The APC/CCdh1 substrates Cdc5, Clb2 and Hsl1 regulate Swe1 degradation. We observed that clb2Dcdh1D double mutant S. cerevisiae does not express any detectable levels of Swe1, presumably due to its constitutive degradation. This effect of Cdh1 inactivation is due to stabilization of Cdc5 and Hsl1, as expression of the non-degradable Cdc5T29A in clb2D cells prevented Swe1 accumulation. Strikingly, expression of non-degradable Hsl1mdb/mkb prevented Swe1 accumulation even in wild type Clb2 cells. Interestingly Swe1 accumulation could be reconstituted in all these mutants by eliciting a replication fork stress with hydroxyurea. Cells expressing the Clb2ME mutant, that cannot bind Swe1, behaved like clb2D cells, and failed to accumulate Swe1 in the absence of Cdh1 or the presence of Cdc5T29A. This suggests that for Swe1 to accumulate it must interact with Clb2. We further show that in the absence of Clb2, Hsl1 is no longer essential for Swe1 degradation. We hypothesize that Clb2-Cdk1 protects Swe1 from premature degradation until its Hsl1 mediated de-protection, which enables its Cdc5 mediated degradation. Swe1 levels are thus regulated by monitoring the levels of three major mitotic regulators.