CDC5-MEDIATED PHOSPHORYLATION OF CLB2, CDC14 AND/OR CFI1 IS REQUIRED FOR THE NUCLEOLAR RELEASE OF CDC14

In budding yeast, the protein phosphatase Cdc14 is a critical regulator of exit from mitosis. Cdc14 activity is regulated by changes in its subcellular localization. The phosphatase is sequestered in the nucleolus from G1 up to metaphase by binding to a competitive inhibitor called Cfi1. During anaphase, Cdc14 is released from its inhibitor by the sequential activation of two signaling networks, the FEAR network and the MEN. Several observations suggest that phosphorylation of Cdc14 and/or Cfi1 is responsible for the dissociation of Cdc14 from Cfi1. Three kinases play a relevant role in regulating Cdc14 release: the polo-like kinase Cdc5, the Clb2-Cdk complex and the MEN kinase Dbf2. The aim of my project was to assess the relevance of the phosphorylation of Cdc14 and Cfi1 for Cdc14 release and to investigate the contribution of the mentioned kinases to this process. By modulating the kinases of interest, we found that a correlation exists between the release of Cdc14 from the nucleolus and the phosphorylation of both Cdc14 and Cfi1. Our results suggest a role for Cdc5 in promoting the phosphorylation of Cdc14 and for Clb-Cdk and MEN in promoting the phosphorylation of Cfi1. Moreover we propose that Cfi1 phosphorylation by Clb-Cdk or MEN serves as a priming step to build up the Cdc5-mediated phosphorylation events. Our data also suggest that Clb2-Cdk is not sufficient to promote Cfi1 phosphorylation and that phosphorylation of Clb2 by Cdc5 could enable the protein to perform its function in the FEAR network, likely to promote Cfi1phosphorylation.