The role of Net1 phosphorylation in regulating Cdc14 release during mitotic exit

Abstract

Exit from mitosis is as an important phase in the cell cycle. The molecular event that triggers the cell cycle transition from anaphase into the G1 state involves the inactivation of the cyclin-dependent kinase complex (Cdk) through multiple mechanisms that lead to both destruction of the cyclin subunit co-activator and direct Cdk kinase inhibition. These multiple mechanisms indicate the importance of regulating the inactivation of Cdk to ensure proper cell cycle progression and cytokinesis. We set out to examine the regulation of the protein phosphatase Cdc14. Cdc14 is thought to act through reversal of phosphorylation on key Cdk substrates that promote mitotic exit by stimulating the destruction and inactivation of Cdk. In Saccharomyces cerevisiae, activation of Cdc14 is achieved via release from its nucleolar inhibitor Net1/Cfi1. This activation is correlated with multi-site phosphorylation of Net1 in cells where Cdc14 appears to be released from the nucleolus. We set out to identify new components of the nucleolar complex known as RENT (Regulator of Nucleolar Silencing and Teleophase) which holds Cdc14 in an inactive state. This led to the identification of Casein Kinase II (CKII) as a new component of RENT. CKII was verified to co-immunoprecipitate with Net1; and mutants in CKII arrest in anaphase with unreleased Cdc14 and unsegregated rDNA. Interestingly, phospho-peptide mapping experiments from in vivo Net1 samples revealed phosphorylation of a CKII consensus sequence within Net1. In vivo mapping also revealed another subset of sites that matched the consensus sequence established for Cdk phosphorylation. Mutational analysis of these sites unveiled their involvement in Cdc14 release during early anaphase and a role for a network of genetically interacting proteins involved in Fourteen Early Anaphase Release (FEAR) in promoting these phosphorylations. In summary, the regulation of Cdc14 release via phosphorylation of its nucleolar inhibitor Net1 as demonstrated by this work highlights the importance of nucleolar sequesteration and regulated release as a mechanism of controlling important cell cycle factors and events. It also points to a fascinating role for Cdk in insuring its own destruction at the end of the cell cycle, thus promoting transition back into the G1 state.