title: Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex.
abstract: BACKGROUND
  In S. cerevisiae, the mitotic exit network (MEN) proteins, including the Polo-like protein kinase Cdc5 and the protein phosphatase Cdc14, are required for exit from mitosis. In pre-anaphase cells, Cdc14 is sequestered to the nucleolus by Net1 as a part of the RENT complex. When cells are primed to exit mitosis, the RENT complex is disassembled and Cdc14 is released from the nucleolus.
  
  
  RESULTS
  Here, we show that Cdc5 is necessary to free nucleolar Cdc14 in late mitosis, that elevated Cdc5 activity provokes ectopic release of Cdc14 in pre-anaphase cells, and that the phosphorylation state of Net1 is regulated by Cdc5 during anaphase. Furthermore, recombinant Cdc5 and Xenopus Polo-like kinase can disassemble the RENT complex in vitro by phosphorylating Net1 and thereby reducing its affinity for Cdc14. Surprisingly, although RENT complexes containing Net1 mutants (Net1(7m) and Net1(19m') lacking sites phosphorylated by Cdc5 in vitro are refractory to disassembly by Polo-like kinases in vitro, net1(7m) and net1(19m') cells grow normally and exhibit only minor defects in releasing Cdc14 during anaphase. However, net1(19m') cells exhibit a synergistic growth defect when combined with mutations in CDC5 or DBF2 (another MEN gene).
  
  
  CONCLUSIONS
  We propose that although Cdc5 potentially disassembles RENT by directly phosphorylating Net1, Cdc5 mediates exit from mitosis primarily by phosphorylating other targets. Our study suggests that Cdc5/Polo is unusually promiscuous and highlights the need to validate Cdc5/Polo in vitro phosphorylation sites by direct in vivo mapping experiments.
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena, California 91125, USA. shouw@its.caltech.edu
authors.forename: Wenying
authors.initials: W
authors.lastname: Shou
authors.forename: Ramzi
authors.initials: R
authors.lastname: Azzam
authors.forename: Susan L
authors.initials: SL
authors.lastname: Chen
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Huddleston
authors.forename: Christopher
authors.initials: C
authors.lastname: Baskerville
authors.forename: Harry
authors.initials: H
authors.lastname: Charbonneau
authors.forename: Roland S
authors.initials: RS
authors.lastname: Annan
authors.forename: Steve A
authors.initials: SA
authors.lastname: Carr
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies