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Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex

Shou, Wenying and Azzam, Ramzi and Chen, Susan L. and Huddleston, Michael J. and Baskerville, Christopher and Charbonneau, Harry and Annan, Roland S. and Carr, Steve A. and Deshaies, Raymond J. (2002) Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex. BMC Molecular Biology, 3 (3). pp. 1-14. ISSN 1471-2199. PMCID PMC113746.

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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.

Item Type:Article
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URLURL TypeDescription CentralArticle
Carr, Steve A.0000-0002-7203-4299
Deshaies, Raymond J.0000-0002-3671-9354
Additional Information:© 2002 Shou et al; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any purpose, provided this notice is preserved along with the article's original URL. Authors' contributions: WS, RA, and RJD contributed to Figures 1 – Figure 6B, Figures 6C-6E, and Figure 7, respectively. Together, RA and WS did many experiments which, although invisible here, are essential to the development of this project. SLC, MJH, RSA and SAC mapped in vitro Cdc5 phosphorylation sites on Net1N and in vivo phosphorylation sites on Net1. CB and HC mapped the Cdc14-interaction domain of Net1 to the first 341 amino acids, and constructed the corresponding Net1N plasmid. Acknowledgements: We thank R. Verma for advice on phospho-site mapping experiments; R. Verma, B. Hay, and D. Chan for discussions; and W. Seufert and E. Traverso for sharing unpublished data. We thank G. Alexandru, D. Chan, R. Lipford, E. Traverso, and R. Verma for critically reading the manuscript. We also thank J. Charles (Morgan lab), A. Kumagai (Dunphy lab), and M. Shirayama (Nasmyth lab) for the Cdc5 baculoviruses, Plx1 baculoviruses, and GALCDC5(DBЊ) strain, respectively; M. Nomura and F. Soloman labs provided antibodies against A190 and tubulin, respectively. WS was an HHMI predoctoral fellow. This research was supported by an NIH grant to RJD. WS dedicates this paper to Maste R. Brain, Rubbe R. Stamp, and their invaluable yet dispensable lessons.
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PubMed Central ID:PMC113746
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ID Code:289
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Deposited On:18 May 2005
Last Modified:09 Mar 2020 13:18

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