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Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex.

Shou W, Azzam R, Chen SL, Huddleston MJ, Baskerville C, Charbonneau H, Annan RS, Carr SA, Deshaies RJ - BMC Mol. Biol. (2002)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California 91125, USA. shouw@its.caltech.edu

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.

No MeSH data available.


Related in: MedlinePlus

Role of Cdc5 in exit from mitosis. Cdc5 may promote Cdc14 early anaphase release (FEAR) in part by phosphorylating Net1 directly (dotted arrow), and by activating an unknown Cdc14 dissociation factor (X). Subsequent activation of the MEN is important to sustain the transient Cdc14 release effected by the FEAR pathway. In the absence of MEN activity, Cdc14 returns to the nucleolus and cells arrest in late anaphase (bottom right). In the presence of MEN activity, Clb/CDK is shut off and cells exit mitosis (top right). The mechanism of action of MEN remains unknown, but activation of MEN correlates with appearance of Cdc14 in the cytoplasm [26], and mutation of known nuclear transport regulators renders mitotic exit independent of CDC15 function [35]. Together, these observations suggest that the MEN may promote exit by biasing the nucleocytoplasmic distribution of Cdc14. In addition to its role in the FEAR pathway, Cdc5 impinges positively on the MEN by several mechanisms, including: (i) Cdc5 promotes Tem1 activation by promoting chromosome segregation [22], which by enabling optimal spindle elongation and penetration into the bud, helps satisfy the spindle positioning checkpoint [12]; (ii) Cdc5 promotes activation of Tem1 GTP-binding protein by inhibiting its negative regulator, the Bfa1/Bub2 GTPase-activating protein [29], and (iii) Cdc5 promotes activation of Dbf2 by an unknown, BUB2-independent mechanism (dashed line) [18].
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Figure 7: Role of Cdc5 in exit from mitosis. Cdc5 may promote Cdc14 early anaphase release (FEAR) in part by phosphorylating Net1 directly (dotted arrow), and by activating an unknown Cdc14 dissociation factor (X). Subsequent activation of the MEN is important to sustain the transient Cdc14 release effected by the FEAR pathway. In the absence of MEN activity, Cdc14 returns to the nucleolus and cells arrest in late anaphase (bottom right). In the presence of MEN activity, Clb/CDK is shut off and cells exit mitosis (top right). The mechanism of action of MEN remains unknown, but activation of MEN correlates with appearance of Cdc14 in the cytoplasm [26], and mutation of known nuclear transport regulators renders mitotic exit independent of CDC15 function [35]. Together, these observations suggest that the MEN may promote exit by biasing the nucleocytoplasmic distribution of Cdc14. In addition to its role in the FEAR pathway, Cdc5 impinges positively on the MEN by several mechanisms, including: (i) Cdc5 promotes Tem1 activation by promoting chromosome segregation [22], which by enabling optimal spindle elongation and penetration into the bud, helps satisfy the spindle positioning checkpoint [12]; (ii) Cdc5 promotes activation of Tem1 GTP-binding protein by inhibiting its negative regulator, the Bfa1/Bub2 GTPase-activating protein [29], and (iii) Cdc5 promotes activation of Dbf2 by an unknown, BUB2-independent mechanism (dashed line) [18].

Mentions: While this manuscript was under consideration, two papers presented new evidence on how Cdc5 contributes to exit from mitosis [29,26]. To summarize our study and these recent findings, we present a model (Figure 7) that is consistent with all of the available data. Upon initiation of chromosome segregation, Cdc5 promotes the transient 'Cdc14 early anaphase release' [26]. Our data suggest that Cdc5 can promote FEAR by directly phosphorylating Net1 as well as other yet-to-be-identified FEAR factors. Cdc5 also helps to activate the MEN by directly phosphorylating and disabling Bfa1 [29], which is thought to inhibit Tem1 by serving as a GTPase-activating protein. Cdc5 presumably impinges on the MEN by other means as well, because Cdc5 is required for maximal Dbf2 protein kinase activity even in the absence of Bfa1's obligate partner Bub2 [18]. Once activated, the MEN in turn sustains the transient Cdc14 release enabled by Cdc5 and other FEAR pathway components. The unique ability of overexpressed Cdc5 to expel Cdc14 from the nucleolus may thus be a consequence of Cdc5's abilities to directly disassemble Net1-Cdc14 complexes, promote FEAR, and activate the MEN. A complete understanding of Cdc5's role in mitotic exit will ultimately require identification of all of its direct substrates in the MEN and FEAR pathways.


Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex.

Shou W, Azzam R, Chen SL, Huddleston MJ, Baskerville C, Charbonneau H, Annan RS, Carr SA, Deshaies RJ - BMC Mol. Biol. (2002)

Role of Cdc5 in exit from mitosis. Cdc5 may promote Cdc14 early anaphase release (FEAR) in part by phosphorylating Net1 directly (dotted arrow), and by activating an unknown Cdc14 dissociation factor (X). Subsequent activation of the MEN is important to sustain the transient Cdc14 release effected by the FEAR pathway. In the absence of MEN activity, Cdc14 returns to the nucleolus and cells arrest in late anaphase (bottom right). In the presence of MEN activity, Clb/CDK is shut off and cells exit mitosis (top right). The mechanism of action of MEN remains unknown, but activation of MEN correlates with appearance of Cdc14 in the cytoplasm [26], and mutation of known nuclear transport regulators renders mitotic exit independent of CDC15 function [35]. Together, these observations suggest that the MEN may promote exit by biasing the nucleocytoplasmic distribution of Cdc14. In addition to its role in the FEAR pathway, Cdc5 impinges positively on the MEN by several mechanisms, including: (i) Cdc5 promotes Tem1 activation by promoting chromosome segregation [22], which by enabling optimal spindle elongation and penetration into the bud, helps satisfy the spindle positioning checkpoint [12]; (ii) Cdc5 promotes activation of Tem1 GTP-binding protein by inhibiting its negative regulator, the Bfa1/Bub2 GTPase-activating protein [29], and (iii) Cdc5 promotes activation of Dbf2 by an unknown, BUB2-independent mechanism (dashed line) [18].
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Figure 7: Role of Cdc5 in exit from mitosis. Cdc5 may promote Cdc14 early anaphase release (FEAR) in part by phosphorylating Net1 directly (dotted arrow), and by activating an unknown Cdc14 dissociation factor (X). Subsequent activation of the MEN is important to sustain the transient Cdc14 release effected by the FEAR pathway. In the absence of MEN activity, Cdc14 returns to the nucleolus and cells arrest in late anaphase (bottom right). In the presence of MEN activity, Clb/CDK is shut off and cells exit mitosis (top right). The mechanism of action of MEN remains unknown, but activation of MEN correlates with appearance of Cdc14 in the cytoplasm [26], and mutation of known nuclear transport regulators renders mitotic exit independent of CDC15 function [35]. Together, these observations suggest that the MEN may promote exit by biasing the nucleocytoplasmic distribution of Cdc14. In addition to its role in the FEAR pathway, Cdc5 impinges positively on the MEN by several mechanisms, including: (i) Cdc5 promotes Tem1 activation by promoting chromosome segregation [22], which by enabling optimal spindle elongation and penetration into the bud, helps satisfy the spindle positioning checkpoint [12]; (ii) Cdc5 promotes activation of Tem1 GTP-binding protein by inhibiting its negative regulator, the Bfa1/Bub2 GTPase-activating protein [29], and (iii) Cdc5 promotes activation of Dbf2 by an unknown, BUB2-independent mechanism (dashed line) [18].
Mentions: While this manuscript was under consideration, two papers presented new evidence on how Cdc5 contributes to exit from mitosis [29,26]. To summarize our study and these recent findings, we present a model (Figure 7) that is consistent with all of the available data. Upon initiation of chromosome segregation, Cdc5 promotes the transient 'Cdc14 early anaphase release' [26]. Our data suggest that Cdc5 can promote FEAR by directly phosphorylating Net1 as well as other yet-to-be-identified FEAR factors. Cdc5 also helps to activate the MEN by directly phosphorylating and disabling Bfa1 [29], which is thought to inhibit Tem1 by serving as a GTPase-activating protein. Cdc5 presumably impinges on the MEN by other means as well, because Cdc5 is required for maximal Dbf2 protein kinase activity even in the absence of Bfa1's obligate partner Bub2 [18]. Once activated, the MEN in turn sustains the transient Cdc14 release enabled by Cdc5 and other FEAR pathway components. The unique ability of overexpressed Cdc5 to expel Cdc14 from the nucleolus may thus be a consequence of Cdc5's abilities to directly disassemble Net1-Cdc14 complexes, promote FEAR, and activate the MEN. A complete understanding of Cdc5's role in mitotic exit will ultimately require identification of all of its direct substrates in the MEN and FEAR pathways.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California 91125, USA. shouw@its.caltech.edu

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.

No MeSH data available.


Related in: MedlinePlus