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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

Overexpression of Cdc5(DB▲) triggers Cdc14 release. (A) Overexpression of Cdc5(DB▲) promotes release of Cdc14 from the nucleolus even in cells with short spindles. CDC14-MYC9 cells with (WY333) or without (WY201) integrated GAL-CDC5(DB▲) were grown in YP + 2% raffinose (GAL promoter uninduced), arrested in G1 by α-factor, supplemented with galactose to 2% for 0.5 hour (GAL promoter induced), and released into YP + 2% galactose at time = 0. At indicated time points after release, samples were fixed, and subjected to indirect immunofluorescence with anti-tubulin and 9E10 antibodies to visualize the mitotic spindles and Cdc14-Myc9, respectively. The percentages of cells with short spindles and diffused Cdc14 were calculated. (B) Overexpression of Cdc5(DB▲) triggers Cdc14 release in cells arrested in mitosis. NET1-MYC9 cells with (WY331) or without (WY53) integrated GAL-CDC5(DB▲) were grown to exponential in YP + 2% raffinose, arrested in mitosis by nocodazole (10 μg/ml final), and supplemented with galactose to 2% for four hours. Samples were subjected to indirect immunofluorescence with anti-Cdc14 and 9E10 (to detect Net1-Myc9).
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Figure 2: Overexpression of Cdc5(DB▲) triggers Cdc14 release. (A) Overexpression of Cdc5(DB▲) promotes release of Cdc14 from the nucleolus even in cells with short spindles. CDC14-MYC9 cells with (WY333) or without (WY201) integrated GAL-CDC5(DB▲) were grown in YP + 2% raffinose (GAL promoter uninduced), arrested in G1 by α-factor, supplemented with galactose to 2% for 0.5 hour (GAL promoter induced), and released into YP + 2% galactose at time = 0. At indicated time points after release, samples were fixed, and subjected to indirect immunofluorescence with anti-tubulin and 9E10 antibodies to visualize the mitotic spindles and Cdc14-Myc9, respectively. The percentages of cells with short spindles and diffused Cdc14 were calculated. (B) Overexpression of Cdc5(DB▲) triggers Cdc14 release in cells arrested in mitosis. NET1-MYC9 cells with (WY331) or without (WY53) integrated GAL-CDC5(DB▲) were grown to exponential in YP + 2% raffinose, arrested in mitosis by nocodazole (10 μg/ml final), and supplemented with galactose to 2% for four hours. Samples were subjected to indirect immunofluorescence with anti-Cdc14 and 9E10 (to detect Net1-Myc9).

Mentions: To identify candidate proteins that directly disrupt the RENT complex, we examined whether overexpression of the MEN proteins could release Cdc14 from the nucleolus. We first tested Cdc5, because Cdc5 is required for Cdc14 release (Figure 1), and because overexpression of Cdc5 triggers ectopic degradation of Clb2 [19,20], which normally depends upon active Cdc14 [3]. These experiments employed a GAL-CDC5(DB▲) strain, in which expression of Cdc5(DB▲), a stabilized version of Cdc5 lacking the destruction box, was driven by the inducible GAL promoter [20]. Cells were arrested in G1 by α-factor, supplemented with galactose to induce the GAL promoter, and released into galactose-containing medium. Cdc14 was focal in almost all cells with short mitotic spindles, but became diffused in a significant fraction of these cells after Cdc5(DB▲) was induced (Figure 2A). A similar result was obtained if the total number of cells with diffused Cdc14 was plotted instead (data not shown). To address whether overexpression of Cdc5 could also force Cdc14 out of the nucleolus in cells arrested at a cell cycle stage where Cdc14 is nucleolar, we arrested cells in metaphase using the microtubule polymerization inhibitor nocodazole. After the expression of Cdc5(DB▲) was induced in nocodazole-arrested cells, Cdc14 diffused beyond the domain of Net1 in ~49% of cells (Figure 2B). In contrast, GAL-driven overexpression of Dbf2/(GST-Mob1) protein kinase complex did not dislodge Cdc14 from the nucleolus (data not shown). Taken together, these data suggest that overexpression of Cdc5 – but not the putative downstream-most MEN component Dbf2/Mob1 – activates premature release of Cdc14 from the nucleolus.


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)

Overexpression of Cdc5(DB▲) triggers Cdc14 release. (A) Overexpression of Cdc5(DB▲) promotes release of Cdc14 from the nucleolus even in cells with short spindles. CDC14-MYC9 cells with (WY333) or without (WY201) integrated GAL-CDC5(DB▲) were grown in YP + 2% raffinose (GAL promoter uninduced), arrested in G1 by α-factor, supplemented with galactose to 2% for 0.5 hour (GAL promoter induced), and released into YP + 2% galactose at time = 0. At indicated time points after release, samples were fixed, and subjected to indirect immunofluorescence with anti-tubulin and 9E10 antibodies to visualize the mitotic spindles and Cdc14-Myc9, respectively. The percentages of cells with short spindles and diffused Cdc14 were calculated. (B) Overexpression of Cdc5(DB▲) triggers Cdc14 release in cells arrested in mitosis. NET1-MYC9 cells with (WY331) or without (WY53) integrated GAL-CDC5(DB▲) were grown to exponential in YP + 2% raffinose, arrested in mitosis by nocodazole (10 μg/ml final), and supplemented with galactose to 2% for four hours. Samples were subjected to indirect immunofluorescence with anti-Cdc14 and 9E10 (to detect Net1-Myc9).
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Related In: Results  -  Collection

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Figure 2: Overexpression of Cdc5(DB▲) triggers Cdc14 release. (A) Overexpression of Cdc5(DB▲) promotes release of Cdc14 from the nucleolus even in cells with short spindles. CDC14-MYC9 cells with (WY333) or without (WY201) integrated GAL-CDC5(DB▲) were grown in YP + 2% raffinose (GAL promoter uninduced), arrested in G1 by α-factor, supplemented with galactose to 2% for 0.5 hour (GAL promoter induced), and released into YP + 2% galactose at time = 0. At indicated time points after release, samples were fixed, and subjected to indirect immunofluorescence with anti-tubulin and 9E10 antibodies to visualize the mitotic spindles and Cdc14-Myc9, respectively. The percentages of cells with short spindles and diffused Cdc14 were calculated. (B) Overexpression of Cdc5(DB▲) triggers Cdc14 release in cells arrested in mitosis. NET1-MYC9 cells with (WY331) or without (WY53) integrated GAL-CDC5(DB▲) were grown to exponential in YP + 2% raffinose, arrested in mitosis by nocodazole (10 μg/ml final), and supplemented with galactose to 2% for four hours. Samples were subjected to indirect immunofluorescence with anti-Cdc14 and 9E10 (to detect Net1-Myc9).
Mentions: To identify candidate proteins that directly disrupt the RENT complex, we examined whether overexpression of the MEN proteins could release Cdc14 from the nucleolus. We first tested Cdc5, because Cdc5 is required for Cdc14 release (Figure 1), and because overexpression of Cdc5 triggers ectopic degradation of Clb2 [19,20], which normally depends upon active Cdc14 [3]. These experiments employed a GAL-CDC5(DB▲) strain, in which expression of Cdc5(DB▲), a stabilized version of Cdc5 lacking the destruction box, was driven by the inducible GAL promoter [20]. Cells were arrested in G1 by α-factor, supplemented with galactose to induce the GAL promoter, and released into galactose-containing medium. Cdc14 was focal in almost all cells with short mitotic spindles, but became diffused in a significant fraction of these cells after Cdc5(DB▲) was induced (Figure 2A). A similar result was obtained if the total number of cells with diffused Cdc14 was plotted instead (data not shown). To address whether overexpression of Cdc5 could also force Cdc14 out of the nucleolus in cells arrested at a cell cycle stage where Cdc14 is nucleolar, we arrested cells in metaphase using the microtubule polymerization inhibitor nocodazole. After the expression of Cdc5(DB▲) was induced in nocodazole-arrested cells, Cdc14 diffused beyond the domain of Net1 in ~49% of cells (Figure 2B). In contrast, GAL-driven overexpression of Dbf2/(GST-Mob1) protein kinase complex did not dislodge Cdc14 from the nucleolus (data not shown). Taken together, these data suggest that overexpression of Cdc5 – but not the putative downstream-most MEN component Dbf2/Mob1 – activates premature release of Cdc14 from the nucleolus.

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