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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

Release of Cdc14 from the nucleolus requires Cdc5. CDC14-HA3 cells in wild type (+) (RJD 1191) or cdc5-1 (RJD 1217) background were grown at 25°C, and a portion of the cultures were further shifted to 33°C for three hours to arrest cdc5-1 in late anaphase. Cells were subjected to indirect immunofluorescence with HA.11 to visualize Cdc14-HA3 (Column 1) and either anti-tubulin or anti-A190 antibodies to visualize the mitotic spindles and the nucleoli, respectively (Column 2). The positions of nuclei, as indicated by DAPI staining, are shown in Column 3.
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Figure 1: Release of Cdc14 from the nucleolus requires Cdc5. CDC14-HA3 cells in wild type (+) (RJD 1191) or cdc5-1 (RJD 1217) background were grown at 25°C, and a portion of the cultures were further shifted to 33°C for three hours to arrest cdc5-1 in late anaphase. Cells were subjected to indirect immunofluorescence with HA.11 to visualize Cdc14-HA3 (Column 1) and either anti-tubulin or anti-A190 antibodies to visualize the mitotic spindles and the nucleoli, respectively (Column 2). The positions of nuclei, as indicated by DAPI staining, are shown in Column 3.

Mentions: In S. cerevisiae, disassembly of the RENT complex and the subsequent release of Cdc14 from the nucleolus drive cells to exit mitosis [8,9]. This event is thought to require two independent signals: an unknown mitosis-specific signal and a signal from the mitotic exit network (MEN) proteins including Tem1, Cdc15, and Dbf2 [8,9]. The Polo-like kinase Cdc5 also belongs to the MEN, thus we tested whether it too is required for release of Cdc14 from the nucleolus by using indirect immunofluorescence to compare Cdc14 localization patterns in wild type and cdc5-1 cells. Wild type cells with short spindles were in pre-anaphase and showed focal nucleolar Cdc14 staining, and those with elongated mitotic spindles were in late anaphase and showed diffused Cdc14 staining (Figure 1, Rows 1 and 4; [8,9]). In contrast, cdc5-1 cells uniformly exhibited focal Cdc14 staining whether they were grown exponentially at permissive temperature (25°C) or arrested in late mitosis at nonpermissive temperature (33°C) (Figure 1, Rows 2 and 5). Furthermore, in all (including late anaphase) cdc5-1 cells, Cdc14 co-localized with A190, a nucleolar protein (Figure 1, Rows 3 and 6), suggesting that when CDC5 is compromised, most or all Cdc14 fails to vacate the nucleolus in late anaphase.


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)

Release of Cdc14 from the nucleolus requires Cdc5. CDC14-HA3 cells in wild type (+) (RJD 1191) or cdc5-1 (RJD 1217) background were grown at 25°C, and a portion of the cultures were further shifted to 33°C for three hours to arrest cdc5-1 in late anaphase. Cells were subjected to indirect immunofluorescence with HA.11 to visualize Cdc14-HA3 (Column 1) and either anti-tubulin or anti-A190 antibodies to visualize the mitotic spindles and the nucleoli, respectively (Column 2). The positions of nuclei, as indicated by DAPI staining, are shown in Column 3.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC113746&req=5

Figure 1: Release of Cdc14 from the nucleolus requires Cdc5. CDC14-HA3 cells in wild type (+) (RJD 1191) or cdc5-1 (RJD 1217) background were grown at 25°C, and a portion of the cultures were further shifted to 33°C for three hours to arrest cdc5-1 in late anaphase. Cells were subjected to indirect immunofluorescence with HA.11 to visualize Cdc14-HA3 (Column 1) and either anti-tubulin or anti-A190 antibodies to visualize the mitotic spindles and the nucleoli, respectively (Column 2). The positions of nuclei, as indicated by DAPI staining, are shown in Column 3.
Mentions: In S. cerevisiae, disassembly of the RENT complex and the subsequent release of Cdc14 from the nucleolus drive cells to exit mitosis [8,9]. This event is thought to require two independent signals: an unknown mitosis-specific signal and a signal from the mitotic exit network (MEN) proteins including Tem1, Cdc15, and Dbf2 [8,9]. The Polo-like kinase Cdc5 also belongs to the MEN, thus we tested whether it too is required for release of Cdc14 from the nucleolus by using indirect immunofluorescence to compare Cdc14 localization patterns in wild type and cdc5-1 cells. Wild type cells with short spindles were in pre-anaphase and showed focal nucleolar Cdc14 staining, and those with elongated mitotic spindles were in late anaphase and showed diffused Cdc14 staining (Figure 1, Rows 1 and 4; [8,9]). In contrast, cdc5-1 cells uniformly exhibited focal Cdc14 staining whether they were grown exponentially at permissive temperature (25°C) or arrested in late mitosis at nonpermissive temperature (33°C) (Figure 1, Rows 2 and 5). Furthermore, in all (including late anaphase) cdc5-1 cells, Cdc14 co-localized with A190, a nucleolar protein (Figure 1, Rows 3 and 6), suggesting that when CDC5 is compromised, most or all Cdc14 fails to vacate the nucleolus in late anaphase.

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