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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

RENT complexes containing phospho-site mutant Net1(7m) is refractory to disassembly by Polo-like kinase in vitro, but exhibit minor phenotype in vivo. (A) RENT complexes from isogenic NET1-myc9 (WY374) and net1(7m)-myc9 (WY347) cells were immunoprecipitated on 9E10 beads. Beads were divided into equal portions, and treated with indicated amounts of Plx1. Cdc14 retained on Net1-bound beads or released into the supernatant was fractionated by SDS-PAGE and detected by immunoblotting with anti-Cdc14 antibodies. (B) Asynchronous NET1-myc9 (+), net1(7m)-myc9 (7m), and net1(19m')-myc9 (19m') cultures were subjected to indirect immunofluorescence using anti-Cdc14 and anti-tubulin antibodies. Cells with long mitotic spindles (~10% of total) were examined further to calculate the percentage that displayed focal Cdc14 staining. (C) NET1-myc9 (+) and net1(7m)-myc9 (7m) cells were arrested in G1 with α factor, and released into YP + 2% glucose media (T = 0) at 25°C. Samples withdrawn and fixed at the indicated time points were double-labeled with anti-Cdc14 and anti-tubulin antibodies. The percentage of cells with long mitotic spindles (L.S.) and the percentage of cells with delocalized Cdc14 (14) were calculated and plotted independently. (D) Cells harboring the dbf2-1 mutation and either a wild-type or mutant net1 allele were grown in YPD at 25°C, arrested in G1 with α-factor (12 μg/ml), released into YPD prewarmed to 37°C, and incubated at 37°C thereafter. Cells collected at 70–110 minutes after α factor release were double-labeled with anti-Cdc14 and anti-tubulin antibodies. Spindle length was measured and localization of Cdc14 was determined to be in one of the three categories: 1. full release (black box): complete release of Cdc14 from the nucleolus into the nucleus; 2. partial release (gray box): Cdc14 was nuclear in one of the DAPI masses and nucleolar in the other DAPI mass in the same cell, or Cdc14 was nuclear but with stronger nucleolar staining; and 3. no release (clear box): Cdc14 was strictly nucleolar. In each of the three panels, more than 300 cells were counted. (E) Starting with 3000 cells, three-fold serial dilutions of NET1-myc9 (+), net1(7m)-myc9 (7m), and net1(19m')-myc9 (19m') cells in msd2-1 or dbf2-1 background were spotted on a YPD plate from right to left, and incubated at the indicated temperature for 2 days before the picture was taken. Two independent isolates for each strain were used.
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Figure 6: RENT complexes containing phospho-site mutant Net1(7m) is refractory to disassembly by Polo-like kinase in vitro, but exhibit minor phenotype in vivo. (A) RENT complexes from isogenic NET1-myc9 (WY374) and net1(7m)-myc9 (WY347) cells were immunoprecipitated on 9E10 beads. Beads were divided into equal portions, and treated with indicated amounts of Plx1. Cdc14 retained on Net1-bound beads or released into the supernatant was fractionated by SDS-PAGE and detected by immunoblotting with anti-Cdc14 antibodies. (B) Asynchronous NET1-myc9 (+), net1(7m)-myc9 (7m), and net1(19m')-myc9 (19m') cultures were subjected to indirect immunofluorescence using anti-Cdc14 and anti-tubulin antibodies. Cells with long mitotic spindles (~10% of total) were examined further to calculate the percentage that displayed focal Cdc14 staining. (C) NET1-myc9 (+) and net1(7m)-myc9 (7m) cells were arrested in G1 with α factor, and released into YP + 2% glucose media (T = 0) at 25°C. Samples withdrawn and fixed at the indicated time points were double-labeled with anti-Cdc14 and anti-tubulin antibodies. The percentage of cells with long mitotic spindles (L.S.) and the percentage of cells with delocalized Cdc14 (14) were calculated and plotted independently. (D) Cells harboring the dbf2-1 mutation and either a wild-type or mutant net1 allele were grown in YPD at 25°C, arrested in G1 with α-factor (12 μg/ml), released into YPD prewarmed to 37°C, and incubated at 37°C thereafter. Cells collected at 70–110 minutes after α factor release were double-labeled with anti-Cdc14 and anti-tubulin antibodies. Spindle length was measured and localization of Cdc14 was determined to be in one of the three categories: 1. full release (black box): complete release of Cdc14 from the nucleolus into the nucleus; 2. partial release (gray box): Cdc14 was nuclear in one of the DAPI masses and nucleolar in the other DAPI mass in the same cell, or Cdc14 was nuclear but with stronger nucleolar staining; and 3. no release (clear box): Cdc14 was strictly nucleolar. In each of the three panels, more than 300 cells were counted. (E) Starting with 3000 cells, three-fold serial dilutions of NET1-myc9 (+), net1(7m)-myc9 (7m), and net1(19m')-myc9 (19m') cells in msd2-1 or dbf2-1 background were spotted on a YPD plate from right to left, and incubated at the indicated temperature for 2 days before the picture was taken. Two independent isolates for each strain were used.

Mentions: Next, we asked if the mutant RENT complex is more resistant toward Plx1. RENT complexes from net1▲ NET1-myc9 and net1▲ net1(7m)-myc9 cell lysates were captured on 9E10 beads and treated with increasing amounts of recombinant Plx1 plus ATP. Whereas ~40% of Cdc14 was released from wild-type RENT complex in the presence of 0.5 μl of Plx1, less than 10% of Cdc14 was released from mutant RENT complex when treated with 15 μl of Plx1 (Figure 6A). Thus, the mutant RENT is at least 30-fold more resistant to Plx1 than wild-type RENT. RENT complexes comprising Net1(19m') yielded nearly identical results (data not shown).


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)

RENT complexes containing phospho-site mutant Net1(7m) is refractory to disassembly by Polo-like kinase in vitro, but exhibit minor phenotype in vivo. (A) RENT complexes from isogenic NET1-myc9 (WY374) and net1(7m)-myc9 (WY347) cells were immunoprecipitated on 9E10 beads. Beads were divided into equal portions, and treated with indicated amounts of Plx1. Cdc14 retained on Net1-bound beads or released into the supernatant was fractionated by SDS-PAGE and detected by immunoblotting with anti-Cdc14 antibodies. (B) Asynchronous NET1-myc9 (+), net1(7m)-myc9 (7m), and net1(19m')-myc9 (19m') cultures were subjected to indirect immunofluorescence using anti-Cdc14 and anti-tubulin antibodies. Cells with long mitotic spindles (~10% of total) were examined further to calculate the percentage that displayed focal Cdc14 staining. (C) NET1-myc9 (+) and net1(7m)-myc9 (7m) cells were arrested in G1 with α factor, and released into YP + 2% glucose media (T = 0) at 25°C. Samples withdrawn and fixed at the indicated time points were double-labeled with anti-Cdc14 and anti-tubulin antibodies. The percentage of cells with long mitotic spindles (L.S.) and the percentage of cells with delocalized Cdc14 (14) were calculated and plotted independently. (D) Cells harboring the dbf2-1 mutation and either a wild-type or mutant net1 allele were grown in YPD at 25°C, arrested in G1 with α-factor (12 μg/ml), released into YPD prewarmed to 37°C, and incubated at 37°C thereafter. Cells collected at 70–110 minutes after α factor release were double-labeled with anti-Cdc14 and anti-tubulin antibodies. Spindle length was measured and localization of Cdc14 was determined to be in one of the three categories: 1. full release (black box): complete release of Cdc14 from the nucleolus into the nucleus; 2. partial release (gray box): Cdc14 was nuclear in one of the DAPI masses and nucleolar in the other DAPI mass in the same cell, or Cdc14 was nuclear but with stronger nucleolar staining; and 3. no release (clear box): Cdc14 was strictly nucleolar. In each of the three panels, more than 300 cells were counted. (E) Starting with 3000 cells, three-fold serial dilutions of NET1-myc9 (+), net1(7m)-myc9 (7m), and net1(19m')-myc9 (19m') cells in msd2-1 or dbf2-1 background were spotted on a YPD plate from right to left, and incubated at the indicated temperature for 2 days before the picture was taken. Two independent isolates for each strain were used.
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Figure 6: RENT complexes containing phospho-site mutant Net1(7m) is refractory to disassembly by Polo-like kinase in vitro, but exhibit minor phenotype in vivo. (A) RENT complexes from isogenic NET1-myc9 (WY374) and net1(7m)-myc9 (WY347) cells were immunoprecipitated on 9E10 beads. Beads were divided into equal portions, and treated with indicated amounts of Plx1. Cdc14 retained on Net1-bound beads or released into the supernatant was fractionated by SDS-PAGE and detected by immunoblotting with anti-Cdc14 antibodies. (B) Asynchronous NET1-myc9 (+), net1(7m)-myc9 (7m), and net1(19m')-myc9 (19m') cultures were subjected to indirect immunofluorescence using anti-Cdc14 and anti-tubulin antibodies. Cells with long mitotic spindles (~10% of total) were examined further to calculate the percentage that displayed focal Cdc14 staining. (C) NET1-myc9 (+) and net1(7m)-myc9 (7m) cells were arrested in G1 with α factor, and released into YP + 2% glucose media (T = 0) at 25°C. Samples withdrawn and fixed at the indicated time points were double-labeled with anti-Cdc14 and anti-tubulin antibodies. The percentage of cells with long mitotic spindles (L.S.) and the percentage of cells with delocalized Cdc14 (14) were calculated and plotted independently. (D) Cells harboring the dbf2-1 mutation and either a wild-type or mutant net1 allele were grown in YPD at 25°C, arrested in G1 with α-factor (12 μg/ml), released into YPD prewarmed to 37°C, and incubated at 37°C thereafter. Cells collected at 70–110 minutes after α factor release were double-labeled with anti-Cdc14 and anti-tubulin antibodies. Spindle length was measured and localization of Cdc14 was determined to be in one of the three categories: 1. full release (black box): complete release of Cdc14 from the nucleolus into the nucleus; 2. partial release (gray box): Cdc14 was nuclear in one of the DAPI masses and nucleolar in the other DAPI mass in the same cell, or Cdc14 was nuclear but with stronger nucleolar staining; and 3. no release (clear box): Cdc14 was strictly nucleolar. In each of the three panels, more than 300 cells were counted. (E) Starting with 3000 cells, three-fold serial dilutions of NET1-myc9 (+), net1(7m)-myc9 (7m), and net1(19m')-myc9 (19m') cells in msd2-1 or dbf2-1 background were spotted on a YPD plate from right to left, and incubated at the indicated temperature for 2 days before the picture was taken. Two independent isolates for each strain were used.
Mentions: Next, we asked if the mutant RENT complex is more resistant toward Plx1. RENT complexes from net1▲ NET1-myc9 and net1▲ net1(7m)-myc9 cell lysates were captured on 9E10 beads and treated with increasing amounts of recombinant Plx1 plus ATP. Whereas ~40% of Cdc14 was released from wild-type RENT complex in the presence of 0.5 μl of Plx1, less than 10% of Cdc14 was released from mutant RENT complex when treated with 15 μl of Plx1 (Figure 6A). Thus, the mutant RENT is at least 30-fold more resistant to Plx1 than wild-type RENT. RENT complexes comprising Net1(19m') yielded nearly identical results (data not shown).

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