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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

Polo-like kinases can phosphorylate and disrupt the RENT complex in vitro. (A) Cdc5 can disrupt recombinant Net1N/Cdc14 complex. GST-Cdc14 was incubated with anti-T7 beads loaded with His6-T7-Net1N (assuming 100% binding efficiency, each reaction contained ~10 pmol of Cdc14 and ~80 pmol of Net1N), where Net1N consists of the N-terminal 341 amino acids of Net1. The beads were divided into equal portions, and treated with indicated amounts of Cdc5 (~10 pmol/μl). Proteins released into the supernatant (sup) or bound to the beads (bead) were fractionated by SDS-PAGE and immunoblotted with anti-T7 and anti-GST to detect His6-T7-Net1N and GST-Cdc14, respectively. (B) The Polo-like kinase Plx1 can also disrupt Net1N/Cdc14 complex. GST-T7-Cdc14 was captured on anti-GST resins, and incubated with His6-T7-Net1N. The resins were divided into two equal portions, and treated with either active (+) or inactive (m) Plx1. Proteins released from or bound to beads were fractionated by SDS-PAGE and immunoblotted with anti-T7 antibodies to detect both GST-T7-Cdc14 and His6-T7-Net1N. Note that phosphorylation by active Plx1 causes both GST-T7-Cdc14 and 1 + 156 - T7 - Metlin to migrate shower in SDS-Page. (C) Plx1 can disassemble immunoprecipitated RENT complex. RENT complex was retrieved from myc9-NET1 CDC14-HA3 cell lysate on a resin coated with 9E10 antibodies. The resin was divided into two equal portions, and treated with active (+) or inactive (m) Plx1. Proteins released into the supernatant or bound to the beads were separated by SDS-PAGE and immunoblotted with 9E10, 12CA5, and anti-Sir2 antibodies to detect Net1, Cdc14, and Sir2 proteins, respectively.
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Figure 4: Polo-like kinases can phosphorylate and disrupt the RENT complex in vitro. (A) Cdc5 can disrupt recombinant Net1N/Cdc14 complex. GST-Cdc14 was incubated with anti-T7 beads loaded with His6-T7-Net1N (assuming 100% binding efficiency, each reaction contained ~10 pmol of Cdc14 and ~80 pmol of Net1N), where Net1N consists of the N-terminal 341 amino acids of Net1. The beads were divided into equal portions, and treated with indicated amounts of Cdc5 (~10 pmol/μl). Proteins released into the supernatant (sup) or bound to the beads (bead) were fractionated by SDS-PAGE and immunoblotted with anti-T7 and anti-GST to detect His6-T7-Net1N and GST-Cdc14, respectively. (B) The Polo-like kinase Plx1 can also disrupt Net1N/Cdc14 complex. GST-T7-Cdc14 was captured on anti-GST resins, and incubated with His6-T7-Net1N. The resins were divided into two equal portions, and treated with either active (+) or inactive (m) Plx1. Proteins released from or bound to beads were fractionated by SDS-PAGE and immunoblotted with anti-T7 antibodies to detect both GST-T7-Cdc14 and His6-T7-Net1N. Note that phosphorylation by active Plx1 causes both GST-T7-Cdc14 and 1 + 156 - T7 - Metlin to migrate shower in SDS-Page. (C) Plx1 can disassemble immunoprecipitated RENT complex. RENT complex was retrieved from myc9-NET1 CDC14-HA3 cell lysate on a resin coated with 9E10 antibodies. The resin was divided into two equal portions, and treated with active (+) or inactive (m) Plx1. Proteins released into the supernatant or bound to the beads were separated by SDS-PAGE and immunoblotted with 9E10, 12CA5, and anti-Sir2 antibodies to detect Net1, Cdc14, and Sir2 proteins, respectively.

Mentions: Both Net1 and Cdc14 are extensively phosphorylated by Cdc5 in vitro (Figure 4A). To test if Cdc5 could dislodge Cdc14 from Net1, we devised a simplified in vitro release assay using GST-Cdc14 and His6-T7-Net1N expressed and purified from bacteria. Net1N includes the N-terminal 341 amino acids of Net1 and is sufficient to bind and inhibit Cdc14 [11]. When GST-Cdc14/His6-T7-Net1N complex anchored to anti-T7 beads was treated with ATP plus increasing amounts of recombinant Cdc5 purified from insect cells, both Cdc14 and Net1N were phosphorylated as indicated by their increased apparent molecular weight. Furthermore, Cdc14 was released from bead-bound Net1N to the supernatant (Figure 4A), and free Cdc14 was active as a protein phosphatase (data not shown), suggesting that Cdc5 is sufficient to disengage active Cdc14 from Net1.


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)

Polo-like kinases can phosphorylate and disrupt the RENT complex in vitro. (A) Cdc5 can disrupt recombinant Net1N/Cdc14 complex. GST-Cdc14 was incubated with anti-T7 beads loaded with His6-T7-Net1N (assuming 100% binding efficiency, each reaction contained ~10 pmol of Cdc14 and ~80 pmol of Net1N), where Net1N consists of the N-terminal 341 amino acids of Net1. The beads were divided into equal portions, and treated with indicated amounts of Cdc5 (~10 pmol/μl). Proteins released into the supernatant (sup) or bound to the beads (bead) were fractionated by SDS-PAGE and immunoblotted with anti-T7 and anti-GST to detect His6-T7-Net1N and GST-Cdc14, respectively. (B) The Polo-like kinase Plx1 can also disrupt Net1N/Cdc14 complex. GST-T7-Cdc14 was captured on anti-GST resins, and incubated with His6-T7-Net1N. The resins were divided into two equal portions, and treated with either active (+) or inactive (m) Plx1. Proteins released from or bound to beads were fractionated by SDS-PAGE and immunoblotted with anti-T7 antibodies to detect both GST-T7-Cdc14 and His6-T7-Net1N. Note that phosphorylation by active Plx1 causes both GST-T7-Cdc14 and 1 + 156 - T7 - Metlin to migrate shower in SDS-Page. (C) Plx1 can disassemble immunoprecipitated RENT complex. RENT complex was retrieved from myc9-NET1 CDC14-HA3 cell lysate on a resin coated with 9E10 antibodies. The resin was divided into two equal portions, and treated with active (+) or inactive (m) Plx1. Proteins released into the supernatant or bound to the beads were separated by SDS-PAGE and immunoblotted with 9E10, 12CA5, and anti-Sir2 antibodies to detect Net1, Cdc14, and Sir2 proteins, respectively.
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Figure 4: Polo-like kinases can phosphorylate and disrupt the RENT complex in vitro. (A) Cdc5 can disrupt recombinant Net1N/Cdc14 complex. GST-Cdc14 was incubated with anti-T7 beads loaded with His6-T7-Net1N (assuming 100% binding efficiency, each reaction contained ~10 pmol of Cdc14 and ~80 pmol of Net1N), where Net1N consists of the N-terminal 341 amino acids of Net1. The beads were divided into equal portions, and treated with indicated amounts of Cdc5 (~10 pmol/μl). Proteins released into the supernatant (sup) or bound to the beads (bead) were fractionated by SDS-PAGE and immunoblotted with anti-T7 and anti-GST to detect His6-T7-Net1N and GST-Cdc14, respectively. (B) The Polo-like kinase Plx1 can also disrupt Net1N/Cdc14 complex. GST-T7-Cdc14 was captured on anti-GST resins, and incubated with His6-T7-Net1N. The resins were divided into two equal portions, and treated with either active (+) or inactive (m) Plx1. Proteins released from or bound to beads were fractionated by SDS-PAGE and immunoblotted with anti-T7 antibodies to detect both GST-T7-Cdc14 and His6-T7-Net1N. Note that phosphorylation by active Plx1 causes both GST-T7-Cdc14 and 1 + 156 - T7 - Metlin to migrate shower in SDS-Page. (C) Plx1 can disassemble immunoprecipitated RENT complex. RENT complex was retrieved from myc9-NET1 CDC14-HA3 cell lysate on a resin coated with 9E10 antibodies. The resin was divided into two equal portions, and treated with active (+) or inactive (m) Plx1. Proteins released into the supernatant or bound to the beads were separated by SDS-PAGE and immunoblotted with 9E10, 12CA5, and anti-Sir2 antibodies to detect Net1, Cdc14, and Sir2 proteins, respectively.
Mentions: Both Net1 and Cdc14 are extensively phosphorylated by Cdc5 in vitro (Figure 4A). To test if Cdc5 could dislodge Cdc14 from Net1, we devised a simplified in vitro release assay using GST-Cdc14 and His6-T7-Net1N expressed and purified from bacteria. Net1N includes the N-terminal 341 amino acids of Net1 and is sufficient to bind and inhibit Cdc14 [11]. When GST-Cdc14/His6-T7-Net1N complex anchored to anti-T7 beads was treated with ATP plus increasing amounts of recombinant Cdc5 purified from insect cells, both Cdc14 and Net1N were phosphorylated as indicated by their increased apparent molecular weight. Furthermore, Cdc14 was released from bead-bound Net1N to the supernatant (Figure 4A), and free Cdc14 was active as a protein phosphatase (data not shown), suggesting that Cdc5 is sufficient to disengage active Cdc14 from Net1.

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