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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.


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Plx1 abolishes the Cdc14-binding activity of Net1N. Combinations of Plx1-phosphorylated or unphosphorylated Net1N and Cdc14 were tested for their abilities to form protein complexes. All four reactions consisted of identical components and permutations of four steps (1. addition of GST-Cdc14 beads; 2. incubation with Plx1 for 45 min at room temperature; 3. depletion of ATP at room temperature for 20 min; and 4. addition of Net1N). The ATP depletion step ensured that only the component(s) present with Plx1 prior to ATP depletion would be phosphorylated. Thus, Lanes 1 and 5 resulted from 1 -> 2 -> 3 -> 4; Lanes 2 and 6 resulted from 4 -> 2 -> 3 -> 1; Lanes 3 and 7 resulted from 1 -> 4 -> 2 -> 3; and Lanes 4 and 8 resulted from 1 -> 4 -> 3 -> 2. Whether Cdc14 or Net1 was phosphorylated by Plx1 in the presence of ATP (+) or unphosphorylated because of depletion of ATP (-), was indicated above each lane. All reactions were terminated by a final binding reaction at 4°C for 1 hr, and proteins in the supernatant (sup.) and bead fractions were separated by SDS-PAGE and immunoblotted with anti-T7 antibodies to detect both GST-T7-Cdc14 and His6-T7-Net1N.
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Figure 5: Plx1 abolishes the Cdc14-binding activity of Net1N. Combinations of Plx1-phosphorylated or unphosphorylated Net1N and Cdc14 were tested for their abilities to form protein complexes. All four reactions consisted of identical components and permutations of four steps (1. addition of GST-Cdc14 beads; 2. incubation with Plx1 for 45 min at room temperature; 3. depletion of ATP at room temperature for 20 min; and 4. addition of Net1N). The ATP depletion step ensured that only the component(s) present with Plx1 prior to ATP depletion would be phosphorylated. Thus, Lanes 1 and 5 resulted from 1 -> 2 -> 3 -> 4; Lanes 2 and 6 resulted from 4 -> 2 -> 3 -> 1; Lanes 3 and 7 resulted from 1 -> 4 -> 2 -> 3; and Lanes 4 and 8 resulted from 1 -> 4 -> 3 -> 2. Whether Cdc14 or Net1 was phosphorylated by Plx1 in the presence of ATP (+) or unphosphorylated because of depletion of ATP (-), was indicated above each lane. All reactions were terminated by a final binding reaction at 4°C for 1 hr, and proteins in the supernatant (sup.) and bead fractions were separated by SDS-PAGE and immunoblotted with anti-T7 antibodies to detect both GST-T7-Cdc14 and His6-T7-Net1N.

Mentions: Polo-like kinases phosphorylated and disrupted Net1N/Cdc14 complex, raising the question whether disassembly of the complex in vitro required phosphorylation of Net1N or Cdc14 or both. To address this question, we tested the binding affinity of combinations of Plx1-phosphorylated or unphosphorylated Net1N and Cdc14. Unmodified Net1N bound equally well to beads harboring phosphorylated or unphosphorylated Cdc14 (Figure 5, Lanes 5 and 8, respectively). To the contrary, phosphorylated Net1N remained in the supernatant (Figure 5, Lanes 2 and 3), and failed to bind to Cdc14 beads (Figure 5, Lanes 6 and 7). Thus, we surmise that Polo-like kinase disassembles RENT in vitro by eliminating the Cdc14-affinity of 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)

Plx1 abolishes the Cdc14-binding activity of Net1N. Combinations of Plx1-phosphorylated or unphosphorylated Net1N and Cdc14 were tested for their abilities to form protein complexes. All four reactions consisted of identical components and permutations of four steps (1. addition of GST-Cdc14 beads; 2. incubation with Plx1 for 45 min at room temperature; 3. depletion of ATP at room temperature for 20 min; and 4. addition of Net1N). The ATP depletion step ensured that only the component(s) present with Plx1 prior to ATP depletion would be phosphorylated. Thus, Lanes 1 and 5 resulted from 1 -> 2 -> 3 -> 4; Lanes 2 and 6 resulted from 4 -> 2 -> 3 -> 1; Lanes 3 and 7 resulted from 1 -> 4 -> 2 -> 3; and Lanes 4 and 8 resulted from 1 -> 4 -> 3 -> 2. Whether Cdc14 or Net1 was phosphorylated by Plx1 in the presence of ATP (+) or unphosphorylated because of depletion of ATP (-), was indicated above each lane. All reactions were terminated by a final binding reaction at 4°C for 1 hr, and proteins in the supernatant (sup.) and bead fractions were separated by SDS-PAGE and immunoblotted with anti-T7 antibodies to detect both GST-T7-Cdc14 and His6-T7-Net1N.
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Figure 5: Plx1 abolishes the Cdc14-binding activity of Net1N. Combinations of Plx1-phosphorylated or unphosphorylated Net1N and Cdc14 were tested for their abilities to form protein complexes. All four reactions consisted of identical components and permutations of four steps (1. addition of GST-Cdc14 beads; 2. incubation with Plx1 for 45 min at room temperature; 3. depletion of ATP at room temperature for 20 min; and 4. addition of Net1N). The ATP depletion step ensured that only the component(s) present with Plx1 prior to ATP depletion would be phosphorylated. Thus, Lanes 1 and 5 resulted from 1 -> 2 -> 3 -> 4; Lanes 2 and 6 resulted from 4 -> 2 -> 3 -> 1; Lanes 3 and 7 resulted from 1 -> 4 -> 2 -> 3; and Lanes 4 and 8 resulted from 1 -> 4 -> 3 -> 2. Whether Cdc14 or Net1 was phosphorylated by Plx1 in the presence of ATP (+) or unphosphorylated because of depletion of ATP (-), was indicated above each lane. All reactions were terminated by a final binding reaction at 4°C for 1 hr, and proteins in the supernatant (sup.) and bead fractions were separated by SDS-PAGE and immunoblotted with anti-T7 antibodies to detect both GST-T7-Cdc14 and His6-T7-Net1N.
Mentions: Polo-like kinases phosphorylated and disrupted Net1N/Cdc14 complex, raising the question whether disassembly of the complex in vitro required phosphorylation of Net1N or Cdc14 or both. To address this question, we tested the binding affinity of combinations of Plx1-phosphorylated or unphosphorylated Net1N and Cdc14. Unmodified Net1N bound equally well to beads harboring phosphorylated or unphosphorylated Cdc14 (Figure 5, Lanes 5 and 8, respectively). To the contrary, phosphorylated Net1N remained in the supernatant (Figure 5, Lanes 2 and 3), and failed to bind to Cdc14 beads (Figure 5, Lanes 6 and 7). Thus, we surmise that Polo-like kinase disassembles RENT in vitro by eliminating the Cdc14-affinity of 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