Ubiquitin-mediated proteolytic degradation is fundamental to eukaryotic cell cycle progression. From late mitosis through early G1, the Anaphase Promoting Complex (APC) is essential for cell-cycle relevant proteolytic degradation, and its activity is targeted to appropriate substrates by the evolutionarily conserved coactivators Cdc20 and Cdh1. After an initial wave of APC-Cdc20 activity, APC-Cdh1 degrades multiple mitotic proteins from mitotic exit through G1; inhibitory phosphorylation of Cdh1 by CDK and Polo kinase may allow accumulation of Cdh1 targets in the subsequent cell cycle. I demonstrate lethality of exact endogenous gene replacement of CDH1 with the CDK-unphosphorylatable CDH1-m11 allele; neither polo kinase sites nor polo interaction motifs are required for Cdh1 regulation. CDH1-m11 cells arrest in the first cycle with replicated DNA; ~30% of these cells have bipolar spindles. Construction of bipolar spindles in these cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. CDH1-m11 cells with bipolar spindles fail to progress to anaphase, suggesting that Cdh1 inhibits multiple spindle-regulatory pathways. Expression of undegradable mitotic cyclin causes spindle pole body separation (a key step in bipolar spindle assembly) in CDH1-m11 cells; thus mitotic cyclins are a significant target for Cdh1 with respect to bipolar spindle assembly, and reciprocally, cyclin-Cdk activity is the most significant mechanism for Cdh1 inactivation. Cdc20 has been proposed to be a Cdh1 target, but regulation of Cdc20 proteolysis has been controversial. My experiments demonstrate that degradation of Cdc20 can be dependent on Cdh1 and Cdc20 destruction boxes, but Cdh1- and db-independent modes of Cdc20 proteolysis are also effective in limiting Cdc20 levels. To better understand the mechanisms by which multisite CDK phosphorylation inhibits Cdh1, I employed a novel recombination approach to create a series of partially unphosphorylatable CDH1 alleles ablating contiguous sites beginning at either the N or C terminus. Strains lacking N-terminal phosphorylation sites were strictly dependent upon ACM1 and S-phase cyclins for viability, and a fraction of cells displayed evidence of hyperactive APC-Cdh1, in contrast to a non-overlapping larger set of C-terminal site ablations.
","abstract_html":"<p>Ubiquitin-mediated proteolytic degradation is fundamental to eukaryotic cell cycle progression. From late mitosis through early G1, the Anaphase Promoting Complex (APC) is essential for cell-cycle relevant proteolytic degradation, and its activity is targeted to appropriate substrates by the evolutionarily conserved coactivators Cdc20 and Cdh1. After an initial wave of APC-Cdc20 activity, APC-Cdh1 degrades multiple mitotic proteins from mitotic exit through G1; inhibitory phosphorylation of Cdh1 by CDK and Polo kinase may allow accumulation of Cdh1 targets in the subsequent cell cycle. I demonstrate lethality of exact endogenous gene replacement of CDH1 with the CDK-unphosphorylatable CDH1-m11 allele; neither polo kinase sites nor polo interaction motifs are required for Cdh1 regulation. CDH1-m11 cells arrest in the first cycle with replicated DNA; ~30% of these cells have bipolar spindles. Construction of bipolar spindles in these cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. CDH1-m11 cells with bipolar spindles fail to progress to anaphase, suggesting that Cdh1 inhibits multiple spindle-regulatory pathways. Expression of undegradable mitotic cyclin causes spindle pole body separation (a key step in bipolar spindle assembly) in CDH1-m11 cells; thus mitotic cyclins are a significant target for Cdh1 with respect to bipolar spindle assembly, and reciprocally, cyclin-Cdk activity is the most significant mechanism for Cdh1 inactivation. Cdc20 has been proposed to be a Cdh1 target, but regulation of Cdc20 proteolysis has been controversial. My experiments demonstrate that degradation of Cdc20 can be dependent on Cdh1 and Cdc20 destruction boxes, but Cdh1- and db-independent modes of Cdc20 proteolysis are also effective in limiting Cdc20 levels. To better understand the mechanisms by which multisite CDK phosphorylation inhibits Cdh1, I employed a novel recombination approach to create a series of partially unphosphorylatable CDH1 alleles ablating contiguous sites beginning at either the N or C terminus. Strains lacking N-terminal phosphorylation sites were strictly dependent upon ACM1 and S-phase cyclins for viability, and a fraction of cells displayed evidence of hyperactive APC-Cdh1, in contrast to a non-overlapping larger set of C-terminal site ablations.</p>","abstract_has_math":false,"creators":["Robbins, Jonathan"],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Thesis","degree_discipline":null,"degree_department":null,"school":null,"contributors":["Frederick R. Cross"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2010,"date_issued":"2010-01-01T08:00:00Z","date_published":"2010-01-01T08:00:00Z","updated_at":"2026-07-24T04:10:47Z","subjects":["anaphase promoting complex","APC-Cdh1","CDK phosphorylation","Cdc20","Life Sciences"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/89","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Frederick R. 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From late mitosis through early G1, the Anaphase Promoting Complex (APC) is essential for cell-cycle relevant proteolytic degradation, and its activity is targeted to appropriate substrates by the evolutionarily conserved coactivators Cdc20 and Cdh1. After an initial wave of APC-Cdc20 activity, APC-Cdh1 degrades multiple mitotic proteins from mitotic exit through G1; inhibitory phosphorylation of Cdh1 by CDK and Polo kinase may allow accumulation of Cdh1 targets in the subsequent cell cycle. I demonstrate lethality of exact endogenous gene replacement of CDH1 with the CDK-unphosphorylatable CDH1-m11 allele; neither polo kinase sites nor polo interaction motifs are required for Cdh1 regulation. CDH1-m11 cells arrest in the first cycle with replicated DNA; ~30% of these cells have bipolar spindles. Construction of bipolar spindles in these cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. CDH1-m11 cells with bipolar spindles fail to progress to anaphase, suggesting that Cdh1 inhibits multiple spindle-regulatory pathways. Expression of undegradable mitotic cyclin causes spindle pole body separation (a key step in bipolar spindle assembly) in CDH1-m11 cells; thus mitotic cyclins are a significant target for Cdh1 with respect to bipolar spindle assembly, and reciprocally, cyclin-Cdk activity is the most significant mechanism for Cdh1 inactivation. Cdc20 has been proposed to be a Cdh1 target, but regulation of Cdc20 proteolysis has been controversial. My experiments demonstrate that degradation of Cdc20 can be dependent on Cdh1 and Cdc20 destruction boxes, but Cdh1- and db-independent modes of Cdc20 proteolysis are also effective in limiting Cdc20 levels. To better understand the mechanisms by which multisite CDK phosphorylation inhibits Cdh1, I employed a novel recombination approach to create a series of partially unphosphorylatable CDH1 alleles ablating contiguous sites beginning at either the N or C terminus. Strains lacking N-terminal phosphorylation sites were strictly dependent upon ACM1 and S-phase cyclins for viability, and a fraction of cells displayed evidence of hyperactive APC-Cdh1, in contrast to a non-overlapping larger set of C-terminal site ablations.
"]},{"key":"dc:title","label":"Title","values":["Regulation of Anaphase Promoting Complex Coactivators"]}]}],"canonical_facts":{"dc:contributor":["Frederick R. Cross"],"dc:creator":["Robbins, Jonathan"],"dc:description.abstract":["Ubiquitin-mediated proteolytic degradation is fundamental to eukaryotic cell cycle progression. From late mitosis through early G1, the Anaphase Promoting Complex (APC) is essential for cell-cycle relevant proteolytic degradation, and its activity is targeted to appropriate substrates by the evolutionarily conserved coactivators Cdc20 and Cdh1. After an initial wave of APC-Cdc20 activity, APC-Cdh1 degrades multiple mitotic proteins from mitotic exit through G1; inhibitory phosphorylation of Cdh1 by CDK and Polo kinase may allow accumulation of Cdh1 targets in the subsequent cell cycle. I demonstrate lethality of exact endogenous gene replacement of CDH1 with the CDK-unphosphorylatable CDH1-m11 allele; neither polo kinase sites nor polo interaction motifs are required for Cdh1 regulation. CDH1-m11 cells arrest in the first cycle with replicated DNA; ~30% of these cells have bipolar spindles. Construction of bipolar spindles in these cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. CDH1-m11 cells with bipolar spindles fail to progress to anaphase, suggesting that Cdh1 inhibits multiple spindle-regulatory pathways. Expression of undegradable mitotic cyclin causes spindle pole body separation (a key step in bipolar spindle assembly) in CDH1-m11 cells; thus mitotic cyclins are a significant target for Cdh1 with respect to bipolar spindle assembly, and reciprocally, cyclin-Cdk activity is the most significant mechanism for Cdh1 inactivation. Cdc20 has been proposed to be a Cdh1 target, but regulation of Cdc20 proteolysis has been controversial. My experiments demonstrate that degradation of Cdc20 can be dependent on Cdh1 and Cdc20 destruction boxes, but Cdh1- and db-independent modes of Cdc20 proteolysis are also effective in limiting Cdc20 levels. To better understand the mechanisms by which multisite CDK phosphorylation inhibits Cdh1, I employed a novel recombination approach to create a series of partially unphosphorylatable CDH1 alleles ablating contiguous sites beginning at either the N or C terminus. Strains lacking N-terminal phosphorylation sites were strictly dependent upon ACM1 and S-phase cyclins for viability, and a fraction of cells displayed evidence of hyperactive APC-Cdh1, in contrast to a non-overlapping larger set of C-terminal site ablations.
"],"dc:identifier":["https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/89"],"dc:subject":["anaphase promoting complex","APC-Cdh1","CDK phosphorylation","Cdc20","Life Sciences"],"dc:title":["Regulation of Anaphase Promoting Complex Coactivators"],"thesis:degree_level":["Thesis"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T04:10:47Z"}