Related Experiment Video
Updated: Jun 3, 2026
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1
Janet L Burton1, Yong Xiong, Mark J Solomon
1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Abstract:
The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40-containing APC co-activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APC(Cdh1) inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A-motif that interacts with Cdh1 and together with the DB and KEN box is required for APC(Cdh1) inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A-motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APC(Cdh1) whereas lysine removal from the APC substrate Hsl1 converted it into a potent APC(Cdh1) inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APC(Cdh1).
Insights
The anaphase promoting complex (APC) controls cell cycle progression. Researchers found that the inhibitor Acm1 binds Cdh1 tightly, preventing substrate ubiquitination and cell cycle regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The anaphase promoting complex (APC) is a crucial ubiquitin ligase regulating the cell cycle.
- APC co-activators Cdc20 and Cdh1 recruit substrates via destruction boxes (DB) and KEN boxes for ubiquitination.
- Acm1 inhibits APC(Cdh1) by binding Cdh1 but is not itself ubiquitinated.
Purpose of the Study:
- To elucidate the molecular mechanisms differentiating APC substrates from inhibitors.
- To identify key interaction motifs and residues involved in APC(Cdh1) inhibition by Acm1.
Main Methods:
- Investigated Acm1-Cdh1 interactions using biochemical and genetic approaches.
- Performed genetic screens to identify Cdh1 residues critical for Acm1 binding and inhibition.
- Utilized site-directed mutagenesis on Acm1 and the substrate Hsl1 to assess ubiquitination and inhibition.
Main Results:
- Identified the Acm1 A-motif, essential with DB and KEN box for APC(Cdh1) inhibition.
- Discovered Cdh1 WD40 domain residues crucial for Acm1 A-motif interaction, distinct from DB recognition sites.
- Demonstrated that specific lysine mutations in Acm1 promote its ubiquitination, while lysine removal from Hsl1 enhances its inhibitory potential.
Conclusions:
- APC(Cdh1) inhibition involves tight Cdh1 binding by pseudosubstrates like Acm1.
- Inaccessibility of ubiquitinatable lysines on the inhibitor contributes to its pseudosubstrate function.
- Understanding these mechanisms offers insights into cell cycle control and potential therapeutic targets.
More Related Videos
Related Concept Videos
00:50Anaphase Promoting Complex
00:50Anaphase Promoting Complex
02:17Separation of Sister Chromatids
At the onset of anaphase, separase, a proteolytic enzyme, is...
02:19The Spindle Assembly Checkpoint
Many proteins function together to control the spindle assembly checkpoint. Mutations affecting these proteins may allow cells to proceed into anaphase prematurely, resulting in the...
02:57Attachment of Sister Chromatids
01:39Anaphase A and B
Plus-end depolymerization releases tubulin heterodimers from the terminal region of the microtubule. As tubulin subunits are lost, the Ndc80 complexes detach...