| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 101 === Cells contain a surveillance mechanism called the “spindle checkpoint” that monitors spindle attachments on kinetochores, and delays the metaphase-anaphase transition to prevent chromosome mis-segregation. The target of spindle checkpoint is Cdc20, which regulates the metaphase-anaphase transition. The spindle checkpoint regulates the flow of Cdc20 into one of two pathways. One pathway leads to the activation of the APC/C (Anaphase-Promoting Complex/Cyclosome), causing the cell to go into anaphase; the other pathway leads to binding with checkpoint proteins to form the MCC (Mitotic Checkpoint Complex), leading to APC/C inactivation and cell cycle arrest. The molecular details have only been partially defined. To induce cell cycle arrest, Mad2 (Mitotic Arrest Deficient 2) competes with APC/C to bind the MB-motif (Mad2-binding motif) on Cdc20 via a KIRL motif (activator region). Here, we reported that the MB-motif has an additional novel function as an APC/C activator. By characterizing the activities of single amino acid mutants, we found L203 and Y205 in Cdc20 are necessary to activate APC/C, and Y205 and P209 are necessary for Mad2 inhibition. These observations suggest a separation of function for Mad2-inhibition and APC/C activation, and an extension of the currently defined activator region. By screening MB-motif peptides in vitro, “PQ-66”, a peptide including the full MB-motif, was identified as a potential APC/CCdc20 inhibitor. From this observation, we also hypothesize amino acids at the C-terminus of the MB-motif play a role in APC/C binding. We conclude that Mad2-mediates APC/C activity as a competitive inhibitor, which binds with MB-motif to sequester the activator region from the potential APC/C binding site.
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