Characterization of Acm1, a novel pseudosubstrate inhibitor of the anaphase-promoting complex

Eunyoung Choi, Purdue University

Abstract

In Eukaryotes, cell cycle-dependent protein proteolysis is a hallmark of the cell cycle and is mediated by the ubiquitin-proteasome pathway. Cdh1 is an important cell cycle regulator that activates the anaphase-promoting complex (APC), an E3 ubiquitin ligase that promotes mitotic exit and establishes a G1 phase by targeting specific substrate proteins for proteolysis. Cdh1 is kept inactive from late G1 until late mitosis by multiple mechanisms, including phosphorylation, subcellular localization, proteolysis, and binding of inhibitors. Our lab discovered the complex that contains Cdh1, Acm1, and 14-3-3 proteins and it forms only when Cdh1 is inactive from S phase until late mitosis. Acm1 is an inhibitor of Cdh1 in vivo and in vitro. Its inhibitory activity is independent of CDK phosphorylation and 14-3-3 protein binding. Acm1 uses two APC degron-like sequences, a destruction box and KEN box, to interact with the substrate recognition domain of Cdh1. Although Acm1 resembles APCCdh1 substrates in several respects including its expression profile during the cell cycle, its ability to associate with Cdh1, and the presence of conserved destruction motifs in its primary sequence, the stability of Acm1 is independent of APC activity, Cdh1, and the destruction motifs. Therefore, I conclude that Acm1 acts as a pseudosubstrate inhibitor of APCCdh1—it mimics a substrate and blocks the association of Cdh1 with true substrates. Acm1 itself is regulated by proteolysis in late mitosis around the time APCCdh1 becomes activated. However Acm1 is not a substrate of APC. The fact that Acm1 is strongly stabilized by CDK phosphorylation and proteasome inhibition suggested another E3 ligase is responsible for Acm1 proteolysis and that it is sensitive to Acm1 phosphorylation status. Surprisingly, my results suggested that no known E3 ubiquitin ligases were required for Acm1 proteolysis. In addition, Acm1 stability was independent of poly-ubiquitin chain formation and ubiquitin acceptor sites, suggesting that Acm1 is recognized directly by the proteasome without the need for the ubiquitin system. To my knowledge, Acm1 represents a unique case of cell cycle-dependent proteolysis that is independent of the conventional ubiquitin-proteasome pathway.

Degree

Ph.D.

Advisors

Hall, Purdue University.

Subject Area

Molecular biology|Cellular biology|Biochemistry

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