PLEKHA5 Regulates Mitotic Progression by Promoting APC/C Localization to Microtubules

Abstract

Progression through major cell cycle checkpoints involves spatiotemporal coordination of cytoskeletal dynamics with changes in protein activities, chromosomal dynamics, and signaling events. A major role of the ubiquitin–proteasome system is to orchestrate cell cycle progression through temporally controlled protein degradation programs. The anaphase‐promoting complex/cyclosome (APC/C) is the primary E3 ligase controlling all aspects of mitosis, including G2/M transition, early mitosis, the metaphase–anaphase transition, and mitotic exit, via polyubiquitination of important mitotic effector proteins. The temporal control of APC/C‐mediated ubiquitination in these processes is well established, including identities of ubiquitination substrates and accessory factors that modulate E3 ligase activity. Much less is known, however, about the spatial organization of APC/C function, including its potential interplay with the microtubule cytoskeleton, which serves as a signaling platform and whose dynamic behavior is central to advancement through mitosis. Here, we investigate and describe pleckstrin homology domain‐containing family A, member 5 (PLEKHA5) as a new regulator of APC/C function and the ubiquitination pathway in mitosis. We found that PLEKHA5 localized to the microtubule network and interacted with APC/C. PLEKHA5 knockdown antagonized mitotic entry and progression, causing a buildup of APC/C substrates implicated in both the G2/M and metaphase–anaphase transitions, in a manner dependent upon the PLEKHA5 interaction with APC/C. In vitro ubiquitination assays showed that APC/C isolated from mitotic cells lacking PLEKHA5 had lower catalytic activity and a decreased association with its key mitotic co‐activator CDC20. We investigated the dynamic localizations of the APC/C and the potential role of PLEKHA5 in its regulation by developing a TurboID proximity biotinylation tool to assess microtubule localization of endogenous proteins. Microtubule‐targeted TurboIDs revealed that PLEKHA5, APC/C subunits, and the co‐activator CDC20 all localized to microtubules both in interphase and in M phase. Importantly, upon PLEKHA5 knockdown, a pool of APC/C, but not CDC20, lost its microtubule localization, which could explain the decreased APC/C‐CDC20 association. We propose that PLEKHA5 functions as a novel APC/C adaptor to promote its subcellular localization to microtubules and enable APC/C to search for its co‐activator CDC20 more efficiently in one dimension along the microtubule network. In this model PLEKHA5 acts to coordinate the spatial regulation of APC/CCDC20 and facilitate its efficient polyubiquitination of key mitotic effector proteins to ensure proper progression of mitosis.