Project Summary/Abstract
Progression through the cell cycle involves spatiotemporal coordination of cytoskeletal and membrane
dynamics with controlled proteolysis events. The anaphase-promoting complex/cyclosome (APC/C) is the main
E3 ubiquitin ligase regulating mitosis. Whereas the temporal control of APC/C-mediated ubiquitination is well
established, the spatial organization of APC/C function is a key uncharacterized dimension to its activity, access
to substrates, and effects on mitosis. We have identified a novel link between phosphoinositides (PIPs), which
form a lipid-based code of membrane identity, the microtubule cytoskeleton, and the APC/C that is mediated by
PLEKHA5, a pleckstrin homology (PH) domain-containing, PIP-binding protein. We discovered PLEKHA5 as a
microtubule- and plasma membrane-localized protein interactor of the APC/C whose depletion by siRNA
antagonizes mitotic progression, causing a buildup of APC/C substrates. We propose that PLEKHA5 regulates
APC/C subcellular localization and thus controls access to key mitotic substrates. Yet, it is unknown which
aspects of PLEKHA5’s molecular properties are required for its cell cycle functions. As well, the localization of
the APC/C at different stages of the cell cycle still remains largely a mystery, as is the effect of PLEKHA5 on
APC/C localization and function. Further, the role of PLEKHA5 in modulating the composition and E3 ligase
activity of the APC/C is unknown. Our long-term research goal is to understand how PIP-sensing proteins read
the dynamically changing lipid composition of membranes and transduce this information to regulate the
localization and function of important proteins in cell signaling. The objective of this proposal is to understand
the molecular events through which PLEKHA5 controls the localization and activity of the APC/C and thus
regulates cell cycle progression. The central hypothesis guiding this work is that PLEKHA5 engages the plasma
membrane and the microtubule cytoskeleton in a spatiotemporally controlled fashion and recruits the APC/C to
these locations to access substrates whose ubiquitination is critical for progression through mitosis. In this
proposal, we will first establish molecular mechanisms governing PLEKHA5 regulation of mitotic entry and
progression by testing the hypothesis that PLEKHA5 localization and interactions with APC/C are important for
its effects on mitosis. In addition, we will determine subcellular localizations of APC/C and the role of PLEKHA5
in controlling APC/C localization and function by developing and applying a suite of “in vivo biochemistry” tools
to assess the localization of APC/C, evaluate its colocalization with PLEKHA5, elucidate the effects of PLEKHA5
depletion on APC/C localization, and ascertain how ectopic localization of PLEKHA5 affects its cell cycle
functions. Finally, we will determine the direct effects of PLEKHA5 on the composition and in vitro E3 ubiquitin
ligase activity of the APC/C. Our studies will establish a new mechanistic framework for understanding how
spatial organization of the ubiquitination machinery affects cellular pathways important for health and disease.