Abstract
Mitosis and cytokinesis are the final stages of the cell cycle where organelles and replicated DNA are equally separated to give rise to two daughter cells. Irreversible progression through these stages is mediated by proteasomal-dependent degradation of critical cell cycle regulatory proteins such as Cyclin A, Cyclin B, and Securin (Peters, 2002). In addition to proteasomal degradation, an important regulator of cytokinesis progression is the small GTPase RhoA (Glotzer, 2001), whose high activity is required for successful completion of cell division. P190 is a RhoA-specific GTPase Activating Protein (GAP) that inhibits RhoA activity by stimulating the hydrolysis of Rho-bound GTP to GDP. Previous work in our laboratory defined a role for p190 in cytokinesis (Su et al., 2003), namely that (1) overexpression of p190 resulted in a multinucleation phenotype, indicative of cytokinesis failure, (2) p190 was localized to the cleavage furrow, where it colocalized with actin and opposed the action of the Rho Guanine Nucleotide Exchange Factor (GEF) Ect2 (Mikawa et al., 2008) and (3) p190 protein levels were observed to decrease drastically in cytokinesis through ubiquitin-mediated proteasomal degradation. Results presented in this dissertation reveal that the GAP activity of p190 is responsible for the observed multinucleation phenotype and that mitotic p190 degradation, and the associated decrease in GAP activity, is required for successful cytokinesis completion, consistent with the requirement for high RhoGTP levels during this stage of the cell cycle. Furthermore, we discovered that four N-terminal lysine residues in p190 iii are required for its mitotic degradation. Preliminary results suggest the involvement of the APC/C cdc20 E3 ligase complex in mitotic p190 ubiquitination. Additionally, p190 was found to affect the localization of the phosphorylated form of myosin II, necessary for contractile activity at the cleavage furrow, likely through association with the molecular scaffold Anillin, which is critical for the organization and stabilization of the contractile ring. Intriguingly, the interaction between these two molecules was contractility dependent, providing an initial indication of a potential mechanosensing mechanism whereby cells can sense appropriate tension along the cleavage furrow and relay that information to RhoA, the master regulator of contractile ring dynamics. Finally, we report the GAP-dependent involvement of p190 in the alignment of metaphase chromosomes at the equator of a dividing cell. This process does not involve RhoA function but rather, appears to be regulated by the Rho family GTPase Cdc42. This observation suggests that p190 may act as a dual-specificity GAP with affinity for Cdc42 during mitosis and for RhoA affinity during cytokinesis. Together, the work presented in this dissertation increases and clarifies our understanding of the role p190 plays in the Rho family-mediated processes necessary for proper cell division.
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