Spatially distinct inputs modulate the amount of active Mitotic-phase GAP to locally restrict RhoA signaling for successful cell division

At the end of mitosis a contractile ring consisting of filamentous actin (F-actin) assembles at the cell equator and ring constriction equally partitions the cellular content. Inhibitory and stimulatory signaling cascades spatially limit RhoA activity to a narrow central zone to promote F-actin polymerization only at the cell equator. While the stimulatory signal is well-characterized, the mechanisms by which inhibitory signals restrict active RhoA to a narrow equatorial zone and prevent improper RhoA activity at the cell poles are not known. Here, we identify two regulatory inputs that control the activation and cortical targeting of the Mitotic-phase GTPase activating protein (MP-GAP) for RhoA which limits RhoA activity both at the cell poles and at the cell equator. We show that at cell poles, MP-GAP is a direct phosphorylation target of spindle-pole associated Aurora A kinase. We identify three Aurora A target residues in a region of MP-GAP that binds directly to its catalytic GAP domain suggesting the Aurora A phosphorylation releases MP-GAP autoinhibition. We show that phosphorylation of these sites is required to keep RhoA activity low at the cell poles. At the cell equator, F-actin facilitates MP-GAP enrichment, which opposes RhoA GEF activity and thereby accelerates RhoA flux through the GTPase cycle for the formation of a narrow equatorial zone of active RhoA. Thus, the amount of active MP-GAP is modulated by two distinct regulatory inputs that function in spatially restricted locations: Aurora A phosphorylation relieves MP-GAP autoinhibition to limit RhoA activity at the cell poles, while F-actin polymerization promotes MP-GAP targeting to prevent distribution of active RhoA in a broad zone at the cell equator. By determining the mechanism of spatially confining RhoA activity at the equator and the cell poles during cytokinesis, our work has broad implications to how Rho activity zones are formed and maintained during cytokinesis and how defects in their formation impact animal development and disease.