Our long-term goal is to understand the molecular mechanism(s) that regulate(s) cytokinesis in mammalian cells, and to use this knowledge to develop novel cancer therapeutic strategies. Dysfunctional regulation of cytokinesis can lead to uncontrolled cell proliferation, a key characteristic of cancer. The integrity of cytokinesis relies on the precise coordinated regulation of the positioning and timing of contractile ring assembly. One of the prevalent models is that signals from the central spindle determine the positioning and timing of contractile ring assembly. Polo-like kinase 1 (Plk1) and aurora kinase B (aurora B) are two important mitotic kinases, which localize to the central spindle and coordinate with small GTPase signaling to regulate contractile ring assembly. The small GTPase proteins, such as RhoA, Rac1, and Cdc42, are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Thus far, at least three GEFs, i.e. ECT2, GEF-H1, and MyoGEF, are implicated in regulating cytokinesis. However, it is still not clear why cells need three GEFs for cytokinesis and how they cooperate to regulate equatorial RhoA activation and localization. The overall objective of this grant application is to understand how MyoGEF regulates cytokinesis and why mammalian cells need more than one GEF for cytokinesis. Our central hypothesis is that Plk1 and aurora B coordinate to recruit MyoGEF to the central spindle and cleavage furrow, where MyoGEF cooperates with ECT2 and/or GEF-H1 to regulate RhoA activation and localization at the cleavage furrow. We propose two aims to test our central hypothesis, by using a combination of biochemistry, cell biology, and molecular biology, with mammalian cells as a model. In Specific Aim #1, we will elucidate how Plk1 and aurora B coordinate with MyoGEF in regulating cytokinesis. This aim will test our working hypothesis that, during anaphase, aurora B creates a Plk1 docking site in MyoGEF and allows Plk1 to bind and phosphorylate MyoGEF, in turn promoting the recruitments of MyoGEF to the central spindle, thus contributing to the regulation of equatorial RhoA activation and myosin contractile ring assembly. In Specific Aim #2, we will determine why mammalian cells need more than one GEF for cytokinesis. This aim will test our working hypothesis: 1) MyoGEF, ECT2, and/or GEF-H1 redundantly regulate cytokinesis in certain cells;2) MyoGEF, ECT2, and/or GEF-H1 are implicated in cell type-specific regulation of cytokinesis;3) MyoGEF, ECT2, and GEF-H1 coordinate the regulation of equatorial RhoA activation and localization.