Although cytokinesis is the only visually observable step in cell cycle and thus a number of studies were reported, its molecular mechanisms are still uncertain. To delineate the signaling pathways regulating cytokinesis, we knocked down all candidate signaling molecules singly or in combination using RNA interference. These analyses have established the following signaling pathway of cytokinesis: The initiation of cytokinesis is triggered by the specific interaction of ECT2 with two known central spindle-associated proteins, MKLP1 and MgcRacGAP. This association results in the translocation of ECT2 to the central spindle, and the translocated ECT2 then induces the accumulation of GTP-bound RhoA by guanine nucleotide exchange. The GTP-bound RhoA binds to and activates a Rho target, citron kinase, which in turn phosphorylates myosin regulatory light chain. This phosphorylation induces contraction of the actomyosin contractile ring, leading to cleavage furrow ingression. It would be very interesting to understand how the initiation of cytokinesis is regulated by the cell cycle machinery. One of the well known biochemical events at metaphase-anaphase transition is degradation of cyclin B, a regulatory subunit of Cdk1. When Cdk1 was pharmacologically inhibited in M phase-synchronized cells, these cells exhibited an amazing phenotype: precautious initiation of cytokinesis even without chromosome segregation. Subsequent studies have established that Cdk1 negatively regulates cytokinesis, and inactivation of Cdk1 is sufficient to induce cytokinesis. To examine whether ECT2 is regulated by Cdk1, we examined ECT2 phosphorylation. We found that ECT2 is phosphorylated by Cdk1 at several sites including Thr-412 and Thr-341. Phosphorylation of ECT2 at Thr-412 induces binding of another mitotic kinase Plk1, which appears to activate ECT2 by phosphorylation. Phosphorylation of ECT2 at another site, Thr-341, results in a conformational change of ECT2, which can induce the association of ECT2 with its effectors to initiate cytokinesis.
