The ECT2 oncogene has been isolated in a search for mitogenic signal transducers in epithelial cells, where a murine keratinocyte expression cDNA library was introduced into fibroblasts to induce foci of morphologically transformed cells. The transforming ECT2 cDNA encodes the C-terminal half of the full-length protein containing Dbl-homology (DH) and pleckstrin homology (PH) domains, which are now found in a number of molecules involved in regulation of the Rho family GTPases. The N-terminal half of full-length ECT2 contains domains related to the cell cycle regulator/checkpoint control proteins including human XRCC1, budding yeast CLB6 and fission yeast Cut5. The Cut5-related domain consists of two BRCT repeats, which are widespread to repair/checkpoint control proteins. We previously found that ECT2 plays a critical role in cytokinesis. In this period, we identified and characterized two signaling molecules that interact with ECT2. Genetic evidence suggested that Pebble, the Drosophila homologue of human ECT2, interacts with Cyk-4, the Drosophila homologue of human MgcRacGAP. We found that expression of a GAP (GTPase activating protein)-deficient mutant (R386A) of MgcRacGAP induces abnormal cortical activity during cytokinesis in U2OS cells. Multiple large blebs were observed in cells expressing MgcRacGAP R386A from the onset of anaphase to the late stage of cell division. When mitotic blebbing was excessive, cytokinesis was inhibited, and cells with micronuclei were generated. It has been reported that blebbing is caused by abnormal cortical activity. The MgcRacGAP R386A-induced abnormal cortical activity was inhibited by the dominant negative form of RhoA, but not Rac1 or Cdc42. Moreover, expression of constitutively active RhoA also induced drastic cortical activity during cytokinesis. Unlike apoptotic blebbing, MgcRacGAP R386A-induced blebbing was not inhibited by the ROCK inhibitor Y-27632, suggesting that MgcRacGAP regulates cortical activity during cytokinesis through a novel signaling pathway. We propose that MgcRacGAP plays a pivotal role in cytokinesis by regulating cortical movement through RhoA. Using a yeast two hybrid system, we isolated a human Kelch-related protein, designated KLEIP, as an ECT2-interacting protein. KLEIP was concentrated at cell-cell contact sites of MDCK cells, where it colocalized with F-actin. KLEIP recruitment and actin assembly were induced around the E-cadherin-coated beads placed on cell surfaces, suggesting the involvement of the E-cadherin ligation signaling in KLEIP recruitment. Constitutively active Rac1 induced, and dominant negative Rac1 inhibited, the recruitment of KLEIP and F-actin to the adhesion sites, suggesting a regulatory role of Rac1 in KLEIP recruitment. Moreover, N-terminal half of KLEIP, that lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited the Rac1-induced actin recruitment. We propose that KLEIP plays a critical role in the establishment of the contact sites by regulating actin assembly during cell-cell adhesion.
