We have isolated several novel oncogenes using an expression cloning system we have developed. These genes include ect2, ost, TIM, NET1 and NTS, which share common motifs for the guanine nucleotide exchange factors of the Rho family of small GTPases. The main purpose of this project is to understand the biological functions of these exchange factors. Animal cells divide into two daughter cells by the formation of an actomyosin-based contractile ring through a process called cytokinesis. Although many of the structural elements of cytokinesis have been identified, little is known about the signaling pathways and molecular mechanisms underlying this process. In this research period, we found that the human ECT2 protooncogene participates a critical role in the regulation of cytokinesis. We have isolated cDNAs for the human homologue, ECT2, of the previously isolated mouse ect2 protooncogene. The N-terminal half of predicted ECT2 protein exhibited structural similarity to two cell-cycle control proteins, fission yeast Rad4/Cut5 and budding yeast cyclin B6. The central domain of ECT2 contained strucutural motifs for the guanine nucleotide exchange factors of the Rho family of small GTPases. Rho family GTPases, represented by RhoA, Rac1 and Cdc42, act as molecular switches of diverse biological functions involving cytoplasmic actin remodeling. We found that ECT2 catalyzes guanine nucleotide exchange on RhoA, Rac1, and Cdc42 in vitro. Interestingly, ECT2 was phosphorylated during G2 and M phases and phosphorylation was required for its exchange activity. Unlike other known guanine nucleotide exchange factors for Rho GTPases, ECT2 exhibited nuclear localization in interphase, spread throughout the cytoplasm in prometaphase, and was condensed in the midbody during cytokinesis. Expression of an ECT2 derivative, containing the amino- terminal domain required for the midbody localization but lacking the carboxyl-terminal catalytic domain, strongly inhibited cytokinesis. Microinjection of affinity-purified anti-ECT2 antibody into interphase cells also specifically inhibited cytokinesis. These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division.This project has transferred from LCMB to BRL. - cell proliferation, guanine nucleotide exchange factor, oncogene, protein function, protein Kinase, protein phosphorylation, transformation,
