We have isolated several novel oncogenes using an efficient functional cloning system we have developed. These genes include ECT2, OST, TIM and NET1, which share common motifs for the guanine nucleotide exchange factors (GEFs) of the Rho family of small GTPases. The main purpose of this project is to understand the biological functions of these exchange factors. The ECT2 oncogene was isolated from epithelial cells. The transforming ECT2 cDNA encodes the C-terminal half of the full-length protein containing the Rho GEF motif. The N-terminal half of full-length ECT2 contains two repeats of the BRCA1 C-terminal (BRCT) motif, which are widespread to repair/checkpoint control proteins. We previously found that ECT2 plays a critical role in cytokinesis. In this period, we identified Par6 as a binding partner of ECT2 using a yeast two hybrid system. Regulation of cell polarity is an important biological event that governs diverse cell functions such as localization of embryonic determinants and establishment of tissue and organ architecture. The Rho family GTPases and the polarity complex Par6/Par3/atypical PKC play a key role in the signaling pathway. However, the molecules that regulate upstream signaling are still not known. We found that ECT2 interacted with Par6 as well as Par3 and PKC zeta. Coexpression of Par6 and ECT2 efficiently activated Cdc42 in vivo. Overexpression of ECT2 also stimulated PKC zeta activity, whereas dominant-negative ECT2 inhibited the increase of PKC zeta activity stimulated by Par6. ECT2 localization was detected at sites of cell-cell contact as well as in the nucleus of MDCK cells. The expression and localization of ECT2 were regulated by calcium, which is a critical regulator of cell-cell adhesion. Together, these results suggest that ECT2 regulates the polarity complex Par6/Par3/PKC zeta, and possibly plays a role in epithelial cell polarity.The TIM oncogene was isolated from a human mammary epithelial cell line, and encodes a predicted 60-kD protein containing the Rho GEF motif, followed by a SH3 domain. TIM expression in NIH 3T3 cells leads to altered growth properties and tumorigenesis when injected into nude mice. Recent studies indicate that TIM isoforms might play a role in breast tumor progression. In this period, we characterized signaling pathway through TIM. We show that TIM exhibits catalytic activity toward Rac1 and Cdc42 in vitro. However, when expressed in cells, TIM was a potent activator of RhoA. Like activated Rho proteins, expression of TIM potentiated the SRF- and AP-1-regualted transcriptional activities, and activated the SAPK/JNK signaling pathway. In NIH 3T3 cells, TIM induced transforming foci, which was inhibited by the ROCK inhibitor Y-27632 or dominant negative mutants of Rho proteins. Expression of TIM led to pronounced changes in cell shape and the organization of actin cytoskeleton, including the formation of thick stress fibers in cell periphery and cell rounding. TIM also promoted redistribution of vinculin-enriched focal adhesions to cell periphery and increased the phosphorylation of myosin light chain. These results suggest that TIM acts as an upstream regulator for the RhoA/ROCK-mediated cellular functions.
