Mechanism of cellular transformation by the GTP-binding protein CDC42 and its activator, the guanine nucleotide exchange factor, DBL.

摘要

Cdc42 is a small GTPase of the Rho family, which regulates multiple signaling pathways that influence cell morphology, polarity, and cell-cycle progression. While an essential role for Cdc42 in cell growth regulation has been suggested by the finding that the Dbl oncoprotein is an upstream activator of Cdc42, expression of GTPase-defective forms of Cdc42 typically inhibits cell growth. Here, I report a novel mutant of Cdc42, Cdc42(F28L), which is capable of spontaneously exchanging GDP for GTP, while maintaining a normal GTP hydrolytic activity, and thus mimics the functional effects of the Dbl oncoprotein. Cdc42(F28L) is constitutively active in vivo . NIH 3T3 cells stably transfected with Cdc42(F28L) exhibit several hall-marks of transformation. These findings are consistent with the importance of Cdc42 undergoing a complete GTP-binding/GTPase cycle in order to mediate cell growth regulation and induce malignant transformation.;I also examine the corresponding mutants in Rac and Rho, and determine how each individual Rho GTPases contribute to Dbl-induced cellular transformation. I find that Cdc42, Rac and RhoA appear to mediate different aspects of Dbl transformation. Specifically, Cdc42 mediates anchorage-independent growth, Rho causes a loss of contact inhibition, and Rac stimulates the formation of lamellipodia.;Comparisons of the biochemical activities and cellular effects of proto- and oncogenic Dbl have been performed in order to better understand how the GEF and transformation activities of Dbl are regulated. The negative regulation of GEF activity by the amino terminal region within the full length proto-Dbl molecule only occurs in vivo, suggesting that this regulatory effect relies on other cellular factor(s). My present studies show that it may be due to the distinct cellular localizations of proto- and oncogenic Dbl.;The downstream targets for Cdc42 which mediate cellular transformation are still unknown. Among the known targets of Cdc42, some earlier studies suggested that IQGAP might be a candidate for mediating Cdc42-induced cell growth regulation. However, my studies show that while IQGAP may play an essential role in Cdc42- (and Rac-) stimulated actin cytoskeleton change necessary for membrane ruffles, this target is not involved in Cdc42-induced malignant transformation.