Comprehensive analysis of Rho GTPase signaling network reveals hidden node for steering cell migration

Abstract

Rho family GTPases is a subfamily of Ras superfamily that regulates myriad of cellular events including cell migration and cell polarity. Even though it has been generally perceived that Rac and Cdc42 promote membrane protrusions at the leading edge, whereas RhoA induces retraction at the trailing edge of migrating cells, accumulating evidences suggest that RhoA is also involved in initial events of protrusion, and Rac functions in contractility. As this confusing opposed roles of GTPases might be mediated by different downstream effectors, we have developed ‘GTPase-Trap’, a super-sensitive method to systematically analyze interactions between small GTPases and effectors in living cells. Among 285 interaction pairs of 15 Rho small GTPases and 19 effector proteins, we identified 51 novel interactions out of 148 positive interactions and found out that many GTPases extensively share downstream effectors. Interestingly, we noticed that all three Rac proteins strongly bind to ROCK proteins which is typical downstream effectors of RhoA. Despite of its discovery two decades ago, physiological roles of the interaction between Rac and ROCK are still puzzling. In this study, we propose that RhoA-independent Rac-ROCK interaction is essential for generating contractile arc stress fibers through MLC phosphorylation at the lamella of protruded membrane. Moreover, we discovered a Rac1 mutant selectively diminishing interaction with ROCK and found out that arc fibers induced by Rac-ROCK-MLC pathway are not only important for stabilizing membrane protrusion, but also for turning frequency in the process of cell migration. From leading edge to trailing edge, symphony of orchestrated regulation of ROCK activity is essential for steering in cell migration.