Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways

yRad and RhoA are members of the Rho family of Ras-related proteins and function as regulators of actin cytoskeletal organization, gene expression, and cell cycle progression, Constitutive activation of Rad and RhoA causes tumorigenic transformation of NIH 3T3 cells, and their functions may be required for full Ras transformation. The effecters by which Rad and RhoA mediate these diverse activities, as well as the interrelationship between these events, remain poorly understood, Rad is distinct from RhoA in its ability to bind and activate the p65 PAK serine/threonine kinase, to induce lamellipodia and membrane ruffling, and to activate the c-Jun NH2-terminal kinase (JNK). To assess the role of PAK in Rad function, we identified effector domain mutants of Rad and Rac1-RhoA chimeric proteins that no longer bound PAK. Surprisingly, PAK binding was dispensable for Rad-induced transformation and lamellipodium formation, as well as activation of JNK, p38, and serum response factor (SRF). However, the ability of Rad to bind to and activate PAK correlated with its ability to stimulate transcription from the cyclin DI promoter, Furthermore, Rad activation of JNK or SRF, or induction of lamellipodia, was neither necessary nor sufficient for Rad transforming activity, Finally, the signaling pathways that mediate Rad activation of SRF or JNK were distinct from those that mediate Rad induction of lamellipodia, Taken together, these observations suggest that Rad regulates at least four distinct effector-mediated functions and that multiple pathways may contribute to Rad-induced cellular transformation.