Function of the Rho family GTPases in Ras-stimulated Raf activation

Ras plays an essential role in activation of Raf kinase which is directly r esponsible for activation of the MEK-ERK kinase pathway. A direct protein-p rotein interaction between Ras and the N-terminal regulatory domain of Raf is critical for Raf activation. However, association with Ras is not suffic ient to activate Raf in vitro, indicating that Ras must activate some other biochemical events leading to activation of Raf. We have observed that Ras V12Y32F and RasV12T35S mutants fail to activate Raf, yet retain the ability to interact with Raf. In this report, we showed that RasV12Y32F and RasV12 T35S can cooperate with members of the Rho family GTPases to activate Raf w hile alone the Rho family GTPase is not effective in Raf activation. A domi nant negative mutant of Rac or RhoA can block Raf activation by Ras. The ef fect of Rac or Cdc42 can be substituted by the Pak kinase, which is a direc t downstream target of Rac/Cdc42. Furthermore, expression of a kinase inact ive mutant of Pak or the N-terminal inhibitory domain of Pak1 can block the effect of Rac or Cdc42. In contrast, Pak appears to play no direct role in relaying the signal from RhoA to Raf, indicating that RhoA utilizes a diff erent mechanism than Rac/Cdc42. Membrane-associated but not cytoplasmic Raf can be activated by Rac or RhoA. Our data support a model by which the Rho family small GTPases play an important role to mediate the activation of R af by Ras. Ras, at least, has two distinct functions in Raf activation, rec ruitment of Raf to the plasma membrane by direct binding and stimulation of Raf activating kinases via the Rho family GTPases.