COASSOCIATION OF RAP1A AND HA-RAS WITH RAF-1 N-TERMINAL REGION INTERFERES WITH RAS-DEPENDENT ACTIVATION OF RAF-1

Raf-1 is a major downstream effector of mammalian Ras. Binding of the effector domain of Ras to the Ras binding domain of Raf-1 is essential for Ras-dependent Raf-1 activation. However, Rap1A, which has an iden tical effector domain to that of Ras, cannot activate Raf-1 and even a ntagonizes several Ras functions in vivo. Recently, we identified the cysteine-rich region (CRR) of Raf-1 as another Ras-binding domain. Ha- Ras proteins carrying mutations N26G and V45E, which failed to bind to CRR, also failed to activate Raf-1, Since these mutations replace Ras residues with those of Rap1A, we examined if Rap1A lacks the ability to bind to CRR, Contrary to the expectation, Rap1A exhibited a greatly enhanced binding to CRR compared with Ha-Ras. Enhanced CRR binding wa s also found with Ha-Ras carrying another Rap1A-type mutation E31K. Bo th Rap1A and Ha Ras(E31K) mutant failed to activate Raf-1 and interfer ed with Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced bi nding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with R af-1 N-terminal region through binding to CRR and Ras-binding domain, respectively. These results suggest that Rap1A interferes with Ras dep endent Raf-1 activation by inhibiting binding of Ras to Raf-1 CRR.