CYSTEINE-RICH REGION OF RAF-1 INTERACTS WITH ACTIVATOR DOMAIN OF POSTTRANSLATIONALLY MODIFIED HA-RAS

The interaction between ''switch I/effector domain'' of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction, However, the importance of the ''activator domai n'' (approximately corresponding to amino acids 26-28 and 40-49) of Ha -Ras and of the ''cysteine-rich region'' (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interact s directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras ca rrying mutations (N26G and V45E) in the activator domain failed to bin d CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carryi ng mutations in the switch I/effector domain exhibited severely reduce d ability to bind RBD, whereas their ability to bind CRR was unaffecte d, Mutants that bound to either RBD or CRR alone failed to activate Ra f-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. T hese results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-tran slational modifications of Ha-Ras are required for this interaction.