The strength of interaction at the Raf cysteine-rich domain is a critical determinant of response of Raf to Ras family small GTPases

To be fully activated at the plasma membrane, Raf-1 must establish two dist inct modes of interactions with Ras, one through its Ras-binding domain and the other through its cysteine-rich domain (CRD). The Ras homologue Rap1A is incapable of activating Raf-1 and even antagonizes Ras-dependent activat ion of Raf-1. We proposed previously that this property of Rap1A may be att ributable to its greatly enhanced interaction with Raf-1 CRD compared to Ra s. On the other hand, B-Raf, another Raf family member, is activatable by b oth Ras and Rap1A. When interactions with Ras and Rap1A were measured, B-Ra f CRD did not exhibit the enhanced interaction with Rap1A, suggesting that the strength of interaction at CRDs may account for the differential action of Rap1A on Raf-1 and B-Raf. The importance of the interaction at the CRD is further supported by a domain-shuffling experiment between Raf-1 and B-R af, which clearly indicated that the nature of CRD determines the specifici ty of response to Rap1A: Raf-1, whose CRD is replaced by B-Raf CRD, became activatable by Rap1A, whereas B-Raf, whose CRD is replaced by Raf-l CRD, lo st its response to Rap1A. Finally, a B-Raf CRD mutant whose interaction wit h Rap1A is selectively enhanced was isolated and found to possess the doubl e mutation K252E/M278T. B-Raf carrying this mutation was not activated by R ap1A but retained its response to Ras. These results indicate that the stre ngth of interaction with Ras and Rap1A at its CRD may be a critical determi nant of regulation of the Raf kinase activity by the Ras family small GTPas es.