The Strength of Interaction at the Raf Cysteine-Rich Domain Is a Critical Determinant of Response of Raf to Ras Family Small GTPases

Abstract

To be fully activated at the plasma membrane, Raf-1 must establish two distinct 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 activation of Raf-1. We proposed previously that this property of Rap1A may be attributable to its greatly enhanced interaction with Raf-1 CRD compared to Ras. On the other hand, B-Raf, another Raf family member, is activatable by both Ras and Rap1A. When interactions with Ras and Rap1A were measured, B-Raf 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-Raf, which clearly indicated that the nature of CRD determines the specificity 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-1 CRD, lost its response to Rap1A. Finally, a B-Raf CRD mutant whose interaction with Rap1A is selectively enhanced was isolated and found to possess the double mutation K252E/M278T. B-Raf carrying this mutation was not activated by Rap1A but retained its response to Ras. These results indicate that the strength of interaction with Ras and Rap1A at its CRD may be a critical determinant of regulation of the Raf kinase activity by the Ras family small GTPases.Raf-1 is a serine/threonine kinase that plays a pivotal role in conveying a signal from receptor tyrosine kinases and Ras to the mitogen-activated protein kinase (MAPK) cascade. Although it is well established that Raf-1 interacts directly with the GTP-bound active form of Ras, the precise mechanism by which Raf-1 is activated by interaction with Ras is not known (for reviews, see references 1, 4, and 27). In addition to Raf-1, which is found in a variety of mammalian tissues, two close homologues, B-Raf and A-Raf, have been identified. These exhibit more limited tissue distribution: B-Raf expression is confined to the brain and testis, and A-Raf is expressed most abundantly in the ovary and epididymis (42). All Raf kinases possess three distinct regions designated conserved region 1 (CR1), CR2, and CR3 (for a review, see reference 10). The N-terminal regulatory domain contains CR1 and CR2, while the C-terminal CR3 corresponds to the protein kinase catalytic domain. CR1 consists of two distinct structural modules called the Ras-binding domain (RBD; residues 51 to 131) and the cysteine-rich domain (CRD; residues 139 to 184). RBD has a ubiquitin superfold (30) and constitutes a principal interface for GTP-dependent interaction with Ras and Rap1A (5, 46, 49). CRD consists of two zinc finger motifs resembling the C1 domain of protein kinase C, a phorbol ester-binding site (28). Constitutive activation of Raf-1 by N-terminal truncations indicated that the N-terminal reg...

Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions

“…When the Raf-1 CRD is replaced by the lower-affinity B-Raf CRD, Rap1 is converted into an activator. Likewise, the Raf-1 CRD incorporated into B-Raf renders B-Raf insensitive to Rap1 activation [48]. These experiments imply that the conformational changes resulting in Raf activation must accommodate dynamic flexibility.…”

“…B-Raf possesses a ' phosphomimetic ' aspartate at the position equivalent to tyrosine-341 in Raf-1 and features constitutive phosphorylation of the serine-338 counterpart [38]. However, an elegant study using domainswapping experiments between Raf-1 and B-Raf has also shown that protein association via the CRD is crucial for this differential response [48]. These experiments were designed to find out why B-Raf is activated by Rap1, a Ras family member that blocks Raf-1 activation by Ras when overexpressed.…”

The Requirement of Specific Membrane Domains for Raf-1 Phosphorylation and Activation

“…Unlike Raf-1, B-Raf can be activated by the small G protein Rap1 (44). Because of this, Rap1 can activate MEK and ERK in B-Raf-expressing cells (45)(46)(47).…”

“…It has been proposed that sequences within the cysteine-rich domain (CRD) of Raf-1 and B-Raf dictated the contrasting actions of Rap1 on each Raf isoform (44). However, the ability of Rap1 to activate RafY341D, as well as the ability of Rap/Ras chimeras to activate wild type Raf-1, both argue strongly that the interactions between Rap1 and Raf-1 are not the only determinants of Raf-1 inhibition.…”

“…phorylating Raf-1, suggesting that Ras also provides an activation function that is distinct from localization (22,59,65). Furthermore, the lack of activation of B-RafYY by Rap1 suggests that interactions between the B-Raf CRD and Rap1 are also not sufficient to promote activation, although they may be important (44). We propose that the carboxyl-terminal domain of Rap1 provides specificity to Rap1 signaling in addition to that provided through the interaction between Rap1's effector loop and the Raf CRDs.…”