Coassociation of Rap1A and Ha-Ras with Raf-1 N-terminal Region Interferes with Ras-dependent Activation of Raf-1

Abstract

Raf-1 is a major downstream effector of mammalian Ras. Binding of the effector domain of Ras to the Rasbinding domain of Raf-1 is essential for Ras-dependent Raf-1 activation. However, Rap1A, which has an identical effector domain to that of Ras, cannot activate Raf-1 and even antagonizes 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 was also found with Ha-Ras carrying another Rap1A-type mutation E31K. Both Rap1A and Ha-Ras(E31K) mutant failed to activate Raf-1 and interfered with Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced binding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with Raf-1 N-terminal region through binding to CRR and Ras-binding domain, respectively. These results suggest that Rap1A interferes with Ras-dependent Raf-1 activation by inhibiting binding of Ras to Raf-1 CRR.Ras belongs to a family of small GTP-binding proteins playing essential roles in cell proliferation and differentiation. Mammalian ras genes carrying activating mutations are found in many types of neoplastic tissue and are able to induce morphological transformation in vitro when transfected into fibroblast cell lines. However, the rap1A gene (1), encoding a 21-kDa GTP-binding protein with high homology to Ras, has been shown to to induce reversion of the transformed phenotype in Ki-ras-transformed NIH3T3 cells (2). In addition to the overall structural homology, Rap1A shares two important structural features with Ras. One is that Rap1A has an identical effector domain (amino acids 32-40) to that of Ras. The effector domain of Ras is essential for the association with and activation of its effectors (3). The other is that Rap1A undergoes similar post-translational modification to Ras at its C terminus except that Ras is farnesylated and Rap1A is geranylgeranylated (4). This modification is essential for the function of Rap1A as observed for Ras (5, 6).Raf-1, a serine/threonine kinase regulating the mitogen-activated protein kinase cascade, is a major mammalian Ras effector and is thought to play a key role in Ras-induced cellular transformation (7). Although the precise mechanism of Rasdependent Raf-1 activation remains unclear, it is known that the effector domain of Ras interacts with the N-terminal RBD 1 (amino acids 51-131) of Raf-1 and that this interaction is essential for physical association between these proteins as well as for the activation of Raf-1 (7). Rap1A, too, has been shown to associate with Raf-1 N-terminal fragment in vivo (8), and a recent x-ray diffraction study of the crystal of the complex between Rap1A and Raf-1 RBD has provided evidence for this associati...

Genomic Mechanisms of p210BCR-ABL Signaling

“…We find that p210 BCR-ABL also strongly induces expression of RAP-GAP, a p21 rap1 -specific GTPase. Rap1 is a member of the Ras family of small GTPases that antagonizes the growth promoting activity of Ras by competing for binding to Ras effectors, such as Raf-1 and p120 Ras-GAP (48). In addition to altering the balance of Ras/Rap GTPase signaling, we note that p210 BCR-ABL reduces abundance of the phospholipase C␥ isoform and up-regulates the phospholipase C␦ isoform by 21-fold (Table II).…”

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

“…The RBD alone is sufficient for the translocation of Raf-1 from the cytosol to the cell membrane, while the CRD is dispensable. However, the CRD is necessary for efficient activation [22][23][24]. The exact role of the CRD and the localization of its binding epitopes in Ras is controversial, but a consistent finding has been that point mutations in the CRD can influence the affinity of binding to Ras and can affect both basal and Rasinduced kinase activity [25,26].…”

“…respectively, the Raf activation complex seems to be locked in a refractory state [22]. When the Raf-1 CRD is replaced by the lower-affinity B-Raf CRD, Rap1 is converted into an activator.…”

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

“…The Ras-related proteins TC21, R-Ras, R-Ras3, and Ral have been relatively neglected, but the available evidence implicates them as collaborators of Ras in signal transduction, and, as suggested for Ral, they may be downstream effectors of Ras which serve to propagate and diversify signalling via GTPase cascades (5,24,29). The Rap GTPases are distinct in that they seem to act as repressors of Ras function, possibly by competing with Ras for Raf binding (32).…”