Cysteine-rich Region of Raf-1 Interacts with Activator Domain of Post-translationally Modified Ha-Ras

Abstract

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 domain" (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 interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.Ras belongs to a family of small GTP-binding proteins and plays essential roles in the regulation of cell proliferation and differentiation (for a review, see Ref. 1). Like other GTP-binding proteins, the GTP-bound form of Ras is active and able to interact with its effectors whereas the GDP-bound form is not. X-ray crystallographic studies showed that, on the protein surface of mammalian Ha-Ras, the conformation of two regions named "switch I" (Asp ) flanking the switch I have also been shown to be critical for effector activation (6 -9). These residues have been proposed to constitute the "activator domain" (Fig. 1A) (11). The activator domain is also exposed on the surface of Ha-Ras but its conformation is not much altered by GDP/GTP exchange (10, 11). In addition, post-translational modifications of Ha-Ras have also been shown to be crucial for its biological function (for a review, see Ref. 12). However, it is presently unclear how these individual structural features are involved in effector activation.Raf-1, a 74-kDa cytoplasmic serine/threonine protein kinase regulating the mitogen-activated protein kinase cascade, is one of the major effectors of Ha-Ras (for a review, see Ref. 13). Raf-1 shares three regions of conservation, termed CR1, 1 CR2, and CR3, with other Raf isoforms and homologs (Fig. 1B) (13). CR1 and CR2 are located in the N-terminal half of Raf-1, and CR3 corresponds to the C-terminal kinase domain. Activation of Raf-1 by N-terminal truncations indicates that the N-terminal portion plays an important regulatory role (14). The minimal region of Raf-1 responsible for the interaction with Ha-Ras has been mapped into 81 amino acids in CR1, RBD (amino acids 51-131) (15), and mutational analyses have suggested that RBD interacts directly with the switch I of . Howeve...

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

“…Interaction with Ras at the plasma membrane probably has multiple e ects on Raf-1. First of all, Ras association is believed to drive the conformational change necessary to reverse the inhibitory e ects of the amino terminus of Raf-1 (Hu et al, 1995;Luo et al, 1997). Second, docking with Ras brings Raf-1 to the plasma membrane which is the primary site of action for vSrc, the EGF receptor and phorbol esters (Traverse et al, 1993).…”

“…It has been demonstrated that activation of Raf by Ras requires multiple contacts with both the RBD and the zincbinding region (Brtva et al, 1995;Chuang et al, 1994). While the RBD provides a high a nity site for Ras-GTP, the zinc binding region interacts with Rasfarnesyl and is critical for the activation of the protein kinase (Chuang et al, 1994;Hu et al, 1995;Luo et al, 1997). Association with Ras dislodges 14.3.3 from CR2, possibly opening up the molecule for activation by further modi®cation (Clark et al, 1997;Rommel et al, 1996).…”

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

“…But, surprisingly, this interaction does not augment Raf-1's catalytic activity, unless Ras is properly localized at the cell membrane [19]. Ras can interact with two domains in the Raf-1 N-terminus : the Ras-binding domain (RBD ; amino acids and the cysteine-rich domain (CRD ; amino acids [20,21]. The RBD alone is sufficient for the translocation of Raf-1 from the cytosol to the cell membrane, while the CRD is dispensable.…”

“…As Ras can spontaneously form dimers in a lipid bilayer [31], and since dimerization can activate Raf-1 [32,33], such a state could comprise Ras inducing Raf-1 dimerization. An alternative, but not mutually exclusive, possibility is that Ras dimerization is needed for simultaneous binding to the RBD and CRD, which have both been shown to be contacted by the Ras effector domain [21]. A Ras dimer would elegantly resolve the steric dilemma of how the tiny Ras effector domain can engage two different domains in Raf at the same time.…”

Mutation of Ha-Ras C Terminus Changes Effector Pathway Utilization

“…Recent studies show that farnesyl modification of Ha-Ras is important for high affinity interaction with (18,19) and full kinase activity of Raf-1 (20 -22). Farnesylation is also reported to increase in vitro binding of Ha-Ras and KRas4B to p110␥, a PI3-kinase catalytic subunit family member (23).…”