Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro

Abstract

The Ras proteins are key regulators of the growth of eukaryotic cells, but their direct target enzymes, or 'effectors', are unknown. The protein encoded by the c-raf-1 proto-oncogene is thought to function downstream of p21ras because disruption of Raf blocks signalling by Ras in a number of systems. Here we report that the amino-terminal cysteine-rich regulatory region of p74c-raf-1 expressed as a glutathione-S-transferase (GST) fusion protein binds directly to Ras with relatively high affinity (50 nM). The binding is strictly dependent on the Ras protein being in the active GTP-bound conformation rather than the inactive GDP-bound state. Raf-GST interacts with wild-type and oncogenic Ras (Val 12) but fails to interact with a biologically inert effector mutant of Ras (Ala 38) and a dominant negative mutant (Asn 17). A peptide based on the effector region of Ras inhibits the interaction. Raf-GST acts as a potent competitive inhibitor of the GTPase-activating proteins p120GAP and neurofibromin. In addition, Raf itself displays weak GTPase-stimulating activity towards Ras. It is therefore likely that Raf is a direct effector of Ras.

Choline kinase inhibitors as a novel approach for antiproliferative drug design

“…Western blot using an anti-MAPK antibody was performed by standard procedures. Migration sites for p42 and p44 and their phosphorylated, activated forms are indicated While the involvement of the Raf/MAPK pathway in the regulation of cell proliferation is well established, resulting in the regulation of several transcription factors (Moodie et al, 1993;Zhang et al, 1993;Vojtek et al, 1993;Warne et al, 1993), the putative participation of the ras-activated lipid-related pathways is still not completely understood. A consequence of the activation of the PLD/ChoK pathway in ras-transformed cells, is a constitutive increase in the basal levels of PCho which seems critical for cell proliferation (Lacal et al, 1987;Cuadrado et al, 1993;Jime nez et al, 1995).…”

“…Although the Raf kinase constitutes one of the best known e ectors for Ras proteins with which it associates after cell stimulation (Moodie et al, 1993;Zhang et al, 1993;Vojtek et al, 1993;Warne et al, 1993), the precise mechanism for its full activation still remains unknown. Recently, it has been proposed that a lipid-derived molecule might be involved in this process (Dent and Sturgill, 1994).…”

Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol

“…The Ras oncogene also activates the MAP kinase pathway, albeit indirectly, via its down-stream e ector, Raf (reviewed in Avruch et al, 1994;Morrison and Cutler, 1997). The importance of Raf in mediating the oncogenic e ects of Ras is well documented (Leevers et al, 1994;Moodie et al, 1993;Vanaelst et al, 1993;Vojtek et al, 1993;Warne et al, 1993;reviewed in Marshall, 1995b), although other studies suggest that additional, Raf-independent events are also important in Ras-mediated transformation (Khosravi-Far et al, 1996; reviewed in Katz and McCormick, 1997). Besides Ras, other proteins that interact with Raf, and perhaps regulate it, include Mitogen-or extracellular-regulated kinase (MEK1/2); proteins of the 14-3-3 family; molecular chaperones such as HSP90 and Cdc37/p50; and the protein Kinase Suppressor of Ras (KSR) (reviewed in Morrison and Cutler, 1997).…”

Contribution of enhanced transcriptional activation by ER to [12Val] K-Ras mediated NIH3T3 cell transformation

“…Ras protein is activated by growth factors, such as EGF, insulin and IGF. It then triggers the activation of a cascade of serine/threonine kinases, which includes Raf-1 and MAPK (mitogenactivated protein kinases) (Vojek et al, 1993;Warne et al, 1993;Zhang et al, 1993). Activated MAPK then translocates to the nucleus where it phosphorylates and activates transcription factors which, in turn, cause changes in gene expression responsible for growth stimulation.…”