Complexes of Ras⋅GTP with Raf-1 and Mitogen-Activated Protein Kinase Kinase

Abstract

The guanosine triphosphate (GTP)-binding protein Ras functions in regulating growth and differentiation; however, little is known about the protein interactions that bring about its biological activity. Wild-type Ras or mutant forms of Ras were covalently attached to an insoluble matrix and then used to examine the interaction of signaling proteins with Ras. Forms of Ras activated either by mutation (Gly12Val) or by binding of the GTP analog, guanylyl-imidodiphosphate (GMP-PNP) interacted specifically with Raf-1 whereas an effector domain mutant, Ile36Ala, failed to interact with Raf-1. Mitogen-activated protein kinase (MAP kinase) activity was only associated with activated forms of Ras. The specific interaction of activated Ras with active MAP kinase kinase (MAPKK) was confirmed by direct assays. Thus the forming of complexes containing MAPKK activity and Raf-1 protein are dependent upon the activity 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).…”

New insights into protein‐tyrosine kinase receptor signaling complexes

“…The remaining black box in this pathway between the cell surface and the nucleus also appears to have been resolved because complexes containing activated (Gly 12 Val mutants) or GTP-bound forms of Ras, together with the downstream protein kinase, Raf-1, and MAP kinase kinase have been isolated (Moodie et al, 1993). It is not clear whether this is a direct interaction between activated Ras proteins and these protein kinases, or whether the interaction is mediated by an as yet unidentified Ras target protein.…”

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).…”