Activation of the Raf/MAP kinase cascade by the Ras-related protein TC21 is required for the TC21-mediated transformation of NIH 3T3 cells

Abstract

TC21 is a member of the Ras superfamily of small GTP-binding proteins and, like Ras, has been implicated in the regulation of growth-stimulating pathways. Point mutations introduced into TC21 based on equivalent H-Ras oncogenic mutations are transforming in cultured cells, and oncogenic mutations in TC21 have been isolated from several human tumours. The mechanism of TC21 signalling in transformation is poorly understood. While activation of the serine/threonine kinases Raf-1 and B-Raf has been implicated in signalling pathways leading to transformation by H-Ras, it has been argued that TC21 does not activate Raf-1 or B-Raf. Since the Raf-signalling pathway is important in transformation by other Ras proteins, we assessed whether the Raf pathway is important to transformation by TC21. Raf-1 and B-Raf are constitutively active in TC21-transformed cells and the ERK/MAPK cascade is required for the maintenance of the transformed state. We demonstrate that oncogenic V23 TC21, like Ras, interacts with Raf-1 and B-Raf (but not with A-Raf), resulting in the translocation of the Raf proteins to the plasma membrane and in their activation. Furthermore, using point mutations in the effector loop of TC21, we show that the interaction of TC21 with Raf-1 is crucial for transformation.

P21Cip1 induced by Raf is associated with increased Cdk4 activity in hematopoietic cells

“…This experiment was performed three times and similar results were observed Even though all three Raf genes can be activated by oncogenic Ras and target the same downstream molecules to pass on their signals, they have di erent biological and biochemical properties and their functions are noncompensatable (Muszynski et al, 1995;Pritchard et al, 1995;Marias et al, 1997). A-Raf and Raf-1 behave in a more similar fashion than B-Raf (Fabian et al, 1993;Marias et al, 1995Marias et al, , 1997Pritchard and McMahon, 1997;Vossler et al, 1997;Hagemann and Rapp, 1999;Rosario et al, 1999). In our studies, we observed that after 24-h of b-estradiol deprivation, ectopic activation of either Raf-1 or A-Raf were able to induce cell proliferation.…”

“…In addition, in murine cells the kinase activities of both Raf-1 and A-Raf were stimulated by Ras and Src. However, B-Raf kinase activity was controlled by Ras and other members of the small G protein family such as Rap1 in PC12 cells (Vossler et al, 1997) or TC21 in NIH3T3 cells (Marias et al, 1997;Rosario et al, 1999).…”

A different function for a critical tryptophan in c-Raf and Hck

“…The Ras to Raf to MEK to MAPK pathway has been thought to be a simple linear pathway. Recently, COT1 has been shown to activate MEK (Hagemann et al, 1999), TC21 has been shown to activate Raf (Rosario et al, 1999), MKP-1 regulates c-Raf and MAPK (Shapiro and Ahn, 1998). Clearly there are`branches' in the pathway and it may be that other molecules may also participate in the MAPK cascade in ways that have not been observed to date.…”

S-Nitrosocysteine Increases Palmitate Turnover on Ha-Ras in NIH 3T3 Cells

“…Both phosphorylated and unphosphorylated ERK1 and ERK2 proteins were detected by immunoblotting with ERK1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The blot membranes were then stripped as described (48) and were reprobed using a monoclonal antibody specific for the phosphorylated forms of ERK1 and ERK2 (New England Biolabs, catalog no. 9106S) followed by development with ECL (Pierce) using the manufacturer's protocol.…”