Two Distinct Raf Domains Mediate Interaction with Ras

Brtva, Teresa R.; Drugan, Jonelle K.; Ghosh, Sujoy; Terrell, Regina S.; Campbell, Sharon L.; Bell, Robert M.; Der, Channing J.

doi:10.1074/jbc.270.17.9809

Cited by 233 publications

(208 citation statements)

References 28 publications

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“…Furthermore, the identi®cation of Ras mutants that bind to Raf-1 with high a nity but are defective in Raf-1 activation, suggested that a direct correlation could not be made between the a nity of Raf-1 to Ras-GTP and Raf-1 activation, as would be expected if the sole role of Ras in Raf activation is its recruitment to the membrane (Akasaka et al, 1996). Hence these observations, coupled with recent ®ndings that Ras interacts with two distinct NH 2 -terminal regions of Raf-1, suggests that Ras promotes more than just membrane translocation of Raf, and instead, may also facilitate subsequent events that lead to Raf-1 activation ( Figure 2) (Brtva et al, 1995;Drugan et al, 1996;. The prevailing data indicate that the ®rst Ras binding site (RBS) in Raf-1 (residues 55 ± 131) is required for Raf-1 translocation by Ras, whereas interactions between processed Ras and the second Ras binding site in the cysteine-rich domain (CRD) of Raf-1 are required for full activation of Raf-1 activity by Ras (Roy et al, 1997;Clark et al, 1995;Mineo et al, 1997).…”

Section: Introductionmentioning

confidence: 53%

Increasing complexity of Ras signaling

et al. 1998

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The initial discovery that ras genes endowed retroviruses with potent carcinogenic properties and the subsequent determination that mutated ras genes were present in a wide variety of human cancers, prompted a strong suspicion that the growth-promoting actions of mutated Ras proteins contribute to their aberrant regulation of growth stimulatory signaling pathways. In 1993, a remarkable convergence of experimental observations from genetic analyses of Drosophila, S. cerevisiae and C. elegans as well as biochemical and biological studies in mammalian cells came together to de®ne a clear role for Ras in signal transduction. What emerged was an elegant linear signaling pathway where Ras functions as a relay switch that is positioned downstream of cell surface receptor tyrosine kinases and upstream of a cytoplasmic cascade of kinases that included the mitogen-activated protein kinases (MAPKs). Activated MAPKs in turn regulated the activities of nuclear transcription factors. Thus, a signaling cascade where every component between the cell surface and the nucleus was de®ned and conserved in worms,¯ies and man. This was a remarkable achievement in our e orts to appreciate how the aberrant function of Ras proteins may contribute to the malignant growth properties of the cancer cell. However, the identi®cation of this pathway has proven to be just the beginning, rather than the culmination, of our understanding of Ras in signal transduction. Instead, we now appreciate that this simple linear pathway represents but a minor component of a very complex signaling circuitry. Ras signaling has emerged to involve a complex array of signaling pathways, where cross-talk, feedback loops, branch points and multi-component signaling complexes are recurring themes. The simplest concept of a signaling cascade, where each component simply relays the same message to the next, is clearly not the case. In this review, we summarize our current understanding of Ras signal transduction with an emphasis on new complexities associated with the recognition and/or activation of cellular e ectors, and the diverse array of signaling pathways mediated by interaction between Ras and Ras-subfamily proteins with multiple e ectors.

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Section: Introductionmentioning

confidence: 53%

Increasing complexity of Ras signaling

et al. 1998

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“…Raf-1 then stimulates MAPK kinases (MEK1 and MEK2), which in turn activate p42 and p44 MAPK. The important role of the Raf/MEK/MAPK pathway in Ras-mediated transformation of ®broblasts is supported by the observations that kinase-de®cient, dominant negative mutants of Raf-1, MEK and MAP kinases are potent inhibitors of Ras signal transduction and transformation (Kolch et al, 1991;Cowley et al, 1994;Meloche et al, 1992;Westwick et al, 1994;Troppmair et al, 1994;Brtva et al, 1995). Furthermore, since constitutively activated mutants of Raf-1 or MEK cause tumorigenic transformation of NIH3T3 cells (Kolch et al, 1991;Leevers et al, 1994;Stokoe et al, 1994;Cowley et al, 1994;Mansour et al, 1994), activation of the Raf-1/MEK/MAPK pathway alone is believed to be su cient to mediate Ras transformation.…”

Section: Introductionmentioning

confidence: 97%

Ras, but not Src, transformation of RIE-1 epithelial cells is dependent on activation of the mitogen-activated protein kinase cascade

et al. 1998

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Src transformation of NIH3T3 mouse ®broblasts has been shown to be dependent on Ras function. Since we recently showed that the signaling pathways that mediate Ras transformation of RIE-1 rat intestinal epithelial cells are distinct from those that cause Ras transformation of ®broblasts, we utilized three approaches to determine if Src transformation of RIE-1 cells is dependent on Ras. First, although both Ras and Src cause upregulation of an epidermal growth factor (EGF) receptor-dependent autocrine growth loop, only Ras transformation required this activity. Second, whereas both Src and Ras caused upregulation of the p42 and p44 mitogen-activated protein kinases (MAPKs), only Ras transformation was blocked by the inhibition of MAPK activation by treatment with the PD 98059 MEK inhibitor. Third, treatment with the farnesyltransferase inhibitor FTI-277 blocked Ras, but not Src, transformation. Taken together, these observations suggest that Src transformation of RIE-1 cells is not dependent on Ras. Finally, we determined that Ras activation of Raf-independent pathways alone is su cient to cause growth transformation of RIE-1 cells. Thus, both Ras and Src cause transformation of RIE-1 cells via pathways distinct from those required to cause transformation of NIH3T3 cells.

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“…These activation steps may include a conformational change which would relieve the inhibition imposed by the Raf-1 amino-terminus, multiple phosphorylations of CR2 and CR3, and the presence of accessory proteins such as hsp90 and 14.3.3 (Heidecker et al, 1992;Morrison and Cutler, 1997). 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).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 1998

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We have uniformly examined the regulatory steps required by oncogenic Ras, Src, EGF and phorbol 12-myristate 13-acetate (PMA) to activate Raf-1. Speci®-cally, we determined the role of Ras binding and the phosphorylation of serines 338/339, tyrosines 340/341 and the activation loop (491 ± 508) in response to these stimuli in COS-7 cells. An intact Ras binding domain was found to be essential for Raf-1 kinase activation by each stimulus, including PMA. Brief treatment of COS-7 cells with PMA was found to rapidly promote accumulation of the active, GTP-bound form of Ras. Furthermore, loss of the serine 338/339 and tyrosine 340/341 phosphorylation sites also blocked Raf-1 activation by all stimuli tested. Loss of the serine 497 and serine 499 PKCa phosphorylation sites failed to signi®cantly reduce Raf-1 activation by any stimulus including PMA. Alanine substitution of all other potential phosphorylation sites within the Raf-1 activation loop had little or no e ect on kinase regulation by Ras[V12] or vSrc although some mutants were less responsive to PMA. These results suggest that in mammalian cells, Raf-1 can be regulated by a variety of di erent stimuli through a common mechanism involving association with Ras-GTP and multiple phosphorylations of the amino-terminal region of the catalytic domain. Phosphorylation of the activation loop does not appear to be a signi®cant mechanism of Raf-1 kinase regulation in COS-7 cells.

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Two Distinct Raf Domains Mediate Interaction with Ras

Cited by 233 publications

References 28 publications

Increasing complexity of Ras signaling

Increasing complexity of Ras signaling

Ras, but not Src, transformation of RIE-1 epithelial cells is dependent on activation of the mitogen-activated protein kinase cascade

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

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