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Principles of Cell Signaling and Biological Consequences

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Journal Club Discussion of Cell 116, 855-967 (2004)

31 January 2005

A. Chan, R. Iyengar, S. Aaronson, A. Caplan, S. Salton, and M. M. Zhou

Students should read the following article, the discussion lead-in summary, and respond to this topic on the questions listed at the end of the summary:

P.T. Wan, M. J. Garnett, S. M. Roe, S. Lee, D. Niculescu-Duvaz, V. M. Good, C. M. Jones, C. J. Marshall, C. J. Springer, D. Barford, R. Marais, Cancer Genome Project, Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116, 855-867 (2004).

Discussion Lead-In Summary

This article illustrates the important concept that the mammalian mitogen-activated protein kinase (MAPK) pathway is a major target for oncogenic activations in human cancer. It was known for nearly two decades that the human N-RAS oncogene is mutated in approximately 20% of human melanomas. The most frequent mutations reside in codon 61, with these lesions found commonly in sun-exposed surfaces. However, the genetic alterations that could account for the remaining melanomas lacking N-RAS mutations had eluded researchers. Clues to this conundrum were provided by Wan et al. (also by Davies et. al. Nature 417, 949-954, 2002) in which B-Raf was identified to be the culprit. Cumulative data from several laboratories have found B-Raf to be mutated to as high as 70% in melanocytic nevi and vertical growth phase melanomas. Intriguingly, unlike N-Ras tumors, these melanomas do not appear to be associated with an etiology involving UV-exposure.

Analyzing the sites of mutation reveal that they are clustered into two major hotspots. These regions represent catalytic motifs that are conserved among various kinases and are absolutely essential for signal transduction. The first mutation cluster accounts for over 90% of all B-Raf mutations in melanomas. It is located at codon Val 599 in the activation segment that spans codon 582-622. Within this 20 amino acid region lies two phosphorylation sites at Thr 598 and Ser 602. In addition, Asp 593 of the conserved DFG motif serves to stabilize an Mg2+ ion important for phosphotransfer. In the inactive state of the B-Raf kinase, Val 599 interacts with the P-loop, causing the DFG motif to attain a conformation that is not amenable for catalysis. Thus, a Val to Glu mutation at codon 599 in melanomas is predicted to release this constrain such that Asp 593 can now direct the transfer of the phosphate to the B-Raf substrate, MEK.

The second finding of this paper illustrates a well-known feature of the MAPK pathway and that is the ability to form signaling complexes. In a very low fraction of melanomas, B-Raf acquires mutations in the P-loop, which is responsible for binding to the phosphate moieties of ATP. The primary amino acid sequence is characterized by the Gly-X-Gly-X-X-Gly motif found in majority of mammalian kinases. Mutations at one of the glycines have the paradoxical effect of reducing the kinase activity of the resulting mutant B-Raf protein. This led the authors to propose a novel mechanism that can explain the oncogenicity of these mutants. It is believed that these kinase-impaired B-Raf proteins first heterodimerize with c-Raf. This induces a conformational change in c-Raf leading to its activation. c-Raf activated through this novel mechanism will in turn stimulate MEK and then ERK. These data raise several intriguing questions.

  • What is the physiological relevance of B-Raf/c-Raf complexes in MAPK signaling?
  • Is B-Raf/c-Raf complex only formed in response to a certain upstream signals?

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