Ras-mutant Cancer Cells Display B-Raf Binding to Ras That Activates Extracellular Signal-regulated Kinase and Is Inhibited by Protein Kinase A Phosphorylation

• Published in: The Journal of biological chemistry Vol. 288; no. 38; pp. 27646 - 27657
• Main Authors: Li, Yanping, Takahashi, Maho, Stork, Philip J.S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.09.2013; American Society for Biochemistry and Molecular Biology
• Subjects: B-Raf; C-Raf; Cell Line, Tumor; Cyclic AMP - genetics; Cyclic AMP - metabolism; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; Enzyme Activation - genetics; ERK; Extracellular Signal-Regulated MAP Kinases - genetics; Extracellular Signal-Regulated MAP Kinases - metabolism; Humans; Lung Neoplasms - genetics; Lung Neoplasms - metabolism; Lung Neoplasms - pathology; MAP Kinases (MAPKs); Mutation; Oncogene Protein p21(ras) - genetics; Oncogene Protein p21(ras) - metabolism; Phosphorylation - genetics; Protein Binding - genetics; Protein Kinase A (PKA); Proto-Oncogene Proteins B-raf - genetics; Proto-Oncogene Proteins B-raf - metabolism; Proto-Oncogene Proteins c-raf - genetics; Proto-Oncogene Proteins c-raf - metabolism; Raf; Ras; Signal Transduction; MAP Kinases (MAPKs); Protein Kinase A (PKA); Ras; Raf; C-Raf; B-Raf; ERK
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M113.463067

The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf. Background: How the cAMP-dependent protein kinase PKA regulates B-Raf binding to Ras is not known. Results: PKA inhibits the binding of B-Raf to active Ras via phosphorylation of serine 365 in B-Raf. Conclusion: B-Raf can participate in the Ras-dependent activation of ERK in Ras-mutant cancers, and this is inhibited by PKA. Significance: PKA can block Ras binding to both Raf isoforms C-Raf and B-Raf.