Abstract
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.
Highlights
How the cAMP-dependent protein kinase PKA regulates B-Raf binding to Ras is not known
We examined a range of Ras-mutant cancer cell lines including H1299 cells, a human non-small cell lung carcinomas (NSCLC) line, harboring an neuroblastoma Ras (NRas) Q61K mutation, Calu-6, a human NSCLC line (KRas Q61K), and HCT116, a human colorectal carcinoma cell line (KRas G13D). These cells generally express B-Raf, the contribution of B-Raf to oncogenic Ras signaling is not known. We show that both B-Raf and C-Raf contribute to the basal ERK activation seen in these cells
This was seen in H1299 cells, as basal ERK activation was blocked by short hairpin RNA (shRNA) for NRas (Fig. 1A)
Summary
How the cAMP-dependent protein kinase PKA regulates B-Raf binding to Ras is not known. This was due to the high basal level of ERK activity in these cancer cells and the subsequent ERK-dependent phosphorylation of B-Raf itself, which prevented B-Raf association with oncogenic Ras [9, 10] Whether this isoform switch from B-Raf to C-Raf occurs in non-melanocytic tumors that harbor oncogenic Ras mutations is unknown. We examined a range of Ras-mutant cancer cell lines including H1299 cells, a human NSCLC line, harboring an NRas Q61K mutation, Calu-6, a human NSCLC line (KRas Q61K), and HCT116, a human colorectal carcinoma cell line (KRas G13D) These cells generally express B-Raf, the contribution of B-Raf to oncogenic Ras signaling is not known.
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