Mechanism of cAMP activation of MAP kinase in neuroendocrine cells

The Mitogen Activated Protein Kinases (MAPKs) are ubiquitous signalling proteins which are activated in response to a variety of stimuli by a cytoplasmic kinase cascade known as the MAPK cascade. MAPK in turn phosphorylates multiple substrates which control cell growth and differentiation. cAMP is well known to inhibit the growth factor induced activation of MAPK in fibroblasts and smooth muscle cells. Previous work by our group and others has shown that cAMP can activate MAPK and can synergize with growth factors to promote differentiation in neuroendocrine cells. The purpose of this study was to determine the components of the signalling pathway between cAMP and MAPK. Using DNA-mediated gene transfer of interfering mutants of proteins in the MAPK cascade we show here that cAMP activation of MAPK dependent gene transcription requires cAMP dependent protein kinase (PKA), raf, and mek, but is independent of ras. PC. 12 cells were transfected with plasmids encoding the gene for a fusion protein of Elk-1/gal-4 and a gal response/luciferase gene as a reporter system for activation of the MAP kinase cascade. Elk-1's activity as a transcription factor has been previously shown to be dependent upon phosphorylation by MAPK. 10μM Forskolin resulted in reproducible Elk-1 activation as assessed by luciferase assay. This effect is blocked by interfering mutants of raf (raf 301) and mek (mek K97R) as well as by the PKA inhibitor PKL However, an interfering mutant of ras (N17 ras) had no effect on forskolin activation of Elk-1. Immune complex assays of the known raf isoforms, Raf-1, B-raf, and A-raf, using recombinant mek as a substrate show no activation by forskolin but robust activation by the growth factors EGF and NGF. We conclude that PKA activates the MAPK cascade at the level of raf but independent of ras and the currently known isoforms of raf. We propose that a novel raf isoform is the intermediate between PKA and the MAPK cascade in neuroendocrine cells.