Cell type-specific regulation of B-Raf kinase by cAMP and 14-3-3 proteins

Cyclic AMP can either activate or inhibit the mitogen-activated protein kin ase (MAPK) pathway in different cell types; MAPK activation has been observ ed in B-Raf-expressing cells and has been attributed to Rap1 activation wit h subsequent B-Raf activation, whereas MAPK inhibition has been observed in cells lacking B-Raf and has been attributed to cAMP-dependent protein kina se (protein kinase A)-mediated phosphorylation and inhibition of Raf-l kina se. We found that cAMP stimulated MAPK activity in CHO-K1 and PC12 cells bu t inhibited MAPK activity in C6 and NB2A cells. In all four cell types, cAM P activated Rap1, and the 95- and 68-kDa isoforms of B-Raf were expressed, cAMP activation or inhibition of MAPK correlated with activation or inhibit ion of endogenous and transfected B-Raf kinase. Although all cell types exp ressed similar amounts of 14-3-3 proteins, approximately B-fold less 14-3-3 was associated with B-Raf in cells in which cAMP was inhibitory than in ce lls in which cAMP was stimulatory. We found that the cell type-specific inh ibition of B-Raf could be completely prevented by overexpression of 14-3-3 isoforms, whereas expression of a dominant negative 14-3-3 mutant resulted in partial loss of B-Raf activity. Our data suggest that 14-3-3 bound to B- Raf protects the enzyme from protein kinase A-mediated inhibition; the amou nt of 14-3-3 associated with B-Raf may explain the tissue-specific effects of cAMP on B-Raf kinase activity.