Abstract
Cyclic AMP can either activate or inhibit the mitogen-activated protein kinase (MAPK) pathway in different cell types; MAPK activation has been observed in B-Raf-expressing cells and has been attributed to Rap1 activation with subsequent B-Raf activation, whereas MAPK inhibition has been observed in cells lacking B-Raf and has been attributed to cAMP-dependent protein kinase (protein kinase A)-mediated phosphorylation and inhibition of Raf-1 kinase. We found that cAMP stimulated MAPK activity in CHO-K1 and PC12 cells but inhibited MAPK activity in C6 and NB2A cells. In all four cell types, cAMP activated Rap1, and the 95- and 68-kDa isoforms of B-Raf were expressed. cAMP activation or inhibition of MAPK correlated with activation or inhibition of endogenous and transfected B-Raf kinase. Although all cell types expressed similar amounts of 14-3-3 proteins, approximately 5-fold less 14-3-3 was associated with B-Raf in cells in which cAMP was inhibitory than in cells in which cAMP was stimulatory. We found that the cell type-specific inhibition 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 amount of 14-3-3 associated with B-Raf may explain the tissue-specific effects of cAMP on B-Raf kinase activity.
Highlights
The serine/threonine protein kinases of the Raf family (Raf-1, A-Raf, and B-Raf) are key regulators of cell growth, differentiation, and apoptosis in eukaryotic cells [1]
Cyclic AMP can either activate or inhibit the mitogenactivated protein kinase (MAPK) pathway in different cell types; MAPK activation has been observed in B-Rafexpressing cells and has been attributed to Rap1 activation with subsequent B-Raf activation, whereas MAPK inhibition has been observed in cells lacking B-Raf and has been attributed to cAMP-dependent protein kinase-mediated phosphorylation and inhibition of Raf-1 kinase
When we transfected the B-Raf catalytic domain into C6 and Chinese hamster ovary (CHO)-K1 cells, we found that 8-pCPTcAMP inhibited its activity in both cell types, inhibition was more pronounced in C6 cells than in CHO-K1 cells; co-transfection of a 14-3-3 expression vector prevented the inhibition in both cell types (Fig. 8)
Summary
The serine/threonine protein kinases of the Raf family (Raf-1, A-Raf, and B-Raf) are key regulators of cell growth, differentiation, and apoptosis in eukaryotic cells [1]. We transfected C6, CHO-K1, NB2A, and PC12 cells with increasing amounts of an expression vector encoding GST-tagged full-length B-Raf and isolated the enzyme on glutathione-Sepharose beads.
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