Cell Type-specific Regulation of B-Raf Kinase by cAMP and 14-3-3 Proteins

• Published in: The Journal of biological chemistry Vol. 275; no. 41; pp. 31921 - 31929
• Main Authors: Qiu, Wansong, Zhuang, Shunhui, von Lintig, Friederike C., Boss, Gerry R., Pilz, Renate B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.10.2000; American Society for Biochemistry and Molecular Biology
• Subjects: 14-3-3 Proteins; Animals; Cell Line; Cricetinae; Cyclic AMP - analogs & derivatives; Cyclic AMP - pharmacology; Cyclic AMP-Dependent Protein Kinases - metabolism; Enzyme Activation - drug effects; Guanosine Triphosphate - metabolism; Isoenzymes - metabolism; MAP Kinase Signaling System - drug effects; Mitogen-Activated Protein Kinases - antagonists & inhibitors; Mitogen-Activated Protein Kinases - metabolism; Mutation; Organ Specificity; Phosphorylation - drug effects; Protein Binding; Proto-Oncogene Proteins c-raf - antagonists & inhibitors; Proto-Oncogene Proteins c-raf - metabolism; Rats; Thionucleotides - pharmacology; Transfection; Tyrosine 3-Monooxygenase - genetics; Tyrosine 3-Monooxygenase - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M003327200

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.