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J. Biol. Chem., Vol. 275, Issue 41, 31921-31929, October 13, 2000
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From the Department of Medicine and Cancer Center, University of
California, San Diego, La Jolla, California 92093-0652
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.
Cell Type-specific Regulation of B-Raf Kinase by cAMP and
14-3-3 Proteins*
§,,
*
This work was supported by Public Health Service Grants
R01-GM55586 (to R. B. P.) and R21-CA81115 (to G. R. B.) and by Tobacco-Related Disease Research Program Grant 91T-0069 (to
R. B. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
These authors contributed equally to this work.
§
Present address: Bellevue Hospital Center, Dept. Pathology,
1st Avenue at 27th St., New York, NY 10016.
¶
To whom correspondence should be addressed. Tel.:
858-534-8805; Fax: 858-534-1421; E-mail: rpilz@ucsd.edu.
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