The Raf-1 kinase domain is kept in an inactive state by the N-terminal regulatory domain. Activation of the kinase domain occurs following release from the N-terminal repression and possible catalytic upregulation. To distinguish the regulatory mechanisms that directly influence the catalytic activity of the enzyme from those which act through the inhibitory domain, the catalytic domain of Raf-1 (CR3) was expressed in COS-7 cells. The role of phosphorylation in the direct regulation of this domain was determined by substituting non-phosphorylatable amino acids for known serine and tyrosine phosphorylation sites. The intrinsic activity of each mutant protein was determined as well as stimulation by v-Src and phorbol esters. Both v-Src and phorbol esters were potent activators of CR3, requiring the serine 338/339 (p21-activated protein kinase, Pak) and tyrosine 340/341 (Src) phosphorylation sites for full stimulation of CR3. In contrast, loss of the serine 497/499 protein kinase C phosphorylation sites had little effect on CR3 activation by either v-Src or phorbol esters. Loss of serine 621, a 14-3-3 adaptor-protein-binding site, prevented activation of CR3 by v-Src or phorbol esters and partially decreased the high basal activity of the kinase fragment. When co-expressed in COS-7 cells, 14-3-3 associated strongly with full-length Raf-1, weakly with wild-type CR3 and not at all with the A621 and D621 CR3 mutants. The role of 14-3-3 in maintaining the activity of the catalytic domain of Raf-1 was investigated further by performing peptide-competition studies with wild-type CR3, wild-type CR3 and v-Src or constitutively active CR3 (CR3[YY340/341DD]). In each case, incubation of the proteins with a phosphoserine-621 Raf-1 peptide, which we show displaced Raf-1 and CR3[YY340/341DD] from 14-3-3, was found to substantially reduce catalytic activity. Taken together, our results support a model of Raf regulation in which the activity of the Raf-1 catalytic domain is directly upregulated by phosphorylation, following relief of inhibition by the N-terminal regulatory domain upon Ras-GTP binding. Moreover, the presence of serine 621 in the free catalytic fragment is required for full CR3 activation by stimulatory factors, and the continuous presence of 14-3-3 at this site is necessary for retaining activity once the kinase is activated.
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October 2000
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Research Article|
September 26 2000
Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association
Michele T. YIP-SCHNEIDER;
Michele T. YIP-SCHNEIDER
1
*Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
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Wenyan MIAO;
Wenyan MIAO
1
†Millennium Pharmaceuticals Inc., Cambridge, MA 02142, U.S.A.
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Amy LIN;
Amy LIN
‡Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
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Darlene S. BARNARD;
Darlene S. BARNARD
§Eli Lilly and Company, Lilly Corporate Center, Drop Code 1543, Indianapolis, IN 46285, U.S.A.
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Guri TZIVION;
Guri TZIVION
¶Diabetes Research Laboratory, Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, U.S.A.
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Mark S. MARSHALL
Mark S. MARSHALL
2
§Eli Lilly and Company, Lilly Corporate Center, Drop Code 1543, Indianapolis, IN 46285, U.S.A.
2To whom correspondence should be addressed (e-mail Marshall_Mark@Lilly.com).
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Publisher: Portland Press Ltd
Received:
February 07 2000
Revision Received:
June 26 2000
Accepted:
July 25 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 351 (1): 151–159.
Article history
Received:
February 07 2000
Revision Received:
June 26 2000
Accepted:
July 25 2000
Citation
Michele T. YIP-SCHNEIDER, Wenyan MIAO, Amy LIN, Darlene S. BARNARD, Guri TZIVION, Mark S. MARSHALL; Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association. Biochem J 1 October 2000; 351 (1): 151–159. doi: https://doi.org/10.1042/bj3510151
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