The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch.
The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein-protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.
Dhillon
Amardeep S
AS
Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Bioscience and Biotechnology, University of Melbourne, 30 Flemington Road, Parkville, VIC 3010, Australia. adhillon@unimelb.edu.au
Yip
Yan Yan
YY
Grindlay
G Joan
GJ
Pakay
Julian L
JL
Dangers
Marc
M
Hillmann
Meike
M
Clark
William
W
Pitt
Andrew
A
Mischak
Harald
H
Kolch
Walter
W
D048948:14-3-3 Proteins / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D048948:14-3-3 Proteins; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases