14-3-3 proteins are required for maintenance of Raf-1 phosphorylation and kinase activity.
By binding to serine-phosphorylated proteins, 14-3-3 proteins function as effectors of serine phosphorylation. The exact mechanism of their action is, however, still largely unknown. Here we demonstrate a requirement for 14-3-3 for Raf-1 kinase activity and phosphorylation. Expression of dominant negative forms of 14-3-3 resulted in the loss of a critical Raf-1 phosphorylation, while overexpression of 14-3-3 resulted in enhanced phosphorylation of this site. 14-3-3 levels, therefore, regulate the stoichiometry of Raf-1 phosphorylation and its potential activity in the cell. Phosphorylation of Raf-1, however, was insufficient by itself for kinase activity. Removal of 14-3-3 from phosphorylated Raf abrogated kinase activity, whereas addition of 14-3-3 restored it. This supports a paradigm in which the effects of phosphorylation on serine as well as tyrosine residues are mediated by inducible protein-protein interactions.
Center for Immunology and Department of Pathology, Washington University School of Medicine, St. Louis, Missouri, USA.
J A
JA
Thorson
L W
LW
Yu
A L
AL
Hsu
N Y
NY
Shih
P R
PR
Graves
J W
JW
Tanner
P M
PM
Allen
H
H
Piwnica-Worms
A S
AS
Shaw
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003001:Cloning, Molecular; D005982:Glutathione Transferase; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010768:Phosphoserine; D019000:Phosphotyrosine; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013057:Spectrum Analysis; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D010768:Phosphoserine; D019000:Phosphotyrosine; D014446:Tyrosine 3-Monooxygenase; D015500:Chloramphenicol O-Acetyltransferase; D005982:Glutathione Transferase; D019908:Proto-Oncogene Proteins c-raf