Activated Ras displaces 14-3-3 protein from the amino terminus of c-Raf-1

• Published in: Oncogene Vol. 12; no. 3; p. 609
• Main Authors: Rommel, C, Radziwill, G, Lovrić, J, Noeldeke, J, Heinicke, T, Jones, D, Aitken, A, Moelling, K
• Format: Journal Article
• Language: English
• Published: England 01.02.1996
• Subjects: 14-3-3 Proteins; Adenoviruses, Human; Animals; Binding Sites; Binding, Competitive; Cell Line, Transformed; Cloning, Molecular; Enzyme Inhibitors - metabolism; Glutathione Transferase; Humans; Kidney; Mutagenesis; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; Phosphopeptides - chemistry; Phosphopeptides - isolation & purification; Phosphorylation; Protein-Serine-Threonine Kinases - isolation & purification; Protein-Serine-Threonine Kinases - metabolism; Proteins - isolation & purification; Proteins - metabolism; Proto-Oncogene Proteins - isolation & purification; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-raf; ras Proteins - metabolism; Rats; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Transfection; Tyrosine 3-Monooxygenase
• ISSN: 0950-9232; 1476-5594

The serine/threonine protein kinase c-Raf-1 interacts with a number of cellular proteins including 14-3-3 isoforms which may be regulators or substrates of c-Raf-1 in signal transduction pathways. In vivo and in vitro binding analyses of c-Raf-1 and mutant proteins with 14-3-3 zeta indicate bivalent binding of 14-3-3 zeta to the amino terminus as well as to the carboxy terminus of c-Raf-1. Although 14-3-3 zeta and Ras use different binding regions on the amino terminal regulatory domain of c-Raf-1 (c-Raf-NT), 14-3-3 zeta is displaced from the amino terminus upon binding of activated Ras. In contrast, if c-Raf-1 full length is analysed instead of the separately expressed c-Raf-NT, binding of 14-3-3 zeta is only slightly effected by co-expression of activated Ras. This is explained by a second binding site of 14-3-3 zeta at the carboxy terminus of c-Raf-1. The mutant c-Raf-NT (S259A) cannot bind 14-3-3 zeta, suggesting a regulatory role of this in vivo phosphorylation site. However, c-Raf-NT phosphorylated or unphosphorylated at S259, is able to bind 14-3-3 zeta. Even though 14-3-3 zeta can be phosphorylated in vivo, only the unphosphorylated form binds to the amino terminus of c-Raf-1. The data presented indicate, that 14-3-3 zeta binds to c-Raf-1 in a bivalent fashion in unstimulated cells. 14-3-3 zeta is displaced from the amino terminus but not from the carboxy terminus of c-Raf-1 by binding of activated Ras to c-Raf-1.