ACTIVATED RAS DISPLACES 14-3-3-PROTEIN FROM THE AMINO-TERMINUS OF C-RAF-1

The serine/threonine protein kinase c-Raf-1 interacts with a number of cellular proteins including 14-3-3 isoforms which may be regulators o r substrates of c-Raf-1 in signal transduction pathways, In vivo and i n vitro binding analyses of c-Raf-1 and mutant proteins with 14-3-3 ze ta indicate bivalent binding of 14-3-3 zeta to the amino terminus as w ell as to the carboxy terminus of c-Raf-1, Although 14-3-3 zeta and Ra s use different binding regions on the amino terminal regulatory domai n of c-Raf-1 (c-Raf-NT), 14-3-3 zeta is displaced from the amino termi nus 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 Ra s, This is explained by a second binding site of 14-3-3 zeta at the ca rboxy 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, i s able to bind 14-3-3 zeta, Even though 14-3-3 zeta can be phosphoryla ted in vivo, only the unphosphorylated form binds to the amino terminu s 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 dis placed from the amino terminus but not from the carboxy terminus of c- Raf-1 by binding of activated Ras to c-Raf-1.