RAF-1 KINASE AND EXOENZYME-S INTERACT WITH 14-3-3-ZETA THROUGH A COMMON SITE INVOLVING LYSINE-49

14-3-3 proteins are a family of conserved dimeric molecules that bind to a range of cellular proteins involved in signal transduction and on cogenesis. Our solution of the crystal structure of 14-3-3 zeta reveal ed a conserved amphipathic groove that may allow the association of 14 -3-3 with diverse ligands (Liu, D., Bienkowska, J., Petosa, C., Collie r, R. J., Fu, H., and Liddington, R. (1995) Nature 376, 191-194). Here , the contributions of three positively charged residues (Lys-49, Arg- 56, and Arg-60) that lie in this Raf-binding groove were investigated. Two of the charge-reversal mutations greatly (K49E) or partially (R56 E) decreased the interaction of 14-3-3 zeta with Raf-1 kinase, whereas R60E showed only subtle effects on the binding. Interestingly, these mutations exhibited similar effects on the functional interaction of 1 4-3-3 zeta with another target protein, exoenzyme S (ExoS), an ADP-rib osyltransferase from Pseudomonas aeruginosa. The EC50 values of 14-3-3 zeta required for ExoS activation increased by similar to 110-, 5-, a nd 2-fold for the K49E, R56E, and R60E mutants, respectively. The dras tic reduction of 14-3-3 zeta ligand affinity by the K49E mutation is d ue to a local electrostatic effect, rather than the result of a gross structural alteration, as evidenced by partial proteoIysis and circula r dichroism analysis. This work identifies the first point mutation (K 49E) that dramatically disrupts 14-3-5 zeta ligand interactions. The p arallel effects of this single point mutation on both Raf-1 binding an d ExoS activation strongly suggest that diverse associated proteins sh are a common structural binding determinant on 14-3-3 zeta.