MUTATIONS IN THE HYDROPHOBIC SURFACE OF AN AMPHIPATHIC GROOVE OF 14-3-3-ZETA DISRUPT ITS INTERACTION WITH RAF-1 KINASE

14-3-3 proteins bind to a diverse group of regulatory molecules such a s Raf-1, Cbl, and c-Bcr that are involved in signal transduction pathw ays. The crystal structure of 14-3-3 zeta reveals a conserved amphipat hic groove that may mediate the association of 14-3-3 with diverse lig ands. Consistently, mutations on the charged surface of the groove (Ly s-49, Arg-56, and Arg-60) decrease the binding of 14-3-3 zeta to the l igands tested (Zhang, L., Wang, H., Liu, D., Liddington, R., and Fu, H . (1997) J. Biol. Chem. 272, 13717-13724). Here we report that mutatio ns that altered the hydrophobic property of the groove, V176D, L216D, L220D, and L227D, disrupted the interaction of 14-3-3 zeta with Raf-l kinase. The reduced binding of the 14-3-3 zeta mutants to Raf-1 was ap parently not because of gross structural changes in the mutants as jud ged by their ability to form dimers, by partial proteolysis profiles, and by circular dichroism analysis. These hydrophobic residues appeare d to be required for the binding of 14-3-3 zeta to distinct activation states of Raf-1 because mutations V176D, L216D, L220D, and L227D redu ced the interaction of 14-3-3 zeta with Raf-1 from both phorbol 12-myr istate 13-acetate-stimulated and unstimulated Jurkat T cells. These sa me mutations also disrupted the association of 14-3-3 zeta with other regulatory molecules such as Cbl and c-Bcr, suggesting that the hydrop hobic surface of the amphipathic groove represents part of a binding s ite shared by a number of 14-3-3-associated proteins. The conservation of the hydrophobic residues Val-176, Leu-216, Leu-220, and Leu-227 am ong known 14-3-3 family members implies their general importance in li gand binding.