Hn. Wang et al., MUTATIONS IN THE HYDROPHOBIC SURFACE OF AN AMPHIPATHIC GROOVE OF 14-3-3-ZETA DISRUPT ITS INTERACTION WITH RAF-1 KINASE, The Journal of biological chemistry, 273(26), 1998, pp. 16297-16304
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.