Raf-1 kinase and exoenzyme S interact with 14-3-3zeta 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 oncogenesis. Our solution of the crystal structure of 14-3-3zeta revealed a conserved amphipathic groove that may allow the association of 14-3-3 with diverse ligands (Liu, D., Bienkowska, J., Petosa, C., Collier, 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 (R56E) decreased the interaction of 14-3-3zeta with Raf-1 kinase, whereas R60E showed only subtle effects on the binding. Interestingly, these mutations exhibited similar effects on the functional interaction of 14-3-3zeta with another target protein, exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. The EC50 values of 14-3-3zeta required for ExoS activation increased by approximately 110-, 5-, and 2-fold for the K49E, R56E, and R60E mutants, respectively. The drastic reduction of 14-3-3zeta/ligand affinity by the K49E mutation is due to a local electrostatic effect, rather than the result of a gross structural alteration, as evidenced by partial proteolysis and circular dichroism analysis. This work identifies the first point mutation (K49E) that dramatically disrupts 14-3-3zeta/ligand interactions. The parallel effects of this single point mutation on both Raf-1 binding and ExoS activation strongly suggest that diverse associated proteins share a common structural binding determinant on 14-3-3zeta.
Zhang
L
L
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Wang
H
H
Liu
D
D
Liddington
R
R
Fu
H
H
D048948:14-3-3 Proteins; D036002:ADP Ribose Transferases; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001427:Bacterial Toxins; D001665:Binding Sites; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D017354:Point Mutation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D008024:Ligands; D011506:Proteins; D011518:Proto-Oncogene Proteins; D001120:Arginine; D014446:Tyrosine 3-Monooxygenase; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D008239:Lysine