Interaction of 14-3-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa.
The 14-3-3 proteins are a family of conserved, dimeric proteins that interact with a diverse set of ligands, including molecules involved in cell cycle regulation and apoptosis. It is well-established that 14-3-3 binds to many ligands through phosphoserine motifs. Here we characterize the interaction of 14-3-3 with a nonphosphorylated protein ligand, the ADP-ribosyltransferase Exoenzyme S (ExoS) from Pseudomonas aeruginosa. By using affinity chromatography and surface plasmon resonance, we show that the zeta isoform of 14-3-3 (14-3-3zeta) can directly bind a catalytically active fragment of ExoS in vitro. The interaction between ExoS and 14-3-3zeta is of high affinity, with an equilibrium dissociation constant of 7 nM. ExoS lacks any known 14-3-3 binding motif, but to address the possibility that 14-3-3 binds a noncanonical phosphoserine site, we assayed ExoS for protein-bound phosphate by using mass spectrometry. No detectable phosphoproteins were found. A phosphopeptide ligand of 14-3-3, pS-Raf-259, was capable of inhibiting the binding of 14-3-3 to ExoS, suggesting that phosphorylated and nonphosphorylated ligands may share a common binding site, the conserved amphipathic groove. It is conceivable that 14-3-3 proteins may bind both phosphoserine and nonphosphoserine ligands in cells, possibly allowing kinase-dependent as well as kinase-independent regulation of 14-3-3 binding.
Masters
S C
SC
Graduate Program in Molecular Therapeutics and Toxicology, Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Pederson
K J
KJ
Zhang
L
L
Barbieri
J T
JT
Fu
H
H
D048948:14-3-3 Proteins; D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D001667:Binding, Competitive / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D008024:Ligands; D010455:Peptides; D020033:Protein Isoforms; D011506:Proteins; D011994:Recombinant Proteins; D014446:Tyrosine 3-Monooxygenase; D036002:ADP Ribose Transferases; C444820:exoenzyme S