The neurotoxic phospholipase A2 associates, through a non-phosphorylated binding motif, with 14-3-3 protein γ and ε isoforms

Two novel acceptors for ammodytoxin C, a presynaptically neurotoxic phospholipase A2 from snake venom, have been purified from porcine cerebral cortex by a toxin-affinity-based procedure. Usingtandem mass spectrometry, the isolated acceptors were identified as 14-3-3c and e isoforms, highly conserved cytoplasmic proteins involved in the regulation of numerous physiological processes. The interaction between ammodytoxin C and 14-3-3 proteins is direct and not mediated by calmodulin, a high-affinity acceptor for both ammodytoxin C and 14-3-3 proteins, as demonstrated in pull-down experiments and by surface plasmon resonance. The latter technique gave an apparent dissociation constant of 1:0 � 0:2lM for the interaction between chip-immobilized 143-3 and ammodytoxin C. 14-3-3 usually interacts with proteins through specific phospho-Ser/Thr motifs. Ammodytoxin C is not a phospho-protein, therefore the interaction must occur through a non-phosphorylated binding site, most probably the KEESEK sequence at its C-terminal end. The interaction we describe suggests an explanation for the pathophysiological effects evoked by some secreted phospholipases A2, such as the inhibition of protein phosphorylation, of terminal ion currents, and of neurotransmission, as well as the initiation of neuronal cell death, all processes regulated by 14-3-3 proteins. 2003 Elsevier Science (USA). All rights reserved.