The neurotoxic phospholipase A2 associates, through a non-phosphorylated binding motif, with 14-3-3 protein gamma and epsilon isoforms.
Two novel acceptors for ammodytoxin C, a presynaptically neurotoxic phospholipase A(2) from snake venom, have been purified from porcine cerebral cortex by a toxin-affinity-based procedure. Using tandem mass spectrometry, the isolated acceptors were identified as 14-3-3 gamma and epsilon 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+/-0.2 microM for the interaction between chip-immobilized 14-3-3 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 A(2), 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.
Sribar
Jernej
J
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Sherman
Nicholas E
NE
Prijatelj
Petra
P
Faure
Grazyna
G
Gubensek
Franc
F
Fox
Jay W
JW
Aitken
Alastair
A
Pungercar
Joze
J
Krizaj
Igor
I
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002540:Cerebral Cortex / Q000737:chemistry / Q000378:metabolism; D054501:Group II Phospholipases A2; D006801:Humans; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014757:Viper Venoms / Q000378:metabolism*
D048948:14-3-3 Proteins; D020033:Protein Isoforms; D014757:Viper Venoms; D014446:Tyrosine 3-Monooxygenase; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; C060161:ammodytoxin C