C-terminal fragment of tetanus toxin heavy chain activates Akt and MEK/ERK signalling pathways in a Trk receptor-dependent manner in cultured cortical neurons

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Abstract
Previous publications from our group [Gil, Chaib, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177-182; Gil, Chaib, Blasi and Aguilera (2001) Biochem. J. 356, 97-103] have reported the activation, in rat brain synaptosomes, of several phosphoproteins, such as neurotrophin tyrosine kinase (Trk) A receptor, phospholipase Cgamma-1, protein kinase C (PKC) isoforms and extracellular-signal-regulated kinases 1 and 2 (ERK-1/2). In the present study, we examined, by means of phospho-specific antibodies, the activation of the signalling cascades involving neurotrophin Trk receptor, Akt kinase and ERK pathway, in cultured cortical neurons from foetal rat brain, by tetanus toxin (TeTx) as well as by the C-terminal part of its heavy chain (H(C)-TeTx). TeTx and H(C)-TeTx induce fast and transient phosphorylation of Trk receptor at Tyr(674) and Tyr(675), but not at Tyr(490), although the potency of TeTx in this action was higher when compared with H(C)-TeTx action. Moreover, H(C)-TeTx and TeTx also induced phosphorylation of Akt (at Ser(473) and Thr(308)) and of ERK-1/2 (Thr(202)/Tyr(204)), in a time- and concentration-dependent manner. The detection of TeTx- and H(C)-TeTx-induced phosphorylation at Ser(9) of glycogen synthase kinase 3beta confirms Akt activation. In the extended analysis of the ERK pathway, phosphorylation of the Raf, mitogen-activated protein kinase kinase (MEK)-1/2 and p90Rsk kinases and phosphorylation of the transcription factor cAMP-response-element-binding protein were detected. The use of tyrphostin AG879, an inhibitor of Trk receptors, demonstrates their necessary participation in the H(C)-TeTx-induced activation of Akt and ERK pathways, as well as in the phosphorylation of phospholipase Cgamma-1. Furthermore, both pathways are totally dependent on phosphatidylinositol 3-kinase action, and they are independent of PKC action, as assessed using wortmannin and Ro-31-8220 as inhibitors. The activation of PKC isoforms was determined by their translocation from the cytosolic compartment to the membranous compartment, showing a clear H(C)-TeTx-induced translocation of PKC-alpha and -beta, but not of PKC- epsilon.