CB1R-dependent activation of Fyn tyrosine kinase and protein kinase C Delta, PKCδ, in lipid rafts

Cannabinoid 1 receptors (CB1Rs) are heptahelical transmembrane receptors which may exert their effects through the activation of the extracellular signal-regulated kinases (ERKs). We have previously shown that stably overexpressed CB1R in neuroblastoma cells (SH-SY5Y-CB1R cell line) is coupled to ERK activation via a mechanism that involves cannabinoid-induced transactivation of the EGF receptor and PKC activation. In a new line of experiments, EGFR transactivation by cannabinoid agonists was further supported by assessments of Ras activity. Ras assays revealed elevated Ras activity after Methanandamide treatment, which was abolished by the EGFR inhibitor AG1478. In analyzing this mechanism, we investigated the subcellular trafficking of the CB1R in basal conditions and in response to agonist stimulation in SH-SY5Y-CB1R cells. We found that under basal conditions, CB1R was mainly distributed in subcellular fractions which contain plasma-membrane, mitochondria or ER membranes, whereas after treatment with the CB1 agonist Methanandamide we observed redistribution of the receptor into the lipid rafts fractions. Moreover, we found that the activated (phosphorylated) species of EGFR also appeared in the lipid rafts after Methanandamide and importantly this effect was completely abolished by AG1478. To address what molecular events couple CB1R activation to ERK activation, we investigated whether members of Src family tyrosine kinases mediate this coupling. We found that PP1 and PP2 inhibitors of the Src family of tyrosine kinases in particular of Fyn kinase, abolish the methanandamide-dependent ERK activation. Furthermore, Methanandamide treatment induced tyrosine phosphorylation, an event that was inhibited by PP1, as well as by inhibitors of novel PKCs. In addition, Methanandamide-induced phosphorylation- activation of PKCs was also partially inhibited by Fyn and PKC inhibitors. Next, using immunoprecipitations we found that the novel PKC isoform delta, PKCδ, was tyrosine-phosphorylated in response to Methanandamide treatment and that this tyrosine phosphorylation was abolished by PP1 and Ro31-8220 (an inhibitor of classic and novel PKCs) but not by Go6976 (an inhibitor of classic PKC isoforms). These results suggest that in CB1R signaling a) Fyn activation may lie upstream of PKCδ, and b) a novel PKC isoform other than PKCδ activates Fyn which in turn activates PKCδ. ©PHARMAKON-Press