As shown in Figure 1B, phoshorylation of bcatenin is decreased and total b-catenin level is slightly increased as a result of CT99021 2-hour treatment. Smad2 phosphorylation at the cluster of serines and Smad3 phosphorylation at serine 204 are decreased after CT99021 treatment, while phosphoSmad3 levels are increased. Phosphorylation at threonines 179 and 220 are not affected by CT99021 treatment. These results strongly suggest that GSK3 is implicated in the Smad2 and Smad3 linker phosphorylation, at the cluster of serines and at serine 204 respectively. We previously reported that AbMole BioScience riluzole inhibited phosphorylation of AKT, suggesting that this agent could negatively affectAKTactivity. As shown in Figure 2A, riluzole decreases AKT phosphorylation on serine 473 and threonine 308.AKTphosphorylates GSK3 on serine 9 for GSK3b or 21 for GSK3a, thereby inactivating GSK3. Therefore, we hypothesized that by inhibiting AKT activity, riluzole could decrease the phosphorylation of GSK3 at the AKT site. In order to determine whether GSK3 phosphorylation at the AKT site was decreased in the presence of riluzole, AB1010 melanoma cells were incubated in the absence or presence of this agent for 4, 8 and 16 hours and GSK3b phosphorylation at the AKT site was analyzed. As shown in Figure 2B, treatment of melanoma cells with riluzole led to a decrease in AKT-mediated GSK3b phosphorylation on serine 9. These results suggest that riluzole could positively regulate GSK3b activity. Since we showed that GSK3b is involved in phosphorylating the Smad linker domain, we investigated whether riluzole was able to induce an increase in Smad linker phosphorylation. In order to determine whether riluzole could increase Smad linker phosphorylation, melanoma cell lines were incubated in the presence of this agent for 9 hours. As shown in Figure 3A, riluzoletreated cells had increased linker phosphorylation of Smad2 at serines 245/250/255 in the five melanoma cell lines tested and of Smad3 at serine 204 in all but the 1205LU cell line. As previously shown, constitutive phosphorylation of serines 245/250/ 255 in Smad2 and serine 204 in Smad3 involves GSK3 activity. To directly demonstrate that GSK3 mediated the Smad linker phosphorylation induced by riluzole, melanoma cell lines were treated with riluzole, in the absence or presence of pharmacological inhibitors of GSK3, LiCl and CT99021. As shown in Figures 3B and 3C, GSK3 inhibition led to reduction of basal and riluzole-induced phosphorylation of Smad2 and Smad3 linker phosphorylation. In addition, siRNA knockdown of GSK3a and GSK3b inhibited the riluzole-induced phosphorylation of Smad2 and Smad3 . Finally, the same sites were robustly phosphorylated by GSK3b in an in vitro kinase assay. These results strongly suggest that riluzole, by successively inhibiting AKT and activating GSK3 activities, increases Smad linker phosphorylation. We previously mentioned that preclinical studies in vitro and animal models pointed to the metabotropic glutamate receptor 1 as a key player in melanoma development.