Supplementary Material for: Regulation of AP-1 by MAPK Signaling in Metal-Stressed Sea Anemone

<strong><em>Background/Aims:</em></strong> AP-1 transcription factor plays a conserved role in the immediate response to stress. Activation of AP-1 members <i>jun</i> and <i>fos</i> is mediated by complex signaling cascades to control cell proliferation and survival. To understand the evolution of this broadly-shared pathway, we studied AP-1 regulation by MAPK signaling in a basal metazoan. <b><i>Methods:</i></b> Metal- stressed cnidarian <i>Nematostella vectensis</i> anemones were tested with kinase inhibitors and analyzed for gene expression levels and protein phosphorylation. <b><i>Results:</i></b> We show that in cnidarian, AP-1 is regulated differently than in bilaterian models. ERK2 and ERK5, the main MAPK drivers of AP-1 activation in Bilateria, down-regulated <i>fos1</i> and <i>jun1</i> transcription in anemones exposed to metal stress, whereas p38 MAPK, triggered transcription of <i>jun1</i> but not <i>fos1</i>. Furthermore, our results reveal that GSK3-β is the main driver of the immediate stress response in <i>Nematostella</i>. GSK3-β triggered transcription of AP-1 and two other stress-related genes, <i>egr1</i> and <i>hsp70</i>. Finally, phylogenetic analysis and protein characterization show that while MAPKs and GSK3-β are evolutionarily conserved, Fos and Jun proteins in <i>Nematostella</i> and other cnidarians lack important regulatory and phosphorylation sites found in Bilateria. <b><i>Conclusion:</i></b> These findings reveal alternative network interactions of conserved signaling kinases, providing insight into the evolutionary plasticity of immediate stress response mechanisms.