Figure 1
From: GSK-3β activation is required for ZIP-induced disruption of learned fear
In vitro kinase assay reveals that GSK-3β is directly activated by ZIP. (a) Activities of various protein kinases and protein phosphatases modulated by 10 µM of myristoylated ZIP or SCR-ZIP (n = 9 for GSK-3α and β, n = 4 for PKBα, n = 2 for PKCζ, and n = 3 for others). Significance of the increase or decrease of kinase activity by ZIP and SCR-ZIP is revealed by one sample t-test. *p < 0.05 (AGK kinases: protein kinases A/G/K families, CAM kinases: calcium/calmodulin kinases, CMGC kinases: cyclin-dependent kinase (CDK), mitogen-activated protein kinase (MAPK), glycogen synthase kinase (GSK), CDC-like kinase (CLK)). The dashed line represents activities of protein kinases and protein phosphatases in the absence of the peptides. Activities of various protein kinases and protein phosphatases with ZIP or SCR-ZIP were normalized to 100%. (b) Activities of protein kinases and protein phosphatases modulated by 10 µM of non-myristoylated ZIP and SCR-ZIP (n = 2 per group). Significance of the increase or decrease of kinase activity by non-myristoylated ZIP and SCR-ZIP is revealed by one sample t-test. *p < 0.05 (c) In vitro experiments shows that ZIP activated purified GSK-3α and GSK-3β in a concentration-dependent manner (n = 3 per group). (d) Synaptosomal and total fractions of each brain regions (LA: lateral amygdala, Cortex: cortical brain area obtained in same coronal plane of brain slices including lateral amygdala, DH: dorsal hippocampus) were sampled and further blotted with anti-GSK-3α or β antibody. GSK-3α was not detected in the synaptosomal fraction, while GSK-3β was expressed in both the synaptosomal and total fractions. Full-length blots are displayed in Fig. S3a and b. (e) Activation of purified GSK-3β (12.5 nM) by ZIP (10 µM) was observed only when pre-phosphorylated substrates were used (n = 3 per group). The phosphorylation level in the presence of ZIP was normalized to that in the presence of vehicle. The concentration of GSK-3β substrate was 20 µM. One sample t-test reveals that ZIP significantly enhanced phosphorylation of prephosphorylated substrates, but not of a non-phosphorylated substrate. The prefix ‘p-’ means that the substrate is pre-phosphorylated. **p < 0.01 (f) ZIP facilitated the reaction kinetics of GSK-3β, which were assayed using the peptide (636–661) derived from human muscle glycogen synthase 1. Reaction curves between GSK-3β and the substrate (upper) and between GSK-3β and ATP (lower) are shown (n = 2 per group). Solid curves indicate the reaction in the presence of ZIP (10 µM), and dashed curves indicate the reaction in the presence of vehicle.
