Figure 5.
HNE modification of ZAK suppresses JNK pathway activation in cells. (a) WT-ZAK-transfected HEK-293T cells show higher basal JNK activation compared to mock-, C22A-ZAK-, or K45M-ZAK-transfected. (b,c) Western blots (b) and normalized phosphorylated JNK levels (c) showing that H2O2 treatment (1 mM, 30 min) increases JNK activation in WT- and C22A-ZAK cells and this increase is blocked or amplified in WT- and C22A-ZAK cells, respectively by pre-treatment with HNE (100 μM, 30 min). (d) A model diagramming ZAK-dependent and ZAK-independent pathways for HNE modulation of JNK activation. Dashed line designates the potential for oxidative stress to generate HNE and initiate a negative feedback loop to limit JNK activation. (e,f) Western blots (e) and normalized phosphorylated JNK levels (f) showing dramatic, concentration-dependent activation of JNK by HNE (50 or 100 μM, 60 min) in C22A-ZAK cells, but not in WT-ZAK cells. Note that mock- and K45M-ZAK transfected cells also show modest, but significant elevations in JNK activity following HNE treatment, which is consistent with previous studies indicating that HNE can activate JNK by multiple pathways40. For a, c and f, data are presented as mean values ± s.e.m.; N >= 4 experiments/group (biological replicates). *, P < 0.05; **, P < 0.01; ##, P < 0.01; t-test, two-sided.