Erk5 S496 Phosphorylation, But Not Erk5 Kinase Or Transcriptional Activity, Is Responsible For Promoting Macrophage Inflammation And Mitochondrial Dysfunction Via Upregulating Novel Site Of Nrf2 K518 Sumoylation

ERK5 is a dual kinase-transcription factor, which contains two transcriptional transactivation domains in the C-terminus and a kinase domain in the N-terminus. Many ERK5 kinase inhibitors have been developed, and are being tested in clinical studies for cancer and inflammatory diseases. Recent data has raised questions regarding the functional role of these ERK5 kinase inhibitors. Specifically, the possible link between blockade of pro-inflammatory ERK5 S496 phosphorylation and the anti-inflammatory effects of ERK5-specific kinase inhibitors has largely been neglected. In this study, we aimed to study the role and regulatory mechanisms of ERK5 S496 phosphorylation on macrophage inflammation and the impact of ERK5-specific kinase inhibitors. ATP binding site deletion mutant of ERK5b (a kinase-dead mutant) inhibited KLF2 induction but not oxidized LDL (oxLDL)-induced ERK5 S496 phosphorylation and TNFα expression. In contrast, both specific ERK5 kinase inhibitors (AX15836 and XMD8-92) and a dual phosphorylation site mutant of ERK5 (AEF) inhibited not only KLF2 but also oxLDL-induced ERK5 S496 phosphorylation and TNFα induction. These data suggested that ERK5 S496 phosphorylation, but not ERK5 kinase activity, plays a crucial role in ERK5-mediated pro-inflammatory effects. We also discovered a key effect of ERK5 S496 phosphorylation on SUMOylation at a novel site of NRF2 (i.e., K518), which inhibited NRF2 transcriptional activity without affecting ERK5 kinase activity, and antagonized oxLDL-induced macrophage inflammation. The role of NRF2 activation on the efficiency of oxidative phosphorylation (OXPHOS) and ATP synthesis had previously been reported, and we found that both ERK5 S496A and NRF2 K518R mutants abolished oxLDL-induced reduction of OXPHOS, ATP, and NAD + levels. In summary, we discovered a novel mechanism in which ERK5 S496 phosphorylation directly inhibited NRF2 activity via SUMOylation of NRF2 at K518 and thereby induced macrophage inflammation and mitochondrial dysfunction. The often-neglected role of ERK S496 signaling should be carefully considered in the interpretation of prior reports of ERK5 knockdown and pharmacological kinase inhibition relative to cellular inflammation and mitochondrial dysfunction.