Doublecortin-like kinase 1 activates NF-κB to induce inflammatory responses by binding directly to IKKβ

Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein kinase, is involved in neurogenesis, and its levels are elevated in various human cancers. Recent studies suggest that DCLK1 may relate to inflammatory responses in the mouse model of colitis. However, cellular pathways engaged by DCLK1, and potential substrates of the kinase remain undefined. To understand how DCLK1 regulates inflammatory responses, we utilized the well-established lipopolysaccharide (LPS)-stimulated macrophages and mouse model. Through a range of macrophage-based and cell-free platforms, we discovered that DCLK1 binds directly with the inhibitor of κB kinase β (IKKβ) and induces IKKβ phosphorylation on Ser177/181 to initiate nuclear factor-κB (NF-κB) pathway. Deficiency in DCLK1, achieved by silencing or through pharmacological inhibition, prevented LPS-induced NF-κB activation and cytokine production in macrophages. We further show that mice with myeloid-specific DCLK1 knockout or DCLK1 inhibitor treatment are protected against LPS-induced acute lung injury and septic death. Our studies report a novel functional role of macrophage DCLK1 as a direct IKKβ regulator in inflammatory signaling and suggest targeted therapy against DCLK1 for inflammatory diseases. Monoclonal cell line RAW 264.7 cell lines which steadily expressed DCLK1 shRNA or negative control shRNA were plated at 200,000 cells/well in six-well plates. Cells were challenged with 500 ng/mL LPS for 6 h. Cells were then homogenized using RNAiso Plus (Takara, Japan) and total RNA was extracted using RNeasy Plus Mini Kit. A reference transcriptome analysis was performed by Lianchuan Bio (LC-Bio) Technologies (Hangzhou, Zhejiang). Paired-end sequencing was performed using the Illumina Novaseq 6000 (California, United States).