IκB kinase (IKK) complex is central regulators of the NF-κB pathway, and dysregulation of IKK phosphorylation leads to hyperactivation of proinflammatory response in various chronic inflammatory diseases, including inflammatory bowel disease (IBD). However, the dynamic modulation of IKK phosphorylation and dephosphorylation in intestinal inflammation remains uncharacterized. Here, we found that autophagy/beclin-1 regulator 1 (AMBRA1) was highly expressed in inflamed colons in a colitis mouse model and in clinical IBD samples. Importantly, AMBRA1 deletion significantly decreased proinflammatory cytokine expression and enhanced the therapeutic effect of infliximab on intestinal inflammation. Mechanistically, the N-term F1 domain of AMBRA1 was required for AMBRA1 to competitively interact with protein phosphatase 4 regulatory subunit 1 (PP4R1) and catalytic protein phosphatase 4 (PP4c) to suppress their interactions with IKK, promote the dissociation of the PP4R1/PP4c complex, and antagonize the dephosphorylation activity of this complex towards the IKK complex. In response to TNF-α stimulation, IKKα phosphorylates AMBRA1 at S1043 to stabilize AMBRA1 expression by impairing its binding to Cullin4A (CUL4A) to decrease its CUL4A-mediated K48-linked ubiquitination. Overall, our study identifies an autophagy-independent function of AMBRA1 as a positive modulator of IKK phosphorylation to promote intestinal inflammation, thus providing a new targeted therapeutic strategy for patients with refractory IBD.

IκB 激酶 (IKK) 复合物是 NF-κB 通路的核心调节因子，IKK 磷酸化失调会导致各种慢性炎症疾病（包括炎症性肠病 (IBD)）中促炎症反应过度激活。然而，肠道炎症中 IKK 磷酸化和去磷酸化的动态调节仍然未知。在这里，我们发现自噬/beclin-1 调节因子 1 (AMBRA1) 在结肠炎小鼠模型和临床 IBD 样本中的发炎结肠中高度表达。重要的是，AMBRA1 缺失显着降低了促炎细胞因子的表达，增强了英夫利昔单抗对肠道炎症的治疗效果。从机制上讲，AMBRA1 的 N 端 F1 结构域是 AMBRA1 与蛋白磷酸酶 4 调节亚基 1 (PP4R1) 和催化蛋白磷酸酶 4 (PP4c) 竞争性相互作用所必需的，以抑制它们与 IKK 的相互作用，促进 PP4R1/PP4c 的解离。复合物，并拮抗该复合物对 IKK 复合物的去磷酸化活性。响应 TNF-α 刺激，IKKα 在 S1043 位点磷酸化 AMBRA1，通过削弱其与 Cullin4A (CUL4A) 的结合来稳定 AMBRA1 的表达，从而减少其 CUL4A 介导的 K48 相关泛素化。总体而言，我们的研究确定了 AMBRA1 的独立自噬功能作为 IKK 磷酸化的正调节剂，促进肠道炎症，从而为难治性 IBD 患者提供了新的靶向治疗策略。