Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and Department of Microbiology and Immunology, Stanford University, California, USA.
Department of Microbiology, University of Chicago, Chicago, Illinois, USA.
J Biol Chem. 2022 Jun;298(6):101956. doi: 10.1016/j.jbc.2022.101956. Epub 2022 Apr 20.
The signaling pathways and cellular functions regulated by the four Numb-associated kinases are largely unknown. We reported that AAK1 and GAK control intracellular trafficking of RNA viruses and revealed a requirement for BIKE in early and late stages of dengue virus (DENV) infection. However, the downstream targets phosphorylated by BIKE have not yet been identified. Here, to identify BIKE substrates, we conducted a barcode fusion genetics-yeast two-hybrid screen and retrieved publicly available data generated via affinity-purification mass spectrometry. We subsequently validated 19 of 47 putative BIKE interactors using mammalian cell-based protein-protein interaction assays. We found that CLINT1, a cargo-specific adapter implicated in bidirectional Golgi-to-endosome trafficking, emerged as a predominant hit in both screens. Our experiments indicated that BIKE catalyzes phosphorylation of a threonine 294 CLINT1 residue both in vitro and in cell culture. Our findings revealed that CLINT1 phosphorylation mediates its binding to the DENV nonstructural 3 protein and subsequently promotes DENV assembly and egress. Additionally, using live-cell imaging we revealed that CLINT1 cotraffics with DENV particles and is involved in mediating BIKE's role in DENV infection. Finally, our data suggest that additional cellular BIKE interactors implicated in the host immune and stress responses and the ubiquitin proteasome system might also be candidate phosphorylation substrates of BIKE. In conclusion, these findings reveal cellular substrates and pathways regulated by the understudied Numb-associated kinase enzyme BIKE, a mechanism for CLINT1 regulation, and control of DENV infection via BIKE signaling, with potential implications for cell biology, virology, and host-targeted antiviral design.
由四个 NUMB 相关激酶调节的信号通路和细胞功能在很大程度上是未知的。我们报道 AAK1 和 GAK 控制 RNA 病毒的细胞内运输，并揭示了 BIKE 在登革热病毒（DENV）感染的早期和晚期阶段的必要性。然而，BIKE 磷酸化的下游靶标尚未确定。在这里，为了鉴定 BIKE 的底物，我们进行了条形码融合遗传学-酵母双杂交筛选，并检索了通过亲和纯化质谱生成的公开可用数据。随后，我们使用基于哺乳动物细胞的蛋白质-蛋白质相互作用测定法验证了 47 个假定 BIKE 相互作用体中的 19 个。我们发现，CLINT1 是一种涉及双向高尔基体到内体运输的货物特异性接头，在两个筛选中均成为主要命中。我们的实验表明，BIKE 在体外和细胞培养中催化 CLINT1 丝氨酸 294 残基的磷酸化。我们的发现表明，CLINT1 磷酸化介导其与 DENV 非结构 3 蛋白结合，随后促进 DENV 组装和出芽。此外，通过活细胞成像，我们揭示了 CLINT1 与 DENV 颗粒共运输，并参与介导 BIKE 在 DENV 感染中的作用。最后，我们的数据表明，参与宿主免疫和应激反应以及泛素蛋白酶体系统的其他细胞 BIKE 相互作用物也可能是 BIKE 的候选磷酸化底物。总之，这些发现揭示了受研究较少的 NUMB 相关激酶 BIKE 调节的细胞底物和途径、CLINT1 调节的机制以及通过 BIKE 信号控制 DENV 感染的机制，这可能对细胞生物学、病毒学和宿主靶向抗病毒设计具有重要意义。
