Rahman Md Mizanur, Kim Jong S, Li Laiji, Feisal M Rafid, Mak Kevin Y L, Tavasoli Mahtab, Wang Zhixiang, Ballermann Barbara J, Hwang Peter M
Department of Medicine, University of Alberta, Edmonton, Canada.
Department of Genetics, University of Alberta, Edmonton, Canada.
J Biol Chem. 2025 Aug 28;301(10):110646. doi: 10.1016/j.jbc.2025.110646.
Ezrin, radixin, and moesin (ERM) proteins regulate assembly of actin-based structures, link membrane-spanning proteins to cortical actin, and are part of cell signaling hubs. The chloride intracellular channel (CLIC) 5A protein is very abundant in radixin-dependent inner ear hair cell stereocilia and in ezrin-dependent kidney glomerular podocyte foot processes and is essential for the structural integrity of these actin-based cellular projections. The functional relationship between ERM proteins and CLIC5A is incompletely understood and whether CLIC5A functions as a chloride channel is controversial. We determined whether CLIC5A is membrane-spanning protein and sought direct CLIC5A binding partners. While CLIC5A localized predominantly to the dorsal plasma membrane domain, we found CLIC5A to be a soluble, intracellular protein, without characteristics expected of a membrane-spanning channel. In the yeast two-hybrid assay, CLIC5A interacted directly with the C-terminal domains of ERM with a hierarchy of ezrin > radixin = moesin. The last 16 amino acids of ezrin were essential but not sufficient for CLIC5A binding, and phosphorylation of ezrin at T567 enhanced the interaction. The affinity of purified CLIC5A for a phosphomimetic ezrin (T567E) C-terminal fragment was in the 30 μM range. Silencing of ERM dislodged CLIC5A from the peripheral location, and the CLIC5A-ezrin interaction augmented Rho guanine nucleotide dissociation inhibitor sequestration by ezrin and Rac1 activity. Thus, CLIC5A functions as a direct binding partner of ezrin, stabilizing its open/active conformation and resulting in localized small GTPase activation.
埃兹蛋白、根蛋白和膜突蛋白（ERM）可调节基于肌动蛋白的结构组装，将跨膜蛋白与皮质肌动蛋白相连，并且是细胞信号枢纽的一部分。氯离子细胞内通道（CLIC）5A蛋白在依赖根蛋白的内耳毛细胞静纤毛以及依赖埃兹蛋白的肾肾小球足细胞足突中含量非常丰富，对于这些基于肌动蛋白的细胞突起的结构完整性至关重要。ERM蛋白与CLIC5A之间的功能关系尚未完全明确，并且CLIC5A是否作为氯离子通道发挥作用也存在争议。我们确定了CLIC5A是否为跨膜蛋白，并寻找CLIC5A的直接结合伴侣。虽然CLIC5A主要定位于背侧质膜结构域，但我们发现CLIC5A是一种可溶性细胞内蛋白，不具备跨膜通道所预期的特征。在酵母双杂交试验中，CLIC5A与ERM的C末端结构域直接相互作用，其相互作用强度顺序为埃兹蛋白>根蛋白 = 膜突蛋白。埃兹蛋白的最后16个氨基酸对于CLIC5A结合是必需的，但并不充分，并且埃兹蛋白在T567位点的磷酸化增强了这种相互作用。纯化的CLIC5A与模拟磷酸化的埃兹蛋白（T567E）C末端片段的亲和力在30 μM范围内。沉默ERM可使CLIC5A从外周位置解离，并且CLIC5A - 埃兹蛋白相互作用增强了埃兹蛋白对Rho鸟嘌呤核苷酸解离抑制剂的隔离作用以及Rac1活性。因此，CLIC5A作为埃兹蛋白的直接结合伴侣发挥作用，稳定其开放/活性构象并导致局部小GTP酶激活。
