Zhang Huaguan, Mao Zhenfeng, Yang Ziwei, Nakamura Fumihiko
School of Pharmaceutical Science and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, China.
Biochemistry. 2023 Mar 21;62(6):1197-1208. doi: 10.1021/acs.biochem.2c00665. Epub 2023 Mar 1.
Filamin A (FLNA) cross-links actin filaments and mediates mechanotransduction by force-induced conformational changes of its domains. FLNA's mechanosensitive immunoglobulin-like repeats (R) interact with each other to create cryptic binding sites, which can be exposed by physiologically relevant mechanical forces. Using the FLNA mechanosensing domains as an affinity ligand followed by stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, we recently identified smoothelin and fimbacin as FLNA mechanobinding proteins. Here, using the mechanosensing domain as an affinity ligand and two labeled amino acids, we identify salvador homologue 1 (SAV1), a component of the Hippo pathway kinase cascade, as a new FLNA mechanobinding partner. We demonstrate that SAV1 specifically interacts with the cryptic C-D cleft of FLNA R21 and map the FLNA-binding site on SAV1. We show that point mutations on the R21 C strand block the SAV1 interaction and find that SAV1 contains a FLNA-binding motif in the central region (Phe-Val). Point mutations F116A and T118A (FT/AA) disrupt the interaction. A proximity ligation assay reveals that their interaction occurs in the cytosol in an actin polymerization-dependent manner. Although SAV1 is typically found in the cytosol, disrupting the interaction between SAV1 and FLNA causes SAV1 to diffuse to the nucleus and YAP1 to diffuse to the cytosol in an inverse relationship. These results suggest that FLNA mediates regulation of the Hippo pathway through actin polymerization-dependent interaction with SAV1.
细丝蛋白A（FLNA）交联肌动蛋白丝，并通过其结构域的力诱导构象变化介导机械转导。FLNA的机械敏感免疫球蛋白样重复序列（R）相互作用形成隐蔽结合位点，这些位点可被生理相关的机械力暴露。我们最近使用FLNA机械传感结构域作为亲和配体，随后通过基于细胞培养的氨基酸稳定同位素标记（SILAC）蛋白质组学，鉴定出平滑肌肌动蛋白和菌毛蛋白为FLNA机械结合蛋白。在此，我们使用机械传感结构域作为亲和配体和两种标记氨基酸，鉴定出Hippo信号通路激酶级联的一个组成部分——萨尔瓦多同源物1（SAV1）为一种新的FLNA机械结合伙伴。我们证明SAV1与FLNA R21隐蔽的C-D裂隙特异性相互作用，并绘制出SAV1上的FLNA结合位点。我们发现R21 C链上的点突变会阻断SAV1的相互作用，并发现SAV1在中央区域（苯丙氨酸-缬氨酸）含有一个FLNA结合基序。点突变F116A和T118A（FT/AA）破坏了这种相互作用。邻近连接分析表明，它们的相互作用以肌动蛋白聚合依赖的方式发生在细胞质中。尽管SAV1通常存在于细胞质中，但破坏SAV1与FLNA之间的相互作用会导致SAV1扩散到细胞核，而YAP1则以相反的关系扩散到细胞质中。这些结果表明，FLNA通过与SAV1的肌动蛋白聚合依赖相互作用介导Hippo信号通路的调节。
