Identification of Filamin A Mechanobinding Partner I: Smoothelin Specifically Interacts with the Filamin A Mechanosensitive Domain 21
Identification of Filamin A Mechanobinding Partner I: Smoothelin Specifically Interacts with the Filamin A Mechanosensitive Domain 21
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细丝蛋白 A 机械结合伴侣 I 的鉴定：Smoothelin 与细丝蛋白 A 机械敏感结构域特异性相互作用 21
DOI：
10.1021/acs.biochem.9b00100
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发表时间：
2019
期刊：
影响因子：
2.9
通讯作者：
Fumihiko Nakamura
中科院分区：
文献类型：
--
作者：
Wang L.;Fumihiko Nakamura
细丝蛋白A（FLNA）是一种广泛表达的肌动蛋白交联蛋白，是调节细胞增殖、迁移和存活的众多结合伴侣的支架。FLNA是同源二聚体，每个亚基具有N-末端肌动蛋白结合结构域，随后是24个免疫球蛋白样重复序列（R）。FLNA通过力诱导的R21上其隐蔽整合素结合位点的构象变化介导机械转导。在这里，我们确定了两个新的FLNA结合伙伴，平滑蛋白（SMTN A和B）和亮氨酸拉链蛋白1（LUZP 1），使用稳定同位素标记的氨基酸在细胞培养（SILAC）为基础的蛋白质组学，然后通过计算机筛选具有共识FLNA结合结构域的蛋白质。我们发现，尽管SMTN不与全长FLNA相互作用，但它与暴露R21的隐蔽CD裂缝的FLNA变体1（FLNAvar-1）结合。破坏整联蛋白结合的C链上的点突变也阻断SMTN相互作用。我们使用诱变鉴定了SMTN上的FLNA结合结构域，并使用突变的SMTN来研究FLNA-SMTN相互作用对SMTN在活细胞中的动力学和定位的作用。GFP标记的SMTN在活细胞中的光漂白后的荧光恢复（FRAP）表明，非FLNA结合突变体SMTN比野生型SMTN扩散更快。此外，使用Y27632抑制Rho激酶也会增加扩散。这些数据表明，SMTN特异性地与细胞中的FLNAvar-1和机械活化的FLNA相互作用。配套报告（Wang和中村，2019）描述了FLNA与LUZP 1基因转录本的相互作用。
Filamin A (FLNA) is a ubiquitously expressed actin cross-linking protein and a scaffold of numerous binding partners to regulate cell proliferation, migration, and survival. FLNA is a homodimer, and each subunit has an N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats (R). FLNA mediates mechanotransduction by force-induced conformational changes of its cryptic integrin-binding site on R21. Here, we identified two novel FLNA-binding partners, smoothelins (SMTN A and B) and leucine zipper protein 1 (LUZP1), using stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics followed by anin silicoscreening for proteins having a consensus FLNA-binding domain. We found that, although SMTN does not interact with full-length FLNA, it binds to FLNA variant 1 (FLNAvar-1) that exposes the cryptic CD cleft of R21. Point mutations on the C strand that disrupt the integrin binding also block the SMTN interaction. We identified FLNA-binding domains on SMTN using mutagenesis and used the mutant SMTN to investigate the role of the FLNA–SMTN interaction on the dynamics and localization of SMTN in living cells. Fluorescence recovery after photobleaching (FRAP) of GFP-labeled SMTN in living cells demonstrated that the non-FLNA-binding mutant SMTN diffuses faster than wild-type SMTN. Moreover, inhibition of Rho-kinase using Y27632 also increases the diffusion. These data demonstrated that SMTN specifically interacts with FLNAvar-1 and mechanically activated FLNA in cells. The companion report (Wang and Nakamura, 2019) describes the interactions of FLNA with the transcript of the LUZP1 gene.