Identification of Filamin A Mechanobinding Partner I: Smoothelin Specifically Interacts with the Filamin A Mechanosensitive Domain 21

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 an in silico screening 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.