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

Abstract

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.