Identification of Filamin A Mechanobinding Partner III: SAV1 Specifically Interacts with Filamin A Mechanosensitive Domain 21.

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 (