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
(116Phe–124Val). 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.