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

Abstract

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 ( 116 Phe− 124 Val). 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 polymerizationdependent interaction with SAV1.

Filamin A mediates embryonical palatal fusion by linking mechanotransduction with β-Catenin/Smad2

“…The MST1-regulated transduction process has been reported to be important in the mechanical regulation of bone remodeling (Wang et al , 2022). Notably, the activation and translocation of MST1 is regulated by the FLNA mechanobinding partner, SAV1 (Zhang et al , 2023).…”

“…For example, mutations in Filamin A ( FLNA ) lead to various severe malformations, including periventricular nodular heterotopia, otopalatodigital spectrum disorders, and sometimes cleft palate in humans (Fennell et al , 2015; Oshina et al , 2022). FLNA, the most abundant member of the filamin protein family, acts as a crosslinker for F-actin and interacts with many binding partners (Zhang et al , 2023). Its mechanosensing domain, responding to mechanical forces through conformational changes, is involved in various mechanotransduction signaling pathways, influencing cellular migration, morphology, and other crucial biological processes (Zhang et al , 2023, 21).…”

“…FLNA, the most abundant member of the filamin protein family, acts as a crosslinker for F-actin and interacts with many binding partners (Zhang et al , 2023). Its mechanosensing domain, responding to mechanical forces through conformational changes, is involved in various mechanotransduction signaling pathways, influencing cellular migration, morphology, and other crucial biological processes (Zhang et al , 2023, 21). One previous study has shown that FLNA-regulated apical epithelial extrusion squeezed out the oncogenic Src-transformed cells (Kajita et al , 2014).…”

The Role of Mechanotransduction in Contact Inhibition of Locomotion and Proliferation

“…Given that a single myosin molecule can generate 3-4 pN force, just one or a few myosins can produce enough force to unfold the domain pairs [100,141], allowing for partner interactions. So far, several molecules, including β integrins, migfilin, CFTR, fimbacin, smoothelin, G3BP1, SAV1, and LARP4, have been shown to interact with the CD face of R21 [99,[212][213][214][215][216]. This suggests that these molecules interact with FLNA in a force-dependent manner.…”

“…However, the molecular mechanism by which cheerio controls the Hippo pathway remains unknown. Recently, SAV1, a component of the Hippo pathway, has been identified as a binding partner of FLNA in mammalian cells [215]. Like other known mechanobinding partners of FLNA, SAV1 interacts with the CD face of R21.…”