Identification Of A New Filamin A Mechanobinding Protein

Filamin A(FLNA)is an actin-crosslinking protein and interacts with numerous binding partners,which have important role in cell process such as cell migration,differentiation,proliferation,and survival.Mutations of FLNA gene lead to human disease such as mental retardation,developmental malformations,and heart failure,highlighting the importance of FLNA-regulated signaling pathway in cells.FLNA protein is a homodimer of 280 k Da subunits which consists of an N-terminal actin-binding domain followed by 24 immunoglobulin(Ig)-like repeats(R).FLNA was recently identified as a mechanosensor and mechanotransducer.Force-induced conformational changes in FLNA “mechanosensing domains” can regulate partner interactions by two distinctive mechanisms: 1)exposing a cryptic(structurally covered)binding site of repeat 21(R21 exposure)and 2)spatial separation of two R23,which interacts with Fil GAP dimer at 1:1 molar ratio,of each FLNA subunit.Since Fil GAP is not ubiquitously expressed and conformational changes of R21 are not localized to integrin-rich focal adhesion,we hypothesized that other FLNA-binding partners interact with FLNA mechanosesening domains in a force-dependent manner.Hence,my thesis consists two parts: Part I is “Identification of a new binding partner for filamin A domains 23-24”;Part II is “Identification of Smoothelin as a new filamin A mechanobinding protein”.To identify protein-protein interaction,affinity purification has been used to pull down binding partners and these proteins were subsequently identified by mass-spec.However,this pull down experiment can often result in nonspecific interaction.To distinguish true interactor and non-specific binders,we employed stable-isotope labeling by amino acids in cell culture(SILAC)followed by affinity-tagged protein purification and quantitative mass spectrometry analysis.In part I,to identify proteins that interact with R23,we used R23-24 as a test affinity ligand.As a negative control,two affinity ligands: One is R23-24(M2474E)that is mutated on the CD face of R23,this mutation can disturb the interaction of FLNA with Fil GAP.Another one is R24 alone without R23.Using these affinity ligands,we pulled down binding partners from lysate of mouse embryonic fibroblast(MEF)cells that were labeled with either light or heavy amino acids.We detected the following molecules: Ankycorbin(Rai14),Guanine nucleotide-binding protein G(I)/G(S)/G(T)subunit beta-2(Gnb2),Myosin phosphatase Rho-interacting protein(Mprip),Guanine nucleotide-binding protein G(k)subunit alpha(Gnai3),Moesin(MSN),Lamina-associated polypeptide 2,isoform alpha(Tmpo)and Polo-like kinase 1(PLK1)as new FLNA binding candidates.Preliminary result showed that PLK1 can directly interact with FLNA R23.However,further experiment is necessary to ensure that this interaction occurs in living cells.In Part II,we identified smoothlin(SMTN)as a new FLNA-binding partner of R21 using R21-23 as affinity ligand.As a negative control,R1-3 was used because CD faces of R1-3 are structurally different from that of R21.We found that although SMTN does not interact with canonical 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 dynamics and localization of SMTN in living cells.Fluorescence recovery after photobleaching of GFP-labeled SMTN in living cells demonstrated that 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.