Recombinant Human FLNA protein, MYC/DDK-tagged

• Cat.No.: FLNA-185H
• Product Overview: Recombinant Human FLNA, transcript variant 1, fused with MYC/DDK tag at C-terminal was expressed in HEK293.

Specification

• Species: Human
• Source: HEK293
• Tag: DDK&Myc
• Description: The protein encoded by this gene is an actin-binding protein that crosslinks actin filaments and links actin filaments to membrane glycoproteins. The encoded protein is involved in remodeling the cytoskeleton to effect changes in cell shape and migration. This protein interacts with integrins, transmembrane receptor complexes, and second messengers. Defects in this gene are a cause of several syndromes, including periventricular nodular heterotopias (PVNH1, PVNH4), otopalatodigital syndromes (OPD1, OPD2), frontometaphyseal dysplasia (FMD), Melnick-Needles syndrome (MNS), and X-linked congenital idiopathic intestinal pseudoobstruction (CIIPX). Two transcript variants encoding different isoforms have been found for this gene.
• Form: 25 mM Tris.HCl, pH 7.3, 100 mM glycine, 10% glycerol.
• Molecular Mass: 280.6 kDa
• Purity: > 80% as determined by SDS-PAGE and Coomassie blue staining
• Concentration: >50 ug/mL as determined by microplate BCA method

Gene Information

• Gene Name: FLNA filamin A, alpha [ Homo sapiens ]
• Official Symbol: FLNA
• Synonyms: FLNA; filamin A, alpha; filamin A, alpha (actin binding protein 280) , FLN, FLN1, OPD1, OPD2; filamin-A; ABP 280; actin binding protein 280; filamin-1; alpha-filamin; non-muscle filamin; endothelial actin-binding protein; FLN; FMD; MNS; OPD; ABPX; CVD1; FLN1; NHBP; OPD1; OPD2; XLVD; XMVD; FLN-A; ABP-280; FLJ43642; DKFZp434P031
• Gene ID: 2316
• mRNA Refseq: NM_001110556
• Protein Refseq: NP_001104026
• MIM: 300017
• UniProt ID: P21333
• Chromosome Location: Xq28
• Pathway: Androgen Receptor Signaling Pathway, organism-specific biosystem; Cell junction organization, organism-specific biosystem; Cell-Cell communication, organism-specific biosystem; Cell-extracellular matrix interactions, organism-specific biosystem; Focal Adhesion, organism-specific biosystem; Focal adhesion, organism-specific biosystem; Focal adhesion, conserved biosystem
• Function: Fc-gamma receptor I complex binding; Rac GTPase binding; Ral GTPase binding; Rho GTPase binding; actin binding; actin filament binding; glycoprotein binding; protein binding; protein homodimerization activity; protein kinase C binding; signal transducer activity; small GTPase binding; transcription factor binding

Citation

Significance of filamin A in mTORC2 function in glioblastoma

Journal: Molecular Cancer    PubMed ID: 26134617    Data: 2015/7/2

Authors: Naphat Chantaravisoot, Piriya Wongkongkathep, Fuyuhiko Tamanoi

Article Snippet:Kinase activities of mTORC2 from U87vIII cells expressing FLAG-RICTOR were examined in various conditions using affinity purified mTORC2 as described above.Kinase activities of mTORC2 from U87vIII cells expressing FLAG-RICTOR were examined in various conditions using affinity purified mTORC2 as described above.. Purified FLNA protein (C-terminal fragment amino acid 1730–2639; Creative BioMart) from E. coli and purified AKT protein (full-length; EMD Millipore) were used as substrates for the kinase assay.. In each reaction, purified mTORC2 and its substrate (FLNA or AKT) were incubated in a buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 0.2 mM ATP for 25 min.In each reaction, purified mTORC2 and its substrate (FLNA or AKT) were incubated in a buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 0.2 mM ATP for 25 min.

Purification, identification, and characterization of mTORC2 and its binding partners. a Silver-stained high molecular weight mTORC2 components purified from U87vIII cells stably expressing FLAG-RICTOR are shown. Four large proteins (numbered as 1-4) were analyzed by mass spectrometry after being run on 7 % SDS-PAGE gel, excised, then digested by Trypsin. Mass spectrometry results are summarized in the table on the right. Filamin A (FLNA) and Myosin-9 (MYH9) represent unknown bands 1 and 3, respectively. ?Mascot protein score; a protein with score value >21 with >2 unique peptides is considered significant (P <0.05). b Immunoblots of purified proteins from U87vIII cells showing main components of mTORC2 (mTOR, RICTOR, SIN1) including phosphorylated and total FLNA. c Immunoprecipitation of U87vIII cell lysate by protein G Dynabeads coupled with antibodies against main components of mTOR complex 1 and 2. Phosphorylated FLNA was co-immunoprecipitated with anti-mTOR and anti-RICTOR coated beads while these two components of mTORC2 were pulled down with anti-pFLNA coated beads. RAPTOR, an mTORC1-specific component, can be co-immunoprecipitated with anti-mTOR coated beads but not with anti-pFLNA beads

Purified mTORC2 phosphorylates FLNA at Ser2152 in vitro . a In vitro kinase assay of mTORC2 purified from U87vIII cells with FLNA and AKT as substrates. Level of phosphorylated FLNA (Ser2152) and phosphorylated AKT (Ser473) were increased in the presence of mTORC2 and ATP. Negative control groups contain either no purified complex or no ATP. Activity of mTORC2 is maximal when Mn 2+ is used instead of Mg 2+ . b In vitro kinase assay of mTORC2 purified from U87vIII cells with FLNA as a substrate. Two concentrations of PP242 (+ = 1.25 μM; ++ = 5.0 μM) and one concentration of IPA3 (+ = 40 μM) were added into three samples to inhibit the kinase activity of mTORC2. Levels of pFLNA and pAKT when treated with PP242 are decreased compared to ones without PP242