Abstract Background The sarcoglycan complex (SC) is part of a network that links the striated muscle cytoskeleton to the basal lamina across the sarcolemma. The SC coordinates changes in phosphorylation and Ca ++ -flux during mechanical deformation, and these processes are disrupted with loss-of-function mutations in gamma-sarcoglycan (Sgcg) that cause Limb girdle muscular dystrophy 2C/R5. Methods To gain insight into how the SC mediates mechano-signaling in muscle, we utilized LC-MS/MS proteomics of SC-associated proteins in immunoprecipitates from enriched sarcolemmal fractions. Criteria for inclusion were co-immunoprecipitation with anti-Sgcg from C57BL/6 control muscle and under-representation in parallel experiments with Sgcg-null muscle and with non-specific IgG. Validation of interaction was performed in co-expression experiments in human RH30 rhabdomyosarcoma cells. Results We identified 19 candidates as direct or indirect interactors for Sgcg, including the other 3 SC proteins. Novel potential interactors included protein-phosphatase-1-catalytic-subunit-beta (Ppp1cb, PP1b) and Na + -K + -Cl − -co-transporter NKCC1 (SLC12A2). NKCC1 co-localized with Sgcg after co-expression in human RH30 rhabdomyosarcoma cells, and its cytosolic domains depleted Sgcg from cell lysates upon immunoprecipitation and co-localized with Sgcg after detergent permeabilization. NKCC1 localized in proximity to the dystrophin complex at costameres in vivo. Bumetanide inhibition of NKCC1 cotransporter activity in isolated muscles reduced SC-dependent, strain-induced increases in phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). In silico analysis suggests that candidate SC interactors may cross-talk with survival signaling pathways, including p53, estrogen receptor, and TRIM25. Conclusions Results support that NKCC1 is a new SC-associated signaling protein. Moreover, the identities of other candidate SC interactors suggest ways by which the SC and NKCC1, along with other Sgcg interactors such as the membrane-cytoskeleton linker archvillin, may regulate kinase- and Ca ++ -mediated survival signaling in skeletal muscle.
Tumor cells use actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II, reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV induces redistribution of lamellipodial cortactin and lamellipodin/RAPH1/PREL1 away from the cell periphery to internal sites and concomitantly increases the numbers of F-actin punctae. Most punctae are highly dynamic and colocalize with the podosome/invadopodial proteins, cortactin, Tks5, and cdc42. Cortactin binds SV sequences in vitro and contributes to the formation of enhanced green fluorescent protein (EGFP)-SV induced punctae. SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases average numbers of ECM holes per cell; RNA interference-mediated knockdown of SV decreases these numbers. Although SV knockdown alone has no effect, simultaneous down-regulation of SV and the closely related protein gelsolin reduces invasion through ECM. Together, our results show that SV is a component of podosomes and invadopodia and that SV plays a role in invadopodial function, perhaps as a mediator of cortactin localization, activation state, and/or dynamics of metalloproteinases at the ventral cell surface.
Abstract Although an immunologically cross-reactive protein of similar size has been observed in human polymorphonuclear leukocytes, only ponticulin from the soil amoeba, Dictyostelium discoideum, has been characterized. Purified Dictyostelium ponticulin consists of six major 17-kDa isoforms with pis ranging from 4.2 to 5.2 on two-dimensional SDS-PAGE. In addition, minor polypeptides at 19 kDa (five isoforms, p/ range 4.4 to 5.2) and 15 kDa (two isoforms, pl’s 4.3 and 4.7) co-isolate with ponticulin on F-actin affinity columns. Because all these isoforms are absent from cells in which the single-copy gene encoding ponticulin has been disrupted, the ponticulin isoforms appear to arise from differential post-translational processing.
Supervillin isoforms have been implicated in cell proliferation, actin filament-based motile processes, vesicle trafficking, and signal transduction. However, an understanding of the roles of these proteins in cancer metastasis and physiological processes has been limited by the difficulty of obtaining specific antibodies against these highly conserved membrane-associated proteins. To facilitate research into the biological functions of supervillin, monoclonal antibodies were generated against the bacterially expressed human supervillin N-terminus. Two chimeric monoclonal antibodies with rabbit Fc domains (clones 1E2/CPTC-SVIL-1; 4A8/CPTC-SVIL-2) and two mouse monoclonal antibodies (clones 5A8/CPTC-SVIL-3; 5G3/CPTC-SVIL-4) were characterized with respect to their binding sites, affinities, and for efficacy in immunoblotting, immunoprecipitation, immunofluorescence microscopy and immunohistochemical staining. Two antibodies (1E2, 5G3) recognize a sequence found only in primate supervillins, whereas the other two antibodies (4A8, 5A8) are specific for a more broadly conserved conformational epitope(s). All antibodies function in immunoblotting, immunoprecipitation and in immunofluorescence microscopy under the fixation conditions identified here. We also show that the 5A8 antibody works on immunohistological sections. These antibodies should provide useful tools for the study of mammalian supervillins.
Abstract A protein from Dictyostelium discoideum with an apparent subunit molecular weight of 95,000 daltons (95K protein) was previously identified as an actin‐binding protein ‘Hellewell and Taylor, 1979’. In this paper, we present a method for purifying the protein, and characterize some important aspects of its structure and function. Purification of the 95K protein is achieved by fractionation with ammonium sulfate followed by chromatography on DEAE‐cellulose, gel filtration on 6% agarose, and final purification on hydroxyapatite. The 95K protein is a dimer, composed of apparently identical subunits. It is a rod‐shaped molecule, 38 nm in length, with a Stokes radius of 74 Å. In these structural properties, the 95K protein is similar to muscle and nonmuscle α‐actinins. The 95K protein and filamin are equally competent, when compared on a weight basis, to enhance the apparent viscosity of actin as determined by falling ball viscometry. The apparent viscosity of mixtures of the 95K protein and actin is dramatically reduced at pH greater than 7.0 or free ‘Ca 2+ ’ greater than 10 −7 M. We also examine the mechanism by which calcium regulates the interaction of the 95K protein and actin. A change in free ‘Ca 2+ ’ induces no detectable change in the quaternary structure of the 95K protein. Our experiments indicate that the 95K protein does not dramatically alter the length distribution of actin filaments in the presence of micromolar free ‘Ca 2+ ’. A large fraction of the 95K protein cosediments with actin in the presence of low free ‘Ca 2+ ’ (ca. 3 × 10 −8 M), but not in the presence of high free ‘Ca 2+ ’ (ca. 4 × 10 −6 M). We conclude that increased free ‘Ca 2+ ’ inhibits gelation of actin by the 95K protein by reducing the affinity of the 95K protein for actin. We propose that 95K protein is an important component of the cytoskeletal/contractile system in D. discoideum amoebae.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIdentification of a Novel, N7-Deoxyguanosine Adduct as the Major DNA Adduct Formed by a Non-Bay-Region Diol Epoxide of Benzo[a]pyrene with Low Mutagenic PotentialMichael C. MacLeod, Frederick E. Evans, Jack Lay, Paul Chiarelli, Nicholas E. Geacintov, K. Leslie Powell, Anne Daylong, Ernestina Luna, and Ronald G. HarveyCite this: Biochemistry 1994, 33, 10, 2977–2987Publication Date (Print):March 15, 1994Publication History Published online1 May 2002Published inissue 15 March 1994https://doi.org/10.1021/bi00176a030RIGHTS & PERMISSIONSArticle Views51Altmetric-Citations5LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts
During cell migration, myosin II modulates adhesion, cell protrusion and actin organization at the leading edge. We show that an F-actin- and membrane-associated scaffolding protein, called supervillin (SV, p205), binds directly to the subfragment 2 domains of nonmuscle myosin IIA and myosin IIB and to the N-terminus of the long form of myosin light chain kinase (L-MLCK). SV inhibits cell spreading via an MLCK- and myosin II-dependent mechanism. Overexpression of SV reduces the rate of cell spreading, and RNAi-mediated knockdown of endogenous SV increases it. Endogenous and EGFP-tagged SV colocalize with, and enhance the formation of, cortical bundles of F-actin and activated myosin II during early cell spreading. The effects of SV are reversed by inhibition of myosin heavy chain (MHC) ATPase (blebbistatin), MLCK (ML-7) or MEK (U0126), but not by inhibiting Rho-kinase with Y-27632. Flag-tagged L-MLCK co-localizes in cortical bundles with EGFP-SV, and kinase-dead L-MLCK disorganizes these bundles. The L-MLCK- and myosin-binding site in SV, SV1-171, rearranges and co-localizes with mono- and di-phosphorylated myosin light chain and with L-MLCK, but not with the short form of MLCK (S-MLCK) or with myosin phosphatase. Thus, the membrane protein SV apparently contributes to myosin II assembly during cell spreading by modulating myosin II regulation by L-MLCK.
Activation of human platelets with thrombin transiently increases phosphorylation at 558 threonine of moesin as determined with phosphorylation state-specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [ 32 P]ATP or 125 I show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified. In the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with α- or β/γ-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (K d , ∼1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. In 0.1% Triton X-100, F-actin interacts with phosphorylated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin’s high-affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages between the actin cytoskeleton and the plasma membrane.
