Tyrosine Phosphorylation Regulates Dystroglycan 1719

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Tyrosine Phosphorylation Regulates Dystroglycan 1719 Journal of Cell Science 113, 1717-1726 (2000) 1717 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0874 Adhesion-dependent tyrosine phosphorylation of β-dystroglycan regulates its interaction with utrophin M. James1,*, A. Nuttall1, J. L. Ilsley1, K. Ottersbach1,‡, J. M. Tinsley2, M. Sudol3 and S. J. Winder1,4,§ 1Institute of Cell and Molecular Biology, University of Edinburgh, King’s Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK 2Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK 3Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, One Gustav Levy Place, New York, NY 10029, USA 4IBLS, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK *Present address: Department of Medicine, University of Leicester, Leicester, UK ‡Beatson Institute for Cancer Research, Garscube Estate, Switchback Rd, Bearsden, Glasgow, UK §Author for correspondence at address 4 (e-mail: [email protected]) Accepted 9 March; published on WWW 18 April 2000 SUMMARY Many cell adhesion-dependent processes are regulated by treated with peroxyvanadate, where endogenous β- tyrosine phosphorylation. In order to investigate the role of dystroglycan was tyrosine phosphorylated, β-dystroglycan tyrosine phosphorylation of the utrophin-dystroglycan was no longer co-immunoprecipitated with utrophin fusion complex we treated suspended or adherent cultures of constructs. Peptide ‘SPOTs’ assays confirmed that tyrosine HeLa cells with peroxyvanadate and immunoprecipitated phosphorylation of β-dystroglycan regulated the binding of β-dystroglycan and utrophin from cell extracts. Western utrophin. The phosphorylated tyrosine was identified as blotting of β-dystroglycan and utrophin revealed adhesion- Y892 in the β-dystroglycan WW domain binding motif and peroxyvanadate-dependent mobility shifts which were PPxY thus demonstrating the physiological regulation of recognised by anti-phospho-tyrosine antibodies. Using the β-dystroglycan/utrophin interaction by adhesion- maltose binding protein fusion constructs to the carboxy- dependent tyrosine phosphorylation. terminal domains of utrophin we were able to demonstrate specific interactions between the WW, EF and ZZ domains of utrophin and β-dystroglycan by co-immunoprecipitation Key words: β-Dystroglycan, Adhesion, Tyrosine phosphorylation, with endogenous β-dystroglycan. In extracts from cells WW domain INTRODUCTION associated with cell-cell and cell-matrix adhesion structures (Belkin and Burridge, 1995a,b; Belkin and Smallheiser, 1996; Utrophin is a ubiquitous cytoskeletal protein forming a link James et al., 1996; Khurana et al., 1995) and may therefore between the actin cytoskeleton and the extracellular protein play a role in the organisation of focal adhesions or adherens laminin via a membrane glycoprotein complex which includes junctions and may exist as distinct cell adhesion complexes in α- and β-dystroglycans (Ervasti and Campbell, 1993; their own right. The assembly and disassembly of many Matsumura et al., 1992; Tinsley et al., 1992; Winder et al., adhesion structures are known to be regulated by tyrosine 1995b). In normal skeletal muscle, where utrophin is restricted phosphorylation (Burridge and Chrzanowska-Wodnicka, to the myotendinous and neuromuscular junctions, utrophin 1996). binds to a multimeric protein complex comprising Both utrophin and dystroglycans have been demonstrated to dystroglycans and sarcoglycans. This complex is play a key role in adhesion-mediated events. The dystroglycan indistinguishable from the dystrophin glycoprotein complex gene encodes both α- and β-dystroglycan as a single found in the rest of the sarcolemma (Matsumura et al., 1992), transcript which is post-translationally cleaved (Ibraghimov- though there is differential distribution of the syntrophins Beskrovnaya et al., 1992). Disruption of dystroglycan function, between these complexes. In non-muscle tissues however, either by antibodies (Durbeej et al., 1995) or gene knockout sarcoglycans, with the possible exception of β-sarcoglycan (Henry and Campbell, 1998; Williamson et al., 1997) has a (Bonnemann et al., 1995; Lim et al., 1995), and dystrophin profound effect on developmental processes that rely on cell (except in neuronal tissues) are not expressed and utrophin adhesion. Antibodies which block α-dystroglycan binding to associates specifically with α- and β-dystroglycan at the cell laminin in epithelial cells inhibit branching morphogenesis membrane (James et al., 1996; Matsumura et al., 1997). In (Durbeej et al., 1995), whilst transgenic disruption of the non-muscle cells in culture, utrophin and dystroglycans are dystroglycan gene (DAG1) in mice leads to embryonic lethality 1718 M. James and others at around day 6.5 due to the failure of Reichert’s membrane to free RPMI media) or 100 nM calyculin A (Calbiochem) for various form (Williamson et al., 1997). Embryonic stem cells deficient times at 37°C. Cells were washed once in cold phosphate buffered for α- and β-dystroglycan are unable to assemble basement saline (50 mM sodium phosphate, pH 7.2, 150 mM NaCl) before membrane (Henry and Campbell, 1998). A significant being harvested in ice-cold radio-immunoprecipitation assay (RIPA) α β buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM ethylene reduction of - and -dystroglycan in the sarcolemma is also β ′ ′ symptomatic of mutations in the dystrophin gene which lead glycol-bis-( -aminoethyl ether) N,N,N ,N -tetraacetic acid (EGTA), 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0.5% to Duchenne and Becker muscular dystrophies (Ervasti et al., sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 100 1990; Matsumura et al., 1993), resulting in the destabilisation µM leupeptin, 1 mM phenylmethylsulphonyl fluoride (PMSF), 100 of the membrane cytoskeleton and loss of sarcolemmal µM N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)) for 30 integrity, see Winder (1997) for review. Additionally, antisense minutes on ice. Cells which were not treated with phosphatase depletion of utrophin in astrocytes markedly reduces cell- inhibitors were incubated for an equivalent time in serum-free media matrix interactions (Khurana et al., 1995). before being harvested in RIPA as above. After harvesting, cells were Utrophin is the autosomal homologue of dystrophin, the briefly sonicated to shear the DNA and stored at −20°C until assay. gene affected in Duchenne muscular dystrophy (Tinsley et al., For studies with cells in suspension, HeLa cells were first removed 1992). Like utrophin, dystrophin interacts with the cytoplasmic from the culture vessel by PBS/EDTA treatment and resuspended in domain of β-dystroglycan through a cluster of domains in the serum free RPMI 1640, with 2% bovine serum albumin to prevent non-specific adhesion, and peroxyvanadate as above. Cells were carboxy terminus of dystrophin known collectively as the maintained at 37°C in a 50 ml tube on a tube roller for 1 hour. Cells cysteine-rich region. This region comprises 3 recognised that were still in suspension after one hour were decanted to a fresh modules, a WW domain (Sudol, 1996b), a pair of EF hands tube, recovered by centrifugation and extracted in RIPA buffer as (Koenig et al., 1988; Tufty and Kretsinger, 1975) and a ZZ above, adapted from Renshaw et al. (1997). domain (Ponting et al., 1996). WW domains are 30 amino acid modules containing conserved tryptophan residues that are Antisera involved in protein-protein interactions through proline-rich The anti-β-dystroglycan antibody 43DAG1/8D5 was a gift from L. motifs in their cognate ligands. EF hands are highly conserved Anderson (University of Newcastle) and MANDAG2 (Helliwell et al., motifs involved in the binding of calcium or magnesium ions 1994) was kindly provided by G. E. Morris (NE Wales Institute, and may play a regulatory or a structural role. The ZZ domain Wrexham). Polyclonal antisera against bacterially expressed maltose binding protein (RAB4) and the utrophin carboxy-terminal coiled coil is a putative zinc finger motif found in various functionally domain (RAB5), residues 3204-3433 of human utrophin (Winder and distinct proteins that is believed to mediate protein-protein Kendrick-Jones, 1995) were raised in rabbits using standard interactions. It has been shown previously that the cysteine-rich techniques. RAB4 and RAB5 antisera were effective on western blots region of dystrophin is required for the binding of dystrophin at dilutions of 1:5,000 and 1:10,000, respectively, and the utrophin to β-dystroglycan and the C-terminal domain further increases antiserum did not recognise dystrophin in western blots of whole rat the binding affinity (Jung et al., 1995; Rosa et al., 1996; Suzuki muscle extracts or in mouse C2C12 mouse myotubes (data not et al., 1994). Most recently it has been documented that the shown). Anti-phospho-tyrosine monoclonal (PY20) was from WW domain in concert with the EF hands binds to the C- Transduction Laboratories. β terminal PPxY motif of -dystroglycan and that this binding is Generation of utrophin fusion-proteins enhanced by the presence of the ZZ domain (Rentschler et al., The limits of the utrophin cysteine rich domains WW, EF and ZZ 1999). were identified by sequence alignment (Schultz et al., 1998; Sudol, In this current study we investigated the binding of the 1996a; Thompson et al., 1997) and secondary structure prediction utrophin carboxy-terminal domains to β-dystroglycan using (Rost
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