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• W2017167286 abstract "βArrestin is a multifunctional signal scaffold protein. Using SPOT immobilized peptide arrays, coupled with scanning alanine substitution and mutagenesis, we show that the MAPK kinase, MEK1, interacts directly with βarrestin1. Asp26 and Asp29 in the N-terminal domain of βarrestin1 are critical for its binding to MEK1, whereas Arg47 and Arg49 in the N-terminal domain of MEK1 are critical for its binding to βarrestin1. Wild-type FLAG-tagged βarrestin1 co-immunopurifies with MEK1 in HEKB2 cells, whereas the D26A/D29A mutant does not. ERK-dependent phosphorylation at Ser412 was compromised in the D26A/D29A-βarrestin1 mutant. A cell-permeable, 25-mer N-stearoylated βarrestin1 peptide that encompassed the N-domain MEK1 binding site blocked βarrestin1/MEK1 association in HEK cells and recapitulated the altered phenotype seen with the D26A/D29A-βarrestin1 in compromising the ERK-dependent phosphorylation of βarrestin1. In addition, the MEK disruptor peptide promoted the ability of βarrestin1 to co-immunoprecipitate with endogenous c-Src and clathrin, facilitating the isoprenaline-stimulated internalization of the β2-adrenergic receptor. βArrestin is a multifunctional signal scaffold protein. Using SPOT immobilized peptide arrays, coupled with scanning alanine substitution and mutagenesis, we show that the MAPK kinase, MEK1, interacts directly with βarrestin1. Asp26 and Asp29 in the N-terminal domain of βarrestin1 are critical for its binding to MEK1, whereas Arg47 and Arg49 in the N-terminal domain of MEK1 are critical for its binding to βarrestin1. Wild-type FLAG-tagged βarrestin1 co-immunopurifies with MEK1 in HEKB2 cells, whereas the D26A/D29A mutant does not. ERK-dependent phosphorylation at Ser412 was compromised in the D26A/D29A-βarrestin1 mutant. A cell-permeable, 25-mer N-stearoylated βarrestin1 peptide that encompassed the N-domain MEK1 binding site blocked βarrestin1/MEK1 association in HEK cells and recapitulated the altered phenotype seen with the D26A/D29A-βarrestin1 in compromising the ERK-dependent phosphorylation of βarrestin1. In addition, the MEK disruptor peptide promoted the ability of βarrestin1 to co-immunoprecipitate with endogenous c-Src and clathrin, facilitating the isoprenaline-stimulated internalization of the β2-adrenergic receptor. The βarrestins are multifunctional signal scaffolding proteins that play a pivotal role in the desensitization process that regulates the functioning of many key heptahelical G protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCR, G protein-coupled receptor; β2-AR, β2-adrenergic receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; GST, glutathione S-transferase; PBS, phosphate-buffered saline. 2The abbreviations used are: GPCR, G protein-coupled receptor; β2-AR, β2-adrenergic receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; GST, glutathione S-transferase; PBS, phosphate-buffered saline. (1.Defea K. Br. J. Pharmacol. 2008; 153: 298-309Crossref PubMed Scopus (136) Google Scholar, 2.DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1142) Google Scholar). The β2-adrenergic receptor (β2-AR) has provided a critical functional paradigm in elucidating this fundamental process, where agonist occupancy triggers its phosphorylation by G-protein-coupled receptor kinase, thereby initiating the recruitment of cytosolic βarrestins (3.Ferguson S.S. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. Pharmacol. 1996; 74: 1095-1110Crossref PubMed Scopus (318) Google Scholar, 4.Premont R.T. Gainetdinov R.R. Annu. Rev. Physiol. 2007; 69: 511-534Crossref PubMed Scopus (392) Google Scholar). By associating with agonist-occupied receptors, βarrestins attenuate GPCR functioning by both regulating interaction with signal-transducing G-proteins and facilitating GPCR internalization, leading to either recycling or degradation of the targeted receptor (5.Baillie G.S. Sood A. McPhee I. Gall I. Perry S.J. Lefkowitz R.J. Houslay M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 940-945Crossref PubMed Scopus (302) Google Scholar). Indeed, the interaction of receptor-recruited βarrestin with clathrin cages provides a key part of the paradigm for the deactivation of select ligand-bound GPCRs (6.Gurevich V.V. Gurevich E.V. Pharmacol. Ther. 2006; 110: 465-502Crossref PubMed Scopus (356) Google Scholar). βarrestins can also deliver sequestered cAMP phosphodiesterase-4 isoforms, particularly PDE4D5, to the site of cAMP synthesis associated with the β2-AR, thereby contributing a key part of the cellular desensitizing system for cAMP (5.Baillie G.S. Sood A. McPhee I. Gall I. Perry S.J. Lefkowitz R.J. Houslay M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 940-945Crossref PubMed Scopus (302) Google Scholar, 7.Bolger G.B. Baillie G.S. Li X. Lynch M.J. Herzyk P. Mohamed A. Mitchell L.H. McCahill A. Hundsrucker C. Klussmann E. Adams D.R. Houslay M.D. Biochem. J. 2006; 398: 23-36Crossref PubMed Scopus (133) Google Scholar, 8.Lynch M.J. Baillie G.S. Mohamed A. Li X. Maisonneuve C. Klussmann E. van Heeke G. Houslay M.D. J. Biol. Chem. 2005; 280: 33178-33189Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar).In the resting state, cytosolic βarrestin1 proteins are constitutively phosphorylated by extracellular signal-regulated kinase (ERK) at Ser412, located within their distal C terminus (9.Lin F.T. Miller W.E. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 15971-15974Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 10.Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (696) Google Scholar). The agonist-stimulated recruitment of βarrestin to GPCRs, such as the β2-AR, leads to the dephosphorylation of βarrestin1 at this site. This event acts as a molecular switch, allowing for the internalization of the β2-AR-sequestered βarrestin complex. Thus, ERK-phosphorylated βarrestin1 is unable to associate with clathrin cages, whereas this constraint is removed upon its dephosphorylation (9.Lin F.T. Miller W.E. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 15971-15974Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 11.Lin F.T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Dephosphorylation of Ser412 is also thought to be a determinant for the association of βarrestin1 with c-Src and the phosphorylation of dynamin, a key feature in receptor internalization (12.Miller W.E. Maudsley S. Ahn S. Khan K.D. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2000; 275: 11312-11319Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Thus, the ERK2-dependent Ser412 phosphorylation and dephosphorylation of βarrestin1 provides a pivotal molecular switch that determines the association of βarrestin1 with the endocytic machinery governing internalization of the β2-AR (11.Lin F.T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar).The activation of ERK critically depends upon its phosphorylation by the MAPK kinase, MEK1. Compartmentalization and fidelity of this action is endowed by the ability of these proteins to interact and dock to each other, where a motif called the CD domain has been shown to play an underpinning role (13.Tanoue T. Nishida E. Pharmacol. Ther. 2002; 93: 193-202Crossref PubMed Scopus (111) Google Scholar). In particular, MEK1 is known to bind to a negatively charged cluster that consists of two aspartate residues separated by any other two amino acids (DXXD) (14.Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (667) Google Scholar). The functioning of MEK1 has been shown to be integral to many cellular processes, such as transcription regulation, proliferation, and differentiation. Therapeutically, inhibitors of MEK1 have been developed as potential therapeutics for cancer and, more recently, have become the focus of development for treating chronic inflammatory disorders, such as rheumatoid arthritis and asthma (15.Pelaia G. Cuda G. Vatrella A. Gallelli L. Caraglia M. Marra M. Abbruzzese A. Caputi M. Maselli R. Costanzo F.S. Marsico S.A. J. Cell. Physiol. 2005; 202: 642-653Crossref PubMed Scopus (84) Google Scholar, 16.English J.M. Cobb M.H. Trends Pharmacol. Sci. 2002; 23: 40-45Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 17.Sweeney S.E. Firestein G.S. Ann. Rheum Dis. 2006; 65: 83-88Google Scholar). It has previously been suggested that MEK may form a complex with ERK and with βarrestin (10.Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (696) Google Scholar, 18.Lefkowitz R.J. Inglese J. Koch W.J. Pitcher J. Attramadal H. Caron M.G. Cold Spring Harb. Symp. Quant. Biol. 1992; 57: 127-133Crossref PubMed Google Scholar) that creates a “signalosome” capable of disseminating MAPK signals to defined intracellular compartments. In other studies, it has been suggested that constitutive βarrestin-ERK1 complexes probably recruit MEK1 through its binding to ERK as a consequence of active Ras-dependent signaling (19.DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (678) Google Scholar, 20.McDonald P.H. Lefkowitz R.J. Cell. Signal. 2001; 13: 683-689Crossref PubMed Scopus (111) Google Scholar, 21.Perry S.J. Lefkowitz R.J. Trends Cell Biol. 2002; 12: 130-138Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 22.Lefkowitz R.J. Whalen E.J. Curr. Opin. Cell Biol. 2004; 16: 162-168Crossref PubMed Scopus (243) Google Scholar, 23.Lefkowitz R.J. Shenoy S.K. Science. 2005; 308: 512-517Crossref PubMed Scopus (1405) Google Scholar). However, a recent report suggests that MEK can bind directly to βarrestin1 (24.Song, X., Coffa, S., Fu, H., and Gurevich, V. V. (2008) J. Biol. Chem.Google Scholar) within sites on both N and C domains. Critically, however, the exact nature of MEK interaction with βarrestin1 is unclear. Given that βarrestin1 is ERK-phosphorylated in the cytosol (9.Lin F.T. Miller W.E. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 15971-15974Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and the critical importance of this event to βarrestin-mediated GPCR internalization, the nature of MEK1 interaction with βarrestin warrants investigation. Here then, we evaluate MEK1 interaction with the signal scaffolding protein, βarrestin1, demonstrating that MEK1 binds directly to βarrestin1, allowing the identification of a functionally active peptide disruptor of this complex.EXPERIMENTAL PROCEDURESMaterials—Bradford reagent was from Bio-Rad. The following antibodies were used at concentrations of 1:2000: MEK1 (2352), MEK1/2 (4694), ERK2 (9108), ERK1/2 (4694), and βarrestin1 phospho-Ser412 (2416) (Cell Signaling Technology, Beverly, MA); β2-AR (sc-569), glutathione S-transferase (GST) (sc-53909), clathrin HC (sc-12734), and hemagglutinin probe (sc-7372) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); βarrestin1 phospho-Ser412 (44-200; lot 0102), anti-Src pan (44-655G), and anti-His-horseradish peroxidase (P/N 56-0707) (Invitrogen); and FLAG-horseradish peroxidase (A8592) (Sigma). Monoclonal antisera specific for the PDE4D subfamily was a kind gift from Dr. S Wolda (ICOS Corp., Seattle, WA). Active GST-MEK1 (SGT-220) and inactive HisMEK1 (14-706) were purchased from Millipore. HEKB2 cells were a kind gift from Prof. Graeme Milligan (University of Glasgow). Cell-permeable peptides were a gift from Prof. Enno Klusmann (Leibniz-Institut für Molekulare Pharmakologie, Berlin).Cell Culture and Drug Additions—HEK293 cells and HEK293 cell lines stably overexpressing FLAG-tagged β2-AR-GFP (HEKB2 cells) were cultured as described previously (25.Bolger G.B. McCahill A. Huston E. Cheung Y.F. McSorley T. Baillie G.S. Houslay M.D. J. Biol. Chem. 2003; 278: 49230-49238Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Transfection of these cells was done using Polyfect (Qiagen), following the manufacturer's instructions. Pretreatments for control experiments were done 10 min (UO126, 10 μm) or 2 h (cell-permeable peptides, 10 μm) prior to the addition of isoproterenol (10 μm).Preparation of Peptide Small Molecule Disruptors—Briefly, all stearyl-peptides were prepared by treatment of the peptide resins with stearic acid and diisopropylcarbodiimide in the presence of N-hydroxybenzotriazole, followed by deprotection using trifluoroacetic acid/ethanedithiol (26.Futaki S. Ohashi W. Suzuki T. Niwa M. Tanaka S. Ueda K. Harashima H. Sugiura Y. Bioconjugate Chem. 2001; 12: 1005-1011Crossref PubMed Scopus (401) Google Scholar).Fluorescent peptides were prepared by adding 2 mg of 5,6-carboxyfluorescein N-hydroxysuccinimide ester (in 0.9 ml of DMSO, 100 μl of 0.1 m NaHCO3, pH 8.5) to 10 mg of peptide to be labeled. After reaction at room temperature in darkness for 20 h, the reaction mix was directly applied to preparative high pressure liquid chromatography. Fractions containing labeled peptide were collected and lyophilized.Peptide Arrays and Alanine Scans—βarrestin1 and MEK1 peptide libraries were produced by automatic SPOT synthesis as described previously (27.Baillie G.S. Adams D.R. Bhari N. Houslay T.M. Vadrevu S. Meng D. Li X. Dunlop A. Milligan G. Bolger G.B. Klussmann E. Houslay M.D. Biochem. J. 2007; 404: 71-80Crossref PubMed Scopus (74) Google Scholar). They were synthesized on continuous cellulose membrane supports on Whatman 50 cellulose membranes using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry with the AutoSpot-Robot ASS 222 (Intavis Bioanalytical Instruments AG, Köln, Germany). Alanine-scanning peptide libraries were constructed by taking the residues in positive spots and sequentially changing each residue to alanine (or, if an alanine was the natural amino acid at that position, to aspartate). The interaction of spotted peptides with purified, recombinant GST and GST-βarrestin1 and GST-MEK1 fusion proteins was determined by overlaying the cellulose membranes with 10 μg/ml recombinant protein. Bound recombinant proteins were then detected following wash steps with rabbit anti-GST, and detection was performed with a secondary anti-rabbit horseradish peroxidase-coupled antibody.Expression of GST Fusions in Escherichia coli—Cultures of E. coli JM109 containing pGEX-β-arrestin1 or pGEX were induced with 1 mm isopropyl-β-d-thiogalactopyranoside (Roche Applied Science) for 4 h at 30 °C. Bacteria were harvested by centrifugation at 6,000 × g for 15 min at 4 °C, and the bacterial pellet was frozen at -80 °C overnight. The bacterial pellets were resuspended in 10 ml of ice-cold resuspension buffer (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 10 mm β-mercaptoethanol, and complete protease inhibitor mixture) and sonicated with four 30-s bursts at the maximal setting. Triton X-100 was added to a final concentration of 0.02%, and cell debris was then removed by centrifugation at 15,000 × g for 10 min at 4 °C. The cleared supernatant was incubated with one-tenth volume of pre-equilibrated glutathione-Sepharose beads on an orbital shaker for 30 min at 4 °C. The beads were collected by centrifugation at 13,000 × g for 1 min and washed three times with ice-cold resuspension buffer. The fusion proteins were eluted by the addition of 5 mm glutathione, 50 mm Tris-HCl, pH 8.0, and dialyzed three times against 50 mm Tris-HCl, pH 8.0, 100 mm NaCl, and 5% glycerol. The purified fusion proteins were stored at -80 °C until required.Plasmid Construction and Site-directed Mutagenesis—FLAG-tagged βarrestin1, which was generated by an insertion of the full open reading frame of βarrestin1 into the Not1 site of pcDNA3 (Invitrogen), was a kind gift from Dr. Graeme B. Bolger (University of Alabama at Birmingham). Primers 5-GGGAAAGCGGGCCTTTGTGGCCCACATCGACCTCGTGGACC-3 and 5-GGTCCACGAGGTCGATGTGGGCCACAAAGGCCCGCTTTCCC-3 were used to generated the FLAG-tagged D26A/D29A-βarrestin1 mutant with the QuikChange site-directed mutagenesis kit (Stratagene). The pCHA-MEK1 construct was a gift from Prof. Walter Kolch (Beatson Institute for Cancer Research, Glasgow, UK). Details of the construct were described previously (28.Catling A.D. Schaeffer H.J. Reuter C.W. Reddy G.R. Weber M.J. Mol. Cell. Biol. 1995; 15: 5214-5225Crossref PubMed Scopus (167) Google Scholar). Primers 5-GGAGCTTGACGAGCAGCAGGCGGCGGCGCTCGAGGCCTTTCTGACG-3 and 5-CGTCAGAAAGGCCTCGAGCGCCGCCGCCTGCTGCTCGTCAAGCTCC-3 were designed to generate R47A/K48A/R49A-MEK1.Microscopy—HEKB2 cells were seeded onto poly(l-lysine)-treated coverslips at ∼20% confluence. After treatment with indicated ligands, cells were fixed for 10 min in 4% (w/v) paraformaldehyde followed by three washes with Tris-buffered saline (150 mm NaCl, 20 mm Tris, pH 7.4). The coverslips were mounted to microscope slides with Immunomount. Cells were visualized using the Zeiss Pascal laser-scanning confocal microscope (Zeiss, Oberkochken, Germany). Fluorescent peptide entry into cells was done using an LSM510 laser-scanning microscope (Zeiss).In Vitro Pull-down Using Purified Proteins—1 nmol of purified GST or GST-βarrestin1 was mixed with an equal amount of HisMEK1 (Millipore) in 0.5 ml of binding buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 2 mm MgCl2, 1 mm dithiothreitol, 0.5% Triton X-100). The mixture was incubated for 1 h at 4 °C, and then 50 μl of anti-polyhistidine-agarose beads (A5713; Sigma) were added in for an overnight incubation. Beads were collected by 10,000 × gav centrifugation for 1 min and washed three times with binding buffer before loading to an SDS-polyacrylamide gel for protein separation.Western Blotting and Protein Estimation—Immunoblotting was done as previously described (5.Baillie G.S. Sood A. McPhee I. Gall I. Perry S.J. Lefkowitz R.J. Houslay M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 940-945Crossref PubMed Scopus (302) Google Scholar), using 25–50 μg of cellular protein/well. After treatment, HEKβ2 cells or HEK cells were washed twice with PBS before being scraped into 3T3 lysis buffer (25 mm HEPES, 2.5 mm EDTA, 50 mm NaCl, 50 mm NaF, 30 mm sodium pyrophosphate, 10% glycerol, 1% Triton X-100, pH 7.5) with added protease inhibitors (Complete Protease Inhibitor Mixture; Roche Applied Science). Proteins were separated by PAGE and transferred to nitrocellulose for Western blotting. Protein concentrations of cell lysates were determined with bovine serum albumin as a standard using Bradford reagent as previously described (25.Bolger G.B. McCahill A. Huston E. Cheung Y.F. McSorley T. Baillie G.S. Houslay M.D. J. Biol. Chem. 2003; 278: 49230-49238Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Protein was routinely measured by the method of Bradford using bovine serum albumin as a standard.Immunoprecipitation of Target Molecules—This was done as described previously by us (29.MacKenzie S.J. Houslay M.D. Biochem. J. 2000; 347: 571-578Crossref PubMed Scopus (127) Google Scholar, 30.Shepherd M.C. Baillie G.S. Stirling D.I. Houslay M.D. Br. J. Pharmacol. 2004; 142: 339-351Crossref PubMed Scopus (73) Google Scholar). Briefly, detergent-soluble proteins were isolated from cells by disruption in lysis buffer (1% (v/v) Triton X-100, 50 mm HEPES buffer, pH 7.2, 10 mm EDTA, 100 mm NaH2PO4, 2H2O) containing complete protease inhibitor mixture (Roche Applied Science) to 8% volume. Detergent-insoluble proteins were removed by centrifugation at 10,000 × gav for 10 min, and the soluble fraction was retained. Equal volumes of cell lysate containing 500 μg of protein were cleared by incubation with 30 μl of preimmune serum and/or 30 μl of protein A slurry. The beads were then removed by centrifugation at 10,000 × gav for 10 min at 4 °C, and cleared lysate was incubated at 4 °C for 2 h with constant agitation with a volume of antiserum. Immunoglobulins were then isolated by incubation with protein A-coated Sepharose beads for 1 h before retrieval by refrigerated centrifugation at 10,000 × gav for 5 min. Target molecule-immunoglobulin conjugates attached to the beads were then washed in phosphate-buffered saline (PBS) three times.Internalization of β2-AR Assay—Briefly, cells were grown to 80% confluence in Petri dishes and treated with 0.3 mg/ml disulfide-cleavable biotin (Pierce) in PBS at 4 °C for 30 min (5 ml/well). Cells were then washed with precooled PBS three times to cease biotinylation. The cells were then treated with the appropriate ligands, with or without peptides for the indicated times. Samples were washed with PBS two times, and the biotinylated receptors were stripped with stripping buffer (0.05 m glutathione, 0.3 m NaCl, 0.075 m NaOH, 1% FBS/newborn calf serum in PBS) at 4 °C for 30 min. Cell extracts were resuspended in radioimmune precipitation buffer (150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl, and 0.1% Triton X-100, pH 7.4), and biotinylated receptors were immunologically isolated using immobilized NeatrAvidin (Pierce) beads at 4 °C overnight. Samples were then washed three times with radioimmune precipitation buffer or PBS, and samples were analyzed via SDS-PAGE. Total receptor amounts were visualized and quantified using anti-β2-AR antiserum (sc-569; Santa Cruz Biotechnology).Analysis by Microscopy—Slides were examined using a fluorescent imaging microscope at a magnification of ×43 and imaged for phase contrast, GFP fluorescence, and 4′,6-diamidino-2-phenylindole staining. Images of three random fields of view were taken from each slide, and all cells within these areas were quantified. After correction for background, areas were outlined to define the intracellular compartment of the cell, using phase contrast to define the plasma membrane, and the number of cells present in each field of view was quantified using 4′,6-diamidino-2-phenylindole staining of the nuclei of the cells. The image analysis software, Metamorph 7.0, was used to find spots within the intracellular compartment of the cell, and the number of spots per cell was quantified and used as an indication of the internalization of the β2-adrenoreceptor. Student's t test was used for statistical analysis.RESULTSPurified MEK1-GST Binds Directly to the βArrestin Peptide Array—MEK1 is a 45-kDa MAPK kinase that activates ERK in a classic amplification cascade. Previous studies (10.Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (696) Google Scholar) utilizing multiple transfection of key intermediates have indicated that MEK1 is able to interact with βarrestin. However, it is unclear from these reports as to whether this occurs directly or involves a bridging molecule, such as ERK, which is well known to bind to MEK and has been shown to associate directly with β-arrestin (19.DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (678) Google Scholar, 31.Tohgo A. Pierce K.L. Choy E.W. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2002; 277: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 32.Xu T.R. Baillie G.S. Bhari N. Houslay T.M. Pitt A.M. Adams D.R. Kolch W. Houslay M.D. Milligan G. Biochem. J. 2008; 413: 51-60Crossref PubMed Scopus (35) Google Scholar). Recently, evidence has emerged suggesting that MEK may bind to both the N and C domains of βarrestin1, although the exact nature of the binding sites remains to be determined (24.Song, X., Coffa, S., Fu, H., and Gurevich, V. V. (2008) J. Biol. Chem.Google Scholar).In order to determine whether MEK1 has the potential to interact directly with βarrestin, we first employed peptide array analysis. This provides a novel and powerful technology for gaining insight into the basis of specific protein-protein interactions. Indeed, we have used this to considerable advantage to exhaustively map sites of interaction between both of the scaffolding proteins, βarrestin (27.Baillie G.S. Adams D.R. Bhari N. Houslay T.M. Vadrevu S. Meng D. Li X. Dunlop A. Milligan G. Bolger G.B. Klussmann E. Houslay M.D. Biochem. J. 2007; 404: 71-80Crossref PubMed Scopus (74) Google Scholar) and RACK1 (7.Bolger G.B. Baillie G.S. Li X. Lynch M.J. Herzyk P. Mohamed A. Mitchell L.H. McCahill A. Hundsrucker C. Klussmann E. Adams D.R. Houslay M.D. Biochem. J. 2006; 398: 23-36Crossref PubMed Scopus (133) Google Scholar), with the cAMP-hydrolyzing PDE4D5 isoform. These were confirmed by mutagenesis analyzed through both pull-down and two-hybrid studies (25.Bolger G.B. McCahill A. Huston E. Cheung Y.F. McSorley T. Baillie G.S. Houslay M.D. J. Biol. Chem. 2003; 278: 49230-49238Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Here we have generated a library of overlapping peptides (25-mers), each shifted by 5 amino acids, which spans the entire sequence of βarrestin1. These were spot-synthesized on cellulose membranes to generate an immobilized peptide library that was then probed with a purified, recombinant GST fusion protein of active MEK1. Binding of MEK1 to individual peptide spots was assessed immunologically, with positive interactions identified as dark spots (Fig. 1a). A cluster of peptides (spots 3–6) was observed for GST-MEK1 binding but not GST alone, and that yielding the strongest signal (spot 6; Fig. 1a) was evaluated further.In order to gain insight into the amino acids involved in allowing MEK1 to bind to this region of βarrestin1, we generated a family of peptides derived from the “spot 6” 25-mer parent peptide whose sequence reflected amino acids Asp26–Glu50 of βarrestin1. The 25-mer peptide progeny of this parent peptide each had a single substitution, to alanine, of successive amino acids in the sequence to form a scanning peptide array (Fig. 1b). Using this analysis, we observed that MEK1 binding to the 25-mer Asp26–Glu50 βarrestin peptide was ablated upon alanine substitution of Asp26 and Asp29 (Fig. 1b). Binding of MEK1 was also ablated when the various amino acids in the region Asp26 to His30 were all replaced with alanine residues but not when the run of amino acids from Tyr46 to Glu50 were similarly substituted with alanine residues.To reinforce our findings from peptide array analysis, we attempted to show that MEK1 and βarrestin1 could directly interact in vitro. After mixing purified fusion proteins of His-MEK1 with GST-βarrestin1 or GST alone, we pulled down the His tag on MEK1 and evaluated co-immunoprecipitating species by Western blotting (Fig. 2a). GST-βarrestin1, but not GST, co-purified with His-MEK1, confirming the notion that the MEK1-βarrestin1 association is direct and is not dependent on other putative members of the signaling complex. In order to determine if this DXXD motif does indeed play an important role in defining the interaction between MEK1 and βarrestin, we mutated Asp26 and Asp29 to alanine in a FLAG epitope-tagged βarrestin1 construct and evaluated whether this altered the ability of MEK1 to co-immunoprecipitate with βarrestin in cell lysates where they are co-expressed (Fig. 2b). Doing this, we see that although anti-FLAG immunoprecipitates of FLAG-tagged βarrestin1 pulled down endogenous MEK1, only 8.3 ± 3.9% (mean; n = 3) of MEK remains associated with the FLAG-tagged D26A/D29A-βarrestin1 mutant construct (Fig. 2b). There is specificity in this interaction, since both the wild-type and D26A/D29A mutant forms of FLAG-tagged βarrestin1 acted to pull-down equal amounts of PDE4D (Fig. 2), whose binding site on βarrestin does not involve either Asp26 or Asp29 (27.Baillie G.S. Adams D.R. Bhari N. Houslay T.M. Vadrevu S. Meng D. Li X. Dunlop A. Milligan G. Bolger G.B. Klussmann E. Houslay M.D. Biochem. J. 2007; 404: 71-80Crossref PubMed Scopus (74) Google Scholar). These analyses confirm the peptide array studies in indicating that both Asp26 and Asp29 play a key role in underpinning the interaction of MEK1 with βarrestin1.FIGURE 2Mutation of βarrestin1 Asp26 and Asp29 to Ala ablates MEK1 binding to βarrestin1. a, fusion proteins of His-MEK1 and GST-βarrestin1 or GST alone were mixed before His-MEK was pulled down using anti-His-agarose beads. Immunopurified samples of His-MEK1 were subjected to Western blot analysis for the presence of both GST and His tags. b, HEK293 cells were transfected with FLAG-βarrestin1 or a mutant form of βarrestin1 where Asp26 and Asp29 had been dually mutated to alanine. Both forms of βarrestin1 were immunopurified using the FLAG epitope, and preparations were immunoblotted for MEK, ERK, and PDE4D. The lower panel shows transfection efficiency of the βarrestin1 constructs. Ab, antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)βArrestin has also been shown to interact with ERK (10.Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (696) Google Scholar). Since MEK1 can interact with ERK, it has been postulated that ERK may act as an adaptor that, in binding to βarrestin, sequesters MEK1 there. We thus set out to determine if loss of MEK1 asso" @default.
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• W2017167286 title "MEK1 Binds Directly to βArrestin1, Influencing Both Its Phosphorylation by ERK and the Timing of Its Isoprenaline-stimulated Internalization" @default.
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