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• W2002879408 abstract "JNK scaffold proteins bind JNK and upstream kinases to activate subsets ofJNK and localize activated JNK to specific subcellular sites. We previouslydemonstrated that the dual specificity phosphatases (DSPs) MKP7 and M3/6 bindthe scaffold JNK-interacting protein-1 (JIP-1) and inactivate the bound subsetof JNK (1Willoughby E.A. Perkins G.R. Collins M.K. Whitmarsh A.J. J. Biol. Chem. 2003; 278: 10731-10736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The Gprotein-coupled receptor (GPCR) adaptor β-arrestin 2 is also a JNK3scaffold. It binds the upstream kinases ASK1 and MKK4 and couples stimulationof the angiotensin II receptor AT1aR to activation of a cytoplasmic pool ofJNK3. Here we report that MKP7 also binds β-arrestin 2 via amino acids394–443 of MKP7, the same region that interacts with JIP-1. This regionof MKP7 interacts with β-arrestin 2 at a central region near the JNKbinding domain. MKP7 dephosphorylates JNK3 bound to β-arrestin 2, eitherfollowing activation by ASK1 overexpression or following AT1aR stimulation.Initial AT1aR stimulation causes a rapid (within 5 min) dissociation of MKP7from β-arrestin 2. MKP7 then reassociates with β-arrestin 2 onendocytic vesicles 30–60 min after initial receptor stimulation. Thisdynamic interaction between phosphatase and scaffold permits signaltransduction through a module that binds both positive and negativeregulators. JNK scaffold proteins bind JNK and upstream kinases to activate subsets ofJNK and localize activated JNK to specific subcellular sites. We previouslydemonstrated that the dual specificity phosphatases (DSPs) MKP7 and M3/6 bindthe scaffold JNK-interacting protein-1 (JIP-1) and inactivate the bound subsetof JNK (1Willoughby E.A. Perkins G.R. Collins M.K. Whitmarsh A.J. J. Biol. Chem. 2003; 278: 10731-10736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The Gprotein-coupled receptor (GPCR) adaptor β-arrestin 2 is also a JNK3scaffold. It binds the upstream kinases ASK1 and MKK4 and couples stimulationof the angiotensin II receptor AT1aR to activation of a cytoplasmic pool ofJNK3. Here we report that MKP7 also binds β-arrestin 2 via amino acids394–443 of MKP7, the same region that interacts with JIP-1. This regionof MKP7 interacts with β-arrestin 2 at a central region near the JNKbinding domain. MKP7 dephosphorylates JNK3 bound to β-arrestin 2, eitherfollowing activation by ASK1 overexpression or following AT1aR stimulation.Initial AT1aR stimulation causes a rapid (within 5 min) dissociation of MKP7from β-arrestin 2. MKP7 then reassociates with β-arrestin 2 onendocytic vesicles 30–60 min after initial receptor stimulation. Thisdynamic interaction between phosphatase and scaffold permits signaltransduction through a module that binds both positive and negativeregulators. Mitogen-activated protein kinases(MAPKs) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK,MAPK kinase; MAPKKK, MAPK kinase kinase; JNK, c-Jun NH2-terminalkinase; GPCR, G protein-coupled receptor; JIP, JNK-interacting protein; AT1aR,angiotensin type 1a receptor; GST, glutathione S-transferase; HA,hemagglutinin; GFP, green fluorescent protein; ERK, extracellularsignal-regulated kinase; DSP, dual specificity phosphatase.1The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK,MAPK kinase; MAPKKK, MAPK kinase kinase; JNK, c-Jun NH2-terminalkinase; GPCR, G protein-coupled receptor; JIP, JNK-interacting protein; AT1aR,angiotensin type 1a receptor; GST, glutathione S-transferase; HA,hemagglutinin; GFP, green fluorescent protein; ERK, extracellularsignal-regulated kinase; DSP, dual specificity phosphatase. are essentialcomponents of many signaling pathways, linking extracellular signals tochanges in transcription factor activity and gene expression(2Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2864) Google Scholar). The JNK MAPKs respond tocell stress, growth factors, cytokines, and hormones and are involved in avariety of responses, including apoptosis, embryonic development, cell growth,and the immune response (3Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3611) Google Scholar).The MAPKKKs, MLK, and mitogen-activated protein kinase/extracellularsignal-regulated kinase kinase kinase (MEKK) families, activate the MAPKKs,MKK4, and MKK7, which in turn activate JNKs(3Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3611) Google Scholar). Once activated, dependingon the cellular environment, JNKs can phosphorylate transcription factors,including c-Jun and ATF2 (4Ip Y.T. Davis R.J. Curr.Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1377) Google Scholar).JNKs can also phosphorylate non-nuclear substrates, including the Bcl-2 familymember BAD (5Yu C. Minemoto Y. Zhang J. Liu J. Tang F. Bui T.N. Xiang J. Lin A. Mol. Cell. 2004; 13: 329-340Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) and the 14-3-3protein (6Tsuruta F. Sunayama J. Mori Y. Hattori S. Shimizu S. Tsujimoto Y. Yoshioka K. Masuyama N. Gotoh Y. EMBO J. 2004; 23: 1889-1899Crossref PubMed Scopus (444) Google Scholar). MAPKs are inactivated by Tyr phosphatases, Ser/Thr phosphatases, andDSPs/MKPs (7Keyse S.M. Curr. Opin. CellBiol. 2000; 12: 186-192Crossref PubMed Scopus (703) Google Scholar). The DSPs make upan evolutionarily conserved family that inactivates MAPKs by dephosphorylationof critical threonine and/or tyrosine residues(8Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (714) Google Scholar,9Theodosiou A. Ashworth A. Genome Biol. 2002; 3: S3009Crossref Google Scholar). DSPs, MKP7(10Masuda K. Shima H. Watanabe M. Kikuchi K. J. Biol. Chem. 2001; 276: 39002-39011Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar,11Tanoue T. Yamamoto T. Maeda R. Nishida E. J. Biol. Chem. 2001; 276: 26629-26639Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), M3/6 (hVH5),(12Muda M. Theodosiou A. Rodrigues N. Boschert U. Camps M. Gillieron C. Davies K. Ashworth A. Arkinstall S. J. Biol. Chem. 1996; 271: 27205-27208Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar) and MKP5(13Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar,14Theodosiou A. Smith A. Gillieron C. Arkinstall S. Ashworth A. Oncogene. 1999; 18: 6981-6988Crossref PubMed Scopus (121) Google Scholar) specifically inactivatethe JNKs. MKP5 null mice have shown that MKP5 is important for regulation ofinnate and adaptive immunity(15Zhang Y. Blattman J.N. Kennedy N.J. Duong J. Nguyen T. Wang Y. Davis R.J. Greenberg P.D. Flavell R.A. Dong C. Nature. 2004; 430: 793-797Crossref PubMed Scopus (195) Google Scholar). The response to particular stimuli and the subsequent cellular localizationof active MAPKs is thought to be maintained by scaffold proteins(16Morrison D.K. Davis R.J. Annu. Rev. Cell Dev. Biol. 2003; 19: 91-118Crossref PubMed Scopus (647) Google Scholar). For example, the JIPfamily of scaffold proteins are kinesin cargo proteins that recruit MLKs,MKK7, and JNK (17Rincon M. Whitmarsh A. Yang D.D. Weiss L. Derijard B. Jayaraj P. Davis R.J. Flavell R.A. J. Exp. Med. 1998; 188: 1817-1830Crossref PubMed Scopus (192) Google Scholar,18Kelkar N. Gupta S. Dickens M. Davis R.J. Mol. Cell. Biol. 2000; 20: 1030-1043Crossref PubMed Scopus (250) Google Scholar,25Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (585) Google Scholar) and also the DSPs, MKP7,and M3/6 (1Willoughby E.A. Perkins G.R. Collins M.K. Whitmarsh A.J. J. Biol. Chem. 2003; 278: 10731-10736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Such binding ofboth kinases and phosphatases has also been observed in the family of proteinkinase A-anchoring proteins (AKAPs)(19Wong W. Scott J.D. Nat. Rev.Mol. Cell Biol. 2004; 5: 959-970Crossref PubMed Scopus (848) Google Scholar); yotiao/AKAP450 bindsboth PKA and phosphatase PP-1 to controlN-methyl-d-aspartate receptor signaling(20Westphal R.S. Tavalin S.J. Lin J.W. Alto N.M. Fraser I.D. Langeberg L.K. Sheng M. Scott J.D. Science. 1999; 285: 93-96Crossref PubMed Scopus (426) Google Scholar). β-Arrestins are adaptors that control G protein-coupled receptor(GPCR) desensitization and internalization via clathrin-coated vesicles(21Luttrell L.M. Lefkowitz R.J. J. Cell Sci. 2002; 115: 455-465Crossref PubMed Google Scholar,22Lefkowitz R.J. Whalen E.J. Curr. Opin. Cell Biol. 2004; 16: 162-168Crossref PubMed Scopus (243) Google Scholar); recently they have alsobeen shown to act as MAPK scaffolds, recruiting signaling complexes toactivated GPCRs. β-arrestin 2 binds JNK3 and upstream kinases ASK1 andMKK4 to stimulate JNK activation after triggering of the angiotensin type 1areceptor (AT1aR) (23McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar,24Miller W.E. McDonald P.H. Cai S.F. Field M.E. Davis R.J. Lefkowitz R.J. J. Biol.Chem. 2001; 276: 27770-27777Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In this report, we showthat β-arrestin 2 also binds the DSP MKP7. After AT1aR stimulation, MKP7transiently dissociates from β-arrestin 2 allowing transient JNK3activation. Plasmids—β-arrestin 2 was amplified by PCR using TurboPfu polymerase (Stratagene) from an expressed sequence tag(Integrated Molecular Analysis of Genomes and Their Expression Consortiumclone number 3028154), and then full-length and all subsequent β-arrestin2 deletions were subcloned into the pEBG vector and fused to glutathioneS-transferase (GST). The MKP7 C terminus-only construct and M3/6 weresubcloned into pCDNA4hisC and fused to an N-terminal Xp/T7 epitope tag.Xp-tagged MKP1, MKP2, hPAC1, MKP7, and MKP7 deletions(1Willoughby E.A. Perkins G.R. Collins M.K. Whitmarsh A.J. J. Biol. Chem. 2003; 278: 10731-10736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) HA-JNK3(1Willoughby E.A. Perkins G.R. Collins M.K. Whitmarsh A.J. J. Biol. Chem. 2003; 278: 10731-10736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and GST-JIP-1(17Rincon M. Whitmarsh A. Yang D.D. Weiss L. Derijard B. Jayaraj P. Davis R.J. Flavell R.A. J. Exp. Med. 1998; 188: 1817-1830Crossref PubMed Scopus (192) Google Scholar) have been describedpreviously. Plasmids expressing FLAG-JNK3, FLAG-β-arrestin 2,GFP-β-arrestin 2, and HA-ASK1 were a kind gift from Alan Whitmarsh(University of Manchester, UK), and the HA-AT1aR construct was a kind giftfrom Stephane LaPorte (McGill University, Montreal, Canada). Cell Culture—293T and COS-7 cells were maintained inDulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.293T and COS-7 cells were transfected using Lipofectamine (Invitrogen)according to the manufacturer's instructions. Pull-downs, Immunoprecipitations, and Immunoblots—293T cellswere washed once with ice-cold buffered saline then lysed in buffer containing50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% IGEPAL CA-630, 5mm EDTA, and a protease inhibitor mixture (Roche Applied Science).After 10 min of incubation on ice, the extracts were centrifuged at 14000× g for 15 min. Supernatants were subject to pull-down orimmunoprecipitation, as described below. Samples were separated by 10%SDS-PAGE and electrotransferred to nitrocellulose membranes (Hybond ECL,Amersham Biosciences). Membranes were subjected to immunoblotting withanti-Xpress (Invitrogen), anti-M3/6 polyclonal (also used to detect GST),anti-FLAG M2 monoclonal (Sigma), or anti-HA High Affinity (Roche) antibodiesand horseradish peroxidase-conjugated secondary antibodies (Dako). Blots weredeveloped using ECL reagents (Amersham Biosciences). For immunoprecipitationor pull-down experiments, extracts were made as described above and incubatedwith either glutathione-Sepharose 4B (Amersham Biosciences) for GST-containingconstructs or with anti-FLAG M2 antibody with protein G-Sepharose beads(Sigma) for 3 h. Beads were then washed three times in lysis buffer andresuspended in an appropriate amount of gel loading buffer (finalconcentration 50 mm Tris, pH 6.8, 2% SDS, 100 mmdithiothreitol, 4% glycerol). For experiments to determine JNKphosphorylation, COS-7 cells were lysed directly into gel-loading buffer andblots probed with anti-phospho-JNK (Promega), anti-HA High Affinity, anti-FLAGM2, or anti-Xpress antibodies. Immunostaining—COS-7 cells were transfected on glasscoverslips. The cells were incubated with 1 μm angiotensin II(Sigma) for the indicated times. The cells were then washed withphosphate-buffered saline solution, fixed with 4% paraformaldehyde (Sigma),and lysed with 0.2% Triton X-100/phosphate-buffered saline. Indirectimmunofluorescence was performed by incubation with the MKP7 polyclonalantiserum in 1% bovine serum albumin/0.5% goat serumalbumin/phosphate-buffered saline for 1 h at 37 °C. Texas Red-conjugatedsecondary antibody (Jackson Immunoresearch) was used and images prepared on aZeiss confocal microscope using Bio-Rad Lasersharp software. Selective Binding of MKP7 to β-Arrestin 2—Werecently reported that the DSPs, MKP7, and M3/6 bind the JNK scaffold proteinsJIP-1 and JIP-2 (1Willoughby E.A. Perkins G.R. Collins M.K. Whitmarsh A.J. J. Biol. Chem. 2003; 278: 10731-10736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Asβ-arrestin 2 has been identified as a scaffold protein for JNK3, theneuronal isoform of JNK (23McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar,24Miller W.E. McDonald P.H. Cai S.F. Field M.E. Davis R.J. Lefkowitz R.J. J. Biol.Chem. 2001; 276: 27770-27777Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), we asked whether any DSPscould bind β-arrestin 2. We co-expressed GST-tagged β-arrestin 2with Xpress-tagged members of the DSP family and examined β-arrestin 2precipitates for presence of the phosphatases using the anti-Xpress tagantibody. Under these conditions, MKP7 bound β-arrestin 2(Fig. 1A, toppanel, lane 2), whereas none of the other DSPs, including the highlyrelated M3/6, was detected in the β-arrestin 2 precipitates(Fig. 1A, bottompanel, lane 2, and Fig.1B, lanes 2, 4, 6, and 8). We next performed co-precipitation analysis of MKP7 deletion mutants withβ-arrestin 2 (Fig. 1, C andD). This identified a deletion mutant containing aminoacids 1–394 that did not bind β-arrestin 2, whereas a mutantcontaining amino acids 1–443 did(Fig. 1D, lanes6 and 8). The sequence between amino acids 394 and 443 is alsocritical for MKP7 binding to JIP-1. The equivalent region of M3/6 is verysimilar to that of MKP7, which suggests either that β-arrestin 2 (unlikeJIP-1) discriminates between the two sequences or that M3/6 binding is notdetected within the sensitivity of our assay. The MKP7 C-terminal fragmentresidues 360–665 were sufficient for binding both JIP-1 andβ-arrestin 2, whereas residues 1–394 of MKP7 did not bind(Fig. 1E, comparelanes 5, 6, 8, and 9). As shown inFig. 1F,MKP7-(1–394), which cannot bind β-arrestin 2, can still bind JNK3(lane 3). Thus MKP7 binding to β-arrestin 2 is dependent onresidues within its C-terminal region. This region is sufficient for theinteraction, demonstrating that the interaction is independent of JNK3 bindingto MKP7. The Regions of β-Arrestin 2 That Interact withMKP7—We then performed co-precipitation analysis of β-arrestin2 deletion constructs with MKP7. MKP7 bound weakly to the N-terminal region(1–164) and full-length β-arrestin 2(Fig. 2B, (ii),lanes 5 and 7); however, MKP7 bound more strongly to a fragmentof amino acids (165–410) of β-arrestin 2(Fig. 2B, (i),lanes 2 and 3). To map the strong interaction site, furtherβ-arrestin 2 constructs were analyzed. Residues between 195 and 202,previously identified as the JNK binding domain, were critical for strongbinding (Fig. 2C,lanes 2 and 4, and Fig.2D, lanes 4 and 6). To assess the roleof JNK in this interaction, we examined the interaction between aβ-arrestin 2 central fragment and the MKP7 C terminus, which does notbind JNK3. Amino acids 165–239 of β-arrestin 2 bound the MKP7residues 360–665 (Fig.2E, compare lane 5); this demonstrates aninteraction between the MKP7 C terminus and the central JNK binding domainregion of β-arrestin 2 that is independent of JNK3. The C terminus ofMKP7 therefore interacts most strongly with a region between amino acids 195and 202 of β-arrestin 2. Removal of the N-terminal region (1–164)of β-arrestin 2 increases MKP7 binding. It is possible that theN-terminal region may regulate MKP7 binding in the intact protein; for examplea conformational change in β-arrestin 2 that displaces the N-terminalregion might further expose the strong binding region between amino acids 195and 202. MKP7 Dephosphorylates JNK Associated with β-Arrestin2—β-Arrestin 2 binds JNK3 and its upstream kinases, ASK1 andindirectly MKK4, and enhances JNK3 phosphorylation(23McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar,24Miller W.E. McDonald P.H. Cai S.F. Field M.E. Davis R.J. Lefkowitz R.J. J. Biol.Chem. 2001; 276: 27770-27777Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). We therefore assessedwhether MKP7 could dephosphorylate β-arrestin 2-bound JNK, as wepreviously described for JIP-1-bound JNK(1Willoughby E.A. Perkins G.R. Collins M.K. Whitmarsh A.J. J. Biol. Chem. 2003; 278: 10731-10736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). We overexpressed JNK3 withβ-arrestin 2 and the MAP3K ASK1 in COS-7 cells and observed enhancementof JNK activation as reported previously(23McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar)(Fig. 3A, lanes2 and 3). The addition of MKP7 inhibited the JNK3 activationstimulated by β-arrestin 2, whereas MKP7 did not affect phosphorylatedJNK in the absence of scaffold (Fig.3A, compare lanes 2 and 4; andlanes 3 and 5). This activity of MKP7 was dependent on itsphosphatase activity, as a catalytically inactive mutant(1Willoughby E.A. Perkins G.R. Collins M.K. Whitmarsh A.J. J. Biol. Chem. 2003; 278: 10731-10736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) did not affect JNK3activation (Fig. 3B,lanes 3 and 5). These data show that MKP7 can inactivate thepool of JNK3 bound to β-arrestin 2. Angiotensin II ligation to the AT1aR stimulates JNK3 phosphorylation in thepresence of β-arrestin 2(23McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar). We co-expressed JNK3,β-arrestin 2, and AT1aR in COS-7 cells and showed that angiotensin IIstimulated phosphorylation of JNK3 at 15 and 30 min, with the signaldisappearing after 60 min (Fig.3C, lanes 3 and 4), similar topreviously published data (3Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3611) Google Scholar).In the absence of β-arrestin 2 and addition of MKP7, stimulation of JNK3phosphorylation remains the same as above(Fig. 3C, lanes8 and 9). However, when MKP7 was expressed along withβ-arrestin 2, JNK3 phosphorylation was equivalent at 15 min afterangiotensin II stimulation but completely abrogated 30 min after stimulation(Fig. 3C, lanes3 and 4; and 13 and 14). This data indicatesMKP7 utilizes the interaction with β-arrestin 2 to specificallydephosphorylate JNK3, thus allowing a more transient JNK3 phosphorylationunder AT1aR activation. MKP7 Interacts with β-Arrestin 2 in Resting Cells but IsReleased after AT1aR Stimulation—To explain the initial equivalentJNK3 activation seen in the presence of MKP7, followed by its rapidinactivation, we postulated that MKP7 interaction with β-arrestin 2 mightbe regulated by AT1aR stimulation. To detect MKP7 location, we used apreviously described polyclonal antiserum(1Willoughby E.A. Perkins G.R. Collins M.K. Whitmarsh A.J. J. Biol. Chem. 2003; 278: 10731-10736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) that detects a cytoplasmicprotein in transfected COS-7 cells (Fig.4A). We used leptomycin B to inhibit the nuclear exportof MKP7 (10Masuda K. Shima H. Watanabe M. Kikuchi K. J. Biol. Chem. 2001; 276: 39002-39011Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar,11Tanoue T. Yamamoto T. Maeda R. Nishida E. J. Biol. Chem. 2001; 276: 26629-26639Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) and demonstrated resultingnuclear fluorescence accumulation to confirm the specificity of this staining(data not shown). We then co-expressed MKP7 with components of the AT1aRsignaling system and β-arrestin 2.Fig. 4A showsoverexpressed GFP-β-arrestin 2 (green) and MKP7 (red)were both present in the cytoplasm of resting cells. Angiotensin II ligationto AT1aR has been reported to cause internalization of JNK3 bound toβ-arrestin 2 on endocytic vesicles(23McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar).Fig. 4A shows thatβ-arrestin 2 (green) moved to the plasma membrane 5 min afteragonist activation of the AT1aR, whereas MKP7 remains cytoplasmic. After 15min, green vesicles containing β-arrestin 2 (but not MKP7) hadformed, but after 30 min, the vesicles appeared yellow, indicatingthe recruitment of MKP7. After 60 min, yellow endosomal structureshad begun clustering at the perinuclear region of the cell. These experimentssuggest that MKP7 is released from β-arrestin 2 after activation of theAT1aR and is then re-recruited after ∼30 min to β-arrestin 2 presenton endocytic vesicles. To confirm this dynamic interaction between β-arrestin 2 and MKP7, weexamined MKP7 binding to β-arrestin 2 after AT1aR stimulation. For theseexperiments, we used 293T cells to express sufficient protein for detectionafter immunoprecipitation rather than the COS-7 cells in which we measuredsubcellular localization and JNK3 dephosphorylation. In the absence of JNK3,the binding of MKP7 to β-arrestin 2 changed relatively little afterangiotensin II stimulation (Fig.4B, lanes 3–7). However with the additionof JNK3, MKP7 binding to β-arrestin was lost rapidly after angiotensin IIstimulation, returning to the initial level after 60 min(Fig. 4B, lanes9–13). The level of JNK3 binding to β-arrestin 2 remained thesame throughout the time course, as previously described(23McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar) (data not shown).Together, these data demonstrate that activation of the AT1aR causes rapiddissociation of MKP7 from β-arrestin 2. This dissociation is dependent onthe presence of JNK3 and is reversed 30–60 min after AT1aR stimulation.Because JNK3 is required for MKP7 dissociation from β-arrestin 2triggered by angiotensin II, we asked whether JNK3 activation per seis sufficient to cause MKP7 dissociation. To do this, we used overexpressedASK1 as a JNK3 activator. Fig.4C shows that ASK1 causes a significant decrease in theamount of MKP7 bound to β-arrestin 2, suggesting that it is thestimulation of JNK3 phosphorylation that leads to MKP7 dissociation fromβ-arrestin 2. β-arrestins were first described as adaptor proteins involved in thedesensitization and internalization of G protein-coupled receptors. After GPCRactivation, the cytoplasmic tail of the receptor becomes phosphorylated by Gprotein-coupled receptor kinases, leading to β-arrestin recruitment.β-arrestin then blocks further G protein interaction and bind proteinsrequired for the internalization of GPCRs, such as clathrin(26Goodman Jr., O.B. Krupnick J.G. 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Chen W. Miller W.E. Vitale N. Moss J. Premont R.T. Lefkowitz R.J. J. Biol.Chem. 2001; 276: 42509-42513Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). In response to agonistbinding, β-arrestin 2 also undergoes rapid ubiquitination by Mdm2, whichis essential for receptor internalization(32Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (706) Google Scholar). In addition to their roles in receptor inhibition, β-arrestins arealso signal transducers and have been shown to interact with intracellularsignaling components, including both tyrosine and MAP kinases. For example,β-arrestins have been shown to recruit the Src family kinases(33Miller 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 (172) Google Scholar) to endothelin ETareceptors (34Imamura T. Huang J. Dalle S. Ugi S. Usui I. Luttrell L.M. Miller W.E. Lefkowitz R.J. Olefsky J.M. J. Biol. Chem. 2001; 276: 43663-43667Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), neurokinin 1receptors (35DeFea K.A. Vaughn Z.D. O'Bryan E.M. Nishijima D. Dery O. Bunnett N.W. Proc. Natl. Acad. Sci.U. S. A. 2000; 97: 11086-11091Crossref PubMed Scopus (351) Google Scholar), and CXCR1receptors (36Barlic J. Andrews J.D. Kelvin A.A. Bosinger S.E. DeVries M.E. Xu L. Dobransky T. Feldman R.D. Ferguson S.S. Kelvin D.J. Nat. Immunol. 2000; 1: 227-233Crossref PubMed Scopus (196) Google Scholar), resulting inthe activation of the MAPK ERK1/2(37Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.L. Lin F.T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1255) Google Scholar). β-arrestin 2 hasalso been shown to directly recruit components of the ERK MAPK signalingmodule to the AT1aR (38Luttrell 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 (699) Google Scholar).Although G protein-dependent ERK signaling is responsible for the activationof transcription factor Egr-1 after AT1aR stimulation, the β-arrestin2-bound pool of ERK is retained in the cytoplasm, targeting activated ERK toother cytoplasmic substrates(39Wei H. Ahn S. Barnes W.G. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 48255-48261Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar,40Pearson G. Robinson F. Beers Gibson T. Xu B.-E. Karandikar M. Berman K. Cobb M.H. Endocr.Rev. 2001; 22: 153-183Crossref PubMed Scopus (3500) Google Scholar). β-arrestin 2 wasalso recently reported to bind the neuronal JNK isoform, JNK3, and upstreamcomponents ASK1, and indirectly MKK4, and to stimulate JNK3 activation throughAT1aR stimulation (23McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar).Similar to ERK, JNK may bind β-arrestin 2 to maintain an extra nuclearlocation close to specific cytosolic substrates(41Scott M.G. Le Rouzic E. Perianin A. Pierotti V. Enslen H. Benichou S. Marullo S. Benmerah A. J. Biol. Chem. 2002; 277: 37693-37701Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). As well as GPCRs,numerous receptors have been found that utilize β-arrestin-type scaffoldproteins to coordinate both the kinases and phosphatases required for receptorfunction. These include the β-adrenergic receptors(42Shih M. Lin F. Scott J.D. Wang H.Y. Malbon C.C. J. Biol. Chem. 1999; 274: 1588-1595Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar,43Fraser I.D. Cong M. Kim J. Rollins E.N. Daaka Y. Lefkowitz R.J. Scott J.D. Curr.Biol. 2000; 10: 409-412Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), AT1R(44Ali M.S. Sayeski P.P. Bernstein K.E. J. Biol. Chem. 2000; 275: 15586-15593Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and mGluRs(45Brakeman P.R. Lanahan A.A. O'Brien R. Roche K. Barnes C.A. Huganir R.L. Worley P.F. Nature. 1997; 386: 284-288Crossref PubMed Scopus (925) Google Scholar). We have shown that the dual specificity phosphatase MKP7 bindsβ-arrestin 2, using residues 394–443 of MKP7 previously identifiedas the JIP binding domain (1Willoughby E.A. Perkins G.R. Collins M.K. Whitmarsh A.J. J. Biol. Chem. 2003; 278: 10731-10736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).The interaction of this region of MKP7 with these two diverse scaffoldproteins suggests that it may function as a general scaffold-binding domain.The interaction between MKP7 and β-arrestin 2 is dynamic, meaning theydissociate after β-arrestin 2 is recruited to the AT1aR by angiotensinstimulation. Recent data have indicated β-arrestin 2, upon activation bybinding to the phosphorylated C terminus of GPCRs, undergoes a conformationalchange (46Xiao K. Shenoy S.K. Nobles K. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 55744-55753Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Thisconformational change could be involved in the triggering of JNK3 activation.We have shown that the presence of JNK3 on the scaffold is necessary for MKP7dissociation and also that ASK1 overexpression leads to MKP7 dissociation. Wetherefore propose that activation of JNK3 triggers MKP7 dissociation fromβ-arrestin 2. After 30–60 min, depending on the cell system, MKP7reassociates with β-arrestin 2 to specifically dephosphorylate JNK3. Thiscycle of MKP7 dissociation from and reassociation with β-arrestin 2explains how a signal can be transmitted through a scaffold protein thatapparently binds both activating and inhibitory components. By binding intothis complex, under these circumstances, MKP7 may play a role in regulatingJNK in the cytoplasm. These studies have been performed in cells overexpressing the signalingcomponents; however, endogenous interaction between β-arrestin 2 and JNK3has been seen in brain lysates(23McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar), where the widelyexpressed MKP7 is also present(10Masuda K. Shima H. Watanabe M. Kikuchi K. J. Biol. Chem. 2001; 276: 39002-39011Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar,11Tanoue T. Yamamoto T. Maeda R. Nishida E. J. Biol. Chem. 2001; 276: 26629-26639Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Data has shownseven-transmembrane receptors Frizzled(47Chen W. ten Berge D. Brown J. Ahn S. Hu L.A. Miller W.E. Caron M.G. Barak L.S. Nusse R. Lefkowitz R.J. Science. 2003; 301: 1391-1394Crossref PubMed Scopus (291) Google Scholar) and Smoothened(48Wilbanks A.M. Fralish G.B. Kirby M.L. Barak L.S. Li Y.X. Caron M.G. Science. 2004; 306: 2264-2267Crossref PubMed Scopus (100) Google Scholar,49Chen W. Ren X.R. Nelson C.D. Barak L.S. Chen J.K. Beachy P.A. de Sauvage F. Lefkowitz R.J. Science. 2004; 306: 2257-2260Crossref PubMed Scopus (232) Google Scholar), as well as non-GPCRs,such as TGFβ (50Chen W. Kirkbride K.C. How T. Nelson C.D. Mo J. Frederick J.P. Wang X.F. Lefkowitz R.J. Blobe G.C. Science. 2003; 301: 1394-1397Crossref PubMed Scopus (207) Google Scholar), arecoupled to β-arrestin scaffold proteins. It is therefore possible thatMKP7 and other DSPs can regulate MAPK signaling in a number of receptorsystems. We thank Alan Whitmarsh (University of Manchester, UK) and Stephane LaPorte(McGill University, Montreal, Canada) for essential reagents." @default.
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• W2002879408 title "Dynamic Interaction between the Dual Specificity Phosphatase MKP7 and theJNK3 Scaffold Protein β-Arrestin2" @default.
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