How Does Arrestin Assemble MAPKs into a Signaling Complex?
2008; Elsevier BV; Volume: 284; Issue: 1 Linguagem: Inglês
10.1074/jbc.m806124200
ISSN1083-351X
AutoresXiufeng Song, Sergio Coffa, Haian Fu, Vsevolod V. Gurevich,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoArrestins bind active phosphorylated G protein-coupled receptors, precluding G protein activation and channeling signaling to alternative pathways. Arrestins also function as mitogen-activated protein kinase (MAPK) scaffolds, bringing together three components of MAPK signaling modules. Here we have demonstrated that all four vertebrate arrestins interact with JNK3, MKK4, and ASK1, but only arrestin3 facilitates JNK3 activation. Thus, the functional specificity of arrestins is not determined by differential binding of the kinases. Using receptor binding-impaired mutant, we have shown that free arrestin3 readily promotes JNK3 phosphorylation. We identified key arrestin-binding elements in JNK3 and ASK1 and investigated the molecular interactions of arrestin2 and arrestin3 and their individual domains with the components of the two MAPK cascades, ASK1-MKK4-JNK3 and c-Raf-1-MEK1-ERK2. We found that both arrestin domains interact with all six kinases. These findings shed new light on the mechanism of arrestin-mediated MAPK activation and the spatial arrangement of the three kinases on arrestin molecule. Arrestins bind active phosphorylated G protein-coupled receptors, precluding G protein activation and channeling signaling to alternative pathways. Arrestins also function as mitogen-activated protein kinase (MAPK) scaffolds, bringing together three components of MAPK signaling modules. Here we have demonstrated that all four vertebrate arrestins interact with JNK3, MKK4, and ASK1, but only arrestin3 facilitates JNK3 activation. Thus, the functional specificity of arrestins is not determined by differential binding of the kinases. Using receptor binding-impaired mutant, we have shown that free arrestin3 readily promotes JNK3 phosphorylation. We identified key arrestin-binding elements in JNK3 and ASK1 and investigated the molecular interactions of arrestin2 and arrestin3 and their individual domains with the components of the two MAPK cascades, ASK1-MKK4-JNK3 and c-Raf-1-MEK1-ERK2. We found that both arrestin domains interact with all six kinases. These findings shed new light on the mechanism of arrestin-mediated MAPK activation and the spatial arrangement of the three kinases on arrestin molecule. Arrestins are multifunctional regulators of cell signaling (1Gurevich E.V. Gurevich V.V. Genome Biol. 2006; 7: 236Crossref PubMed Scopus (227) Google Scholar, 2Shenoy S.K. Lefkowitz R.J. Biochem. J. 2003; 375: 503-515Crossref PubMed Scopus (333) Google Scholar). Arrestins, which bind active phosphorylated G protein-coupled receptors (GPCRs), 2The abbreviations used are: GPCR, G protein-coupled receptor; ASK, apoptosis signal-regulating kinase; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; MEK, MAPK/ERK kinase; NES, nuclear export signal; NLS, nuclear localization signal; HEK, human embryonic kidney; PBS, phosphate-buffered saline; WT, wild type.2The abbreviations used are: GPCR, G protein-coupled receptor; ASK, apoptosis signal-regulating kinase; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; MEK, MAPK/ERK kinase; NES, nuclear export signal; NLS, nuclear localization signal; HEK, human embryonic kidney; PBS, phosphate-buffered saline; WT, wild type. which play a major role in receptor desensitization and internalization (3Gurevich V.V. Gurevich E.V. Trends Pharmacol. Sci. 2004; 25: 59-112Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 4Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (233) Google Scholar). With the identification of numerous non-receptor binding partners, the classical paradigm of arrestin function has been expanded, implicating arrestins in mitogen-activated protein kinase (MAPK) activation, protein ubiquitination, chemotaxis, apoptosis, and other cellular functions (2Shenoy S.K. Lefkowitz R.J. Biochem. J. 2003; 375: 503-515Crossref PubMed Scopus (333) Google Scholar, 5DeFea K.A. Annu. Rev. Physiol. 2007; 69: 535-560Crossref PubMed Scopus (91) Google Scholar, 6Gurevich V.V. Gurevich E.V. Pharmacol. Ther. 2006; 110: 465-502Crossref PubMed Scopus (358) Google Scholar, 7Hunton D.L. Barnes W.G. Kim J. Ren X.R. Violin J.D. Reiter E. Milligan G. Patel D.D. Lefkowitz R.J. Mol. Pharmacol. 2005; 67: 1229-1236Crossref PubMed Scopus (106) Google Scholar, 8Luttrell 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 (697) Google Scholar, 9McDonald 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, 10Miller 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, 11Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (706) Google Scholar).The first indication that arrestins function as signaling adapters came from the studies of arrestin-dependent c-Src recruitment to the receptors, which results in the activation of extracellular signal-regulated kinases (ERK1/2) (10Miller 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, 12DeFea 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 (350) Google Scholar, 13Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1253) Google Scholar). Subsequently, arrestin2 and arrestin3 in complex with different receptors were reported to scaffold JNK3 (9McDonald 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), ERK1/2 (8Luttrell 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 (697) Google Scholar, 14DeFea 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), and p38 (15Bruchas M.R. Macey T.A. Lowe J.D. Chavkin C. J. Biol. Chem. 2006; 281: 18081-18089Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 16Sun Y. Cheng Z. Ma L. Pei G. J. Biol. Chem. 2002; 277: 49212-49219Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar) activation cascades. Although arrestins play an important role in regulating different MAPK pathways, the mechanism of arrestin-dependent assembly of MAP kinases into a signaling complex remains largely unexplored. Existing models have limited predictive value. For example, the idea that JNK3 is activated solely by arrestin3 because this arrestin subtype has unique ability to bind JNK3 (9McDonald 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, 17Miller 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) was not supported by further experimentation (18Scott 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, 19Song X. Raman D. Gurevich E.V. Vishnivetskiy S.A. Gurevich V.V. J. Biol. Chem. 2006; 281: 21491-21499Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Song X. Gurevich E.V. Gurevich V.V. J. Neurochem. 2007; 103: 1053-1062Crossref PubMed Scopus (48) Google Scholar). Similarly, the hypothesis that only receptor-bound arrestins interact with MAP kinases (8Luttrell 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 (697) Google Scholar, 9McDonald 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) was not confirmed (17Miller 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, 18Scott 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, 19Song X. Raman D. Gurevich E.V. Vishnivetskiy S.A. Gurevich V.V. J. Biol. Chem. 2006; 281: 21491-21499Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Song X. Gurevich E.V. Gurevich V.V. J. Neurochem. 2007; 103: 1053-1062Crossref PubMed Scopus (48) Google Scholar).Here we addressed several key mechanistic issues in arrestin-dependent MAPK signaling. First, we show that the scaffolding function is not limited to receptor-bound arrestin; free arrestin3 facilitates ASK1-mediated JNK3 activation, indicating that arrestins are not exclusively receptor-regulated adapters as thought previously. Second, we show that all four mammalian arrestins bind each component of the JNK3 cascade with comparable affinity, demonstrating that binding does not necessarily translate into activation. This finding establishes the mechanistic basis of the "dominant-negative" effect of certain arrestin subtypes. Third, using truncated forms of ASK1 and JNK3, we identified the major arrestin-binding elements of these two kinases. Finally, we show that every kinase in JNK3 and ERK2 activation cascades binds both arrestin domains. Based on these findings, we propose a functional model of arrestin-dependent regulation of MAPK activity and a new structural model of the arrestin-MAPK multiprotein signaling complex.EXPERIMENTAL PROCEDURESPlasmid Constructs—The coding sequences of bovine arrestin1 (rod), arrestin2, arrestin3, and human arrestin4 (cone) 3Note that here we have used the systematic names of arrestin proteins: arrestin1 (also known as visual or rod arrestin, 48-kDa protein, or S-antigen), arrestin2 (β-arrestin or β-arrestin1), arrestin3 (β-arrestin2), and arrestin4 (cone or X-arrestin). with the C-terminal FLAG tag were subcloned into pcDNA3. Arrestin2 with engineered nuclear export signal (NES) (Q394L), NES-less arrestin3 (L393Q), and "inactive" (D7) mutants with a 7-residue deletion in the interdomain hinge were described previously (19Song X. Raman D. Gurevich E.V. Vishnivetskiy S.A. Gurevich V.V. J. Biol. Chem. 2006; 281: 21491-21499Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 21Hanson S.M. Cleghorn W.M. Francis D.J. Vishnivetskiy S.A. Raman D. Song S. Nair K.S. Slepak V.Z. Klug C.S. Gurevich V.V. J. Mol. Biol. 2007; 368: 375-387Crossref PubMed Scopus (101) Google Scholar). Separated N and C domains of arrestin3 (residues 1-181 and 180-408) and arrestin2 (residues 1-180 and 179-418) were engineered with C-terminal NES and FLAG tag. All constructs were verified by dideoxy sequencing. Expression constructs for GFP-JNK3, HA-JNK3, and HA-MKK4 were gifts from Drs. Louis Luttrell (Medical University of South Carolina), Robert J. Lefkowitz (Duke University), and Jia Le Dai (The University of Texas M. D. Anderson Cancer Center), respectively. HA-ASK was modified by oligo 5′-CCG AAG AAA AAG CGC AAG GTC-3′ to introduce a nuclear localization signal (NLS), and GFP-JNK3 was modified by deleting 39 N-terminal residues, 20 C-terminal residues, or both termini.Cell Culture and Transient Transfection—Adenovirus-transformed human embryonic kidney cells (HEK-293A) and COS-7 African green monkey cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) plus penicillin and streptomycin at 37 °C in a humidified incubator with 5% CO2. The cells were plated at 80-90% confluence and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were serum-starved overnight before all experiments and used 48 h post-transfection.Nuclear Exclusion Assay—The day after transfection HEK-293A cells were seeded onto Lab-Tek CC2 chambered slides coated with fibronectin (20 μg/ml in PBS) (Sigma) for microscopy and onto poly-d-lysine (15 μg/ml)-coated 24-well plates for Western blot. For microscopy cells were fixed in 4% paraformaldehyde on ice (15 min), permeabilized with 0.1% Triton in PBS, and blocked with 3% bovine serum albumin in PBS for 1 h at room temperature. FLAG-tagged arrestins were visualized with M2 anti-FLAG antibody (Sigma) followed by the Alexa 593 (red) anti-mouse secondary antibodies (Molecular Probes, Eugene, OR). GFP-JNK3 was visualized by its intrinsic green fluorescence, using an epifluorescence microscope equipped with a charge-coupled device camera. The slides were air-dried and mounted in the medium containing 4,6-diamidino-2-phenylindole to visualize the nuclei. The images were acquired using a Nikon EC2000 inverted fluorescent microscope. The distribution of full-length and truncated JNK3, ASK1-NLS, and the indicated arrestin in at least 20 cells was scored based on the subcellular distribution of the green fluorescence signal (nucleus > cytoplasm; nucleus = cytoplasm; nucleus < cytoplasm).Western Blot—COS-7 cells were incubated with phosphatase inhibitors (50 mm NaF, 10 mm Na3VO4) in serum-free medium for 15 min at 37 °C, washed with cold PBS, and lysed with SDS sample buffer containing 10 mm NaF, 100 μm Na3VO4, 2 mm EDTA, 2 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride. Whole cell lysates were boiled for 5 min and then centrifuged at 10,000 × g for 10 min, and the supernatants were used for Western blot. The proteins were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). Mouse monoclonal antibodies against FLAG (Sigma), HA (Sigma), GFP (Clontech), and phospho-JNK (Cell Signaling Technology Inc.) were used at 1:1000 or 1:2000 dilution followed by horseradish peroxidase-conjugated anti-mouse secondary antibody. Protein bands were detected by enhanced chemiluminescence (ECL, Pierce) followed by exposure to x-ray film. Immunoblots were quantified using QuantityOne software (Bio-Rad Laboratories).Immunoprecipitation—Cells (60-mm plates) were lysed in 0.75 ml of lysis buffer (50 mm Tris, 2 mm EDTA, 250 mm NaCl, 10% glycerol, 0.5% Nonidet P-40, 20 mm NaF, 1 mm Na orthovanadate, 10 mm N-ethylmaleimide, 2 mm benzamidine, and 1 mm phenylmethylsulfonyl fluoride) for 30-60 min at 4 °C. In experiments involving ERK2, prior to lysis the cells were treated with 1 mm cross-linking reagent dithiobis(succinimidyl propionate) (DSP; Pierce) for 30 min followed by 2 mm Tris-HCl, pH 7.5, for 15 min at room temperature. After centrifugation, supernatants were precleared by 20 μl of protein G-agarose. Then, 600 μl of supernatant was incubated with primary antibodies for 2 h followed by the addition of 12 μl of protein G-agarose beads for 2 h or overnight. The beads were washed three times with 1 ml of lysis buffer, and the proteins were eluted with 50 μl of sample buffer, boiled for 5 min, and analyzed by Western blot as described above.Statistical Analysis—Quantitative data from at least three experiments were analyzed by one-way analysis of variance with arrestin as a main factor (with Bonferroni-Dunn correction for multiple comparisons).RESULTSInteraction Does Not Always Mean Activation, as All Four Mammalian Arrestins Bind ASK1, MKK4, and JNK3, but Only Arrestin3 Enhances JNK3 Phosphorylation—Mammals have four arrestin isoforms (nonvisual arrestin2 and arrestin3, arrestin1 (expressed in rods and cones), and arrestin4 (cone-specific) (1Gurevich E.V. Gurevich V.V. Genome Biol. 2006; 7: 236Crossref PubMed Scopus (227) Google Scholar, 22Nikonov S.S. Brown B.M. Davis J.A. Zuniga F.I. Bragin A. Pugh Jr., E.N. Craft C.M. Neuron. 2008; 59: 462-474Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar)), which share a high degree of structural homology (23Sutton R.B. Vishnivetskiy S.A. Robert J. Hanson S.M. Raman D. Knox B.E. Kono M. Navarro J. Gurevich V.V. J. Mol. Biol. 2005; 354: 1069-1080Crossref PubMed Scopus (148) Google Scholar). Previously we showed that all four arrestins bind JNK3 (19Song X. Raman D. Gurevich E.V. Vishnivetskiy S.A. Gurevich V.V. J. Biol. Chem. 2006; 281: 21491-21499Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Song X. Gurevich E.V. Gurevich V.V. J. Neurochem. 2007; 103: 1053-1062Crossref PubMed Scopus (48) Google Scholar). However, we found that arrestin3 is the only isoform that enhances JNK3α2 phosphorylation in COS-7 cells (Fig. 1), in agreement with previous reports (9McDonald 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, 17Miller 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). Because JNK3 activation requires the assembly of all three components of the cascade, we tested whether other arrestins bind upstream kinases ASK1 and MKK4. Arrestin interaction with ASK1 was tested in a nuclear exclusion assay based on the ability of arrestins equipped with the NES to remove their interaction partners from the nucleus (19Song X. Raman D. Gurevich E.V. Vishnivetskiy S.A. Gurevich V.V. J. Biol. Chem. 2006; 281: 21491-21499Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Song X. Gurevich E.V. Gurevich V.V. J. Neurochem. 2007; 103: 1053-1062Crossref PubMed Scopus (48) Google Scholar). The addition of a short C-terminal NLS, KKKRK, was sufficient to ensure nuclear localization of ASK1 (Fig. 2C). Co-expression of each of the four arrestins relocalized ASK1-NLS to the cytoplasm, demonstrating that all isoforms bind ASK1 (Fig. 2C). Similarly, we found that all four arrestins co-immunoprecipitate MKK4 (Fig. 2B). Among the three members of the ASK1-MKK4-JNK3 cascade the interaction with MKK4 appears to have the lowest affinity; it was detectable only at higher levels of MKK4 expression. However, both nonvisual arrestins bind MKK4 similarly, whereas arrestins 1 and 4 demonstrate higher binding (Fig. 2B). Thus, all members of the arrestin family interact with each kinase in the ASK1-MKK4-JNK3 cascade, so that the binding per se does not explain the unique ability of arrestin3 to facilitate JNK3 activation.FIGURE 2ASK1, MKK4, and JNK3 bind arrestin3. A, COS-7 cells were transfected with FLAG-arrestin3 (FLAG-Arr3) with GFP-JNK3, HA-ASK1, HA-MKK4, or with indicated combinations of these kinases. Cell lysates were immunoprecipitated with rabbit anti-FLAG antibody, and then immunoblotted with mouse anti-HA, anti-GFP and anti-FLAG antibodies. B, COS-7 cells were transfected with HA-MKK4 with indicated FLAG-tagged arrestins. Cell lysates were immunoprecipitated and immunoblotted as in panel A. C, HEK-293A cells were transfected with HA-ASK-NLS alone or with indicated FLAG-tagged arrestins. Arrestins were visualized with M2 anti-FLAG, HA-ASK1 with rat anti-HA high affinity antibody, followed by Alexa 593 anti-mouse (Red) and Alexa 488 anti-rat (Green) secondary antibodies, respectively. The representative images show arrestins (red), HA-ASK1-NLS (green), and both channels merged. At least 20 cells expressing indicated proteins were scored for the subcellular distribution of HA-ASK1-NLS. Means ± S.D. (n = 3) of the fraction of cells with more ASK1 in the cytoplasm than in the nucleus are shown. ****, p < 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because arrestin3, in contrast to arrestin2, has leucine-rich NES promoting its transport to the cytoplasm (18Scott 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, 24Wang P. Wu Y. Ge X. Ma L. Pei G. J. Biol. Chem. 2003; 278: 11648-11653Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), we tested whether the presence of NES affects arrestin-dependent JNK3 activation. All four mammalian arrestins are predominantly cytoplasmic in most cell types that express them endogenously, as well as in overexpressing HEK-293A cells (19Song X. Raman D. Gurevich E.V. Vishnivetskiy S.A. Gurevich V.V. J. Biol. Chem. 2006; 281: 21491-21499Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Song X. Gurevich E.V. Gurevich V.V. J. Neurochem. 2007; 103: 1053-1062Crossref PubMed Scopus (48) Google Scholar). The elimination of a putative NES or the addition of an engineered NES in arrestin1 or arrestin4 did not change their subcellular localization or their ability to redistribute JNK3 (19Song X. Raman D. Gurevich E.V. Vishnivetskiy S.A. Gurevich V.V. J. Biol. Chem. 2006; 281: 21491-21499Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Song X. Gurevich E.V. Gurevich V.V. J. Neurochem. 2007; 103: 1053-1062Crossref PubMed Scopus (48) Google Scholar). Although wild type (WT) arrestin2 did not move JNK3 from the nucleus, an engineered NES (point mutation Q394L) enabled it to redistribute JNK3 as efficiently as arrestin3 (18Scott 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, 19Song X. Raman D. Gurevich E.V. Vishnivetskiy S.A. Gurevich V.V. J. Biol. Chem. 2006; 281: 21491-21499Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The elimination of NES in arrestin3 by the L393Q mutation only partially reduced its ability to recruit JNK3 to the cytoplasm (19Song X. Raman D. Gurevich E.V. Vishnivetskiy S.A. Gurevich V.V. J. Biol. Chem. 2006; 281: 21491-21499Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). To test the effect of NES, we compared the ability of arrestin2-Q394L (NES+) and arrestin3-L393Q (NES-) to promote JNK3 activation with their corresponding parental proteins (Fig. 3A). The elimination of NES in arrestin3 reduced but did not abolish its ability to activate JNK3, whereas arrestin2-NES+ remained as ineffective as WT arrestin2. Thus, NES has only a minor effect on arrestin-dependent JNK3 activation (Fig. 3A).FIGURE 3Free arrestin scaffolds ASK1-MKK4-JNK3 module. A, COS-7 cells were transfected with HA-JNK3 alone, or with HA-ASK1, or together with arrestin3 (Arr3), arrestin3-NES- (Arr3-NES-), arrestin2 (Arr2), or arrestin2-NES+ (Arr2-NES+). Cell lysates were analyzed as in Fig. 1 (untagged arrestin3 was detected with F4C1 mouse monoclonal antibody). Means ± S.D. (n = 3) of the intensity of phospho-JNK3 band are shown. Both arrestin3 (****, p < 0.0001) and arrestin3-NES- (**, p < 0.01) enhance JNK3 activation, as compared with cells expressing ASK1+JNK3 without arrestin; JNK3 activation enhanced by arrestin3-NES- was significantly different from arrestin3 (###, p = 0.0004). B, COS-7 cells were transfected with HA-JNK3 + HA-ASK1, or together with FLAG-tagged arrestin3, arrestin3-D7, arrestin3-N-domain, or arrestin3-C-domain. The lysates were analyzed as in Fig. 1. Means ± S.D. (n = 3) of the intensity of phospho-JNK3 band are shown. Statistical analysis shows that arrestin3 (**, p = 0.0053) and arrestin3-D7 (**, p = 0.0018) enhance JNK3 activation, whereas arrestin3-N (p = 0.5769) and arrestin3-C (p = 0.3629) do not.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Co-expression of arrestin3 with JNK3 and ASK1 increases phospho-JNK3 ∼7-fold in COS-7 cells. To rule out a possible contribution of arrestin binding to endogenous receptors, we used arrestin mutant with a large deletion in the interdomain "hinge," which "freezes" the molecule in the basal conformation and severely impairs receptor binding (21Hanson S.M. Cleghorn W.M. Francis D.J. Vishnivetskiy S.A. Raman D. Song S. Nair K.S. Slepak V.Z. Klug C.S. Gurevich V.V. J. Mol. Biol. 2007; 368: 375-387Crossref PubMed Scopus (101) Google Scholar, 25Vishnivetskiy S.A. Hirsch J.A. Velez M.-G. Gurevich Y.V. Gurevich V.V. J. Biol. 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Separately expressed N and C domains of arrestin3 only marginally increased phospho-JNK3 level (∼2-3-fold) (Fig. 3B). Thus, the two domains in proper relative orientation are required for optimal JNK3 activation.FIGURE 4ASK1, MKK4, and JNK3 interact with both domains of arrestin. A-C, HEK-293A cells were transfected with HA-ASK-NLS or GFP-JNK3 individually or with FLAG-tagged full-length arrestin3 (FLAG-Arr3), its N- (FLAG-Arr3-N) or C-domain (FLAG-Arr3-C), arrestin2-NES+ (Arr2-NES), arrestin2 N-domain-NES (Arr2-N-NES), or arrestin2 C-domain-NES (Arr2-C-NES). Immunocytochemistry was done as described in Fig. 2C. GFP-JNK3 was visualized by its intrinsic fluorescence. Representative images are shown. The localization of GFP-JNK and HA-ASK1-NLS was quantified, as described in the legend to in Fig. 2C. Means ± S.D. (n = 3) of the fraction of cells with more JNK3 or ASK1 in the cytoplasm than in the nucleus also shown. (****, p < 0.0001). D-F, COS-7 cells were transfected with HA-ASK1, HA-MKK4, or GFP-JNK3 alone, or together with FLAG-tagged arrestin3 and its domains. Immunoprecipitation and Western blot analysis were performed as described in the legend to Fig. 2A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate the possible inter-dependence of their binding to arrestin3, we expressed ASK1, MKK4, and JNK3 individually and in different combinations, immunoprecipitated FLAG-arrestin3, and immunoblotted for individual kinases (Fig. 2A). Among the three kinases, ASK1 demonstrated the highest level of binding and MKK4 the lowest. Similar amounts of ASK1 were found in complex with arrestin3 in all cases, indicating that downstream
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