Visual and Both Non-visual Arrestins in Their “Inactive” Conformation Bind JNK3 and Mdm2 and Relocalize Them from the Nucleus to the Cytoplasm

Arrestins bind active phosphorylated G protein-coupled receptors, terminating G protein activation. Receptor-bound non-visual arrestins interact with numerous partners, redirecting signaling to alternative pathways. Arrestins also have nuclear localization and nuclear exclusion signals and shuttle between the nucleus and the cytoplasm. Constitutively shuttling proteins often redistribute their interaction partners between the two compartments. Here we took advantage of the nucleoplasmic shuttling of free arrestins and used a “nuclear exclusion assay” to study their interactions with two proteins involved in “life-and-death” decisions in the cell, the kinase JNK3 and the ubiquitin ligase Mdm2. In human embryonic kidney 293 cells green fluorescent protein (GFP)-JNK3 and GFP-Mdm2 predominantly localize in the nucleus, whereas visual arrestin, arrestin2(Q394L) mutant equipped with the nuclear exclusion signal, and arrestin3 localize exclusively to the cytoplasm. Coexpression of arrestins moves both GFP-JNK3 and GFP-Mdm2 to the cytoplasm. Arrestin mutants “frozen” in the basal conformation are the most efficacious. Thus, arrestins in their basal state interact with JNK3 and Mdm2, suggesting that arrestins are likely “preloaded” with their interaction partners when they bind the receptor. Robust interaction of free arrestins with JNK3 and Mdm2 and their ability to regulate subcellular localization of these proteins may play an important role in the survival of photoreceptors and other neurons, as well as in retinal and neuronal degeneration. Arrestins bind active phosphorylated G protein-coupled receptors, terminating G protein activation. Receptor-bound non-visual arrestins interact with numerous partners, redirecting signaling to alternative pathways. Arrestins also have nuclear localization and nuclear exclusion signals and shuttle between the nucleus and the cytoplasm. Constitutively shuttling proteins often redistribute their interaction partners between the two compartments. Here we took advantage of the nucleoplasmic shuttling of free arrestins and used a “nuclear exclusion assay” to study their interactions with two proteins involved in “life-and-death” decisions in the cell, the kinase JNK3 and the ubiquitin ligase Mdm2. In human embryonic kidney 293 cells green fluorescent protein (GFP)-JNK3 and GFP-Mdm2 predominantly localize in the nucleus, whereas visual arrestin, arrestin2(Q394L) mutant equipped with the nuclear exclusion signal, and arrestin3 localize exclusively to the cytoplasm. Coexpression of arrestins moves both GFP-JNK3 and GFP-Mdm2 to the cytoplasm. Arrestin mutants “frozen” in the basal conformation are the most efficacious. Thus, arrestins in their basal state interact with JNK3 and Mdm2, suggesting that arrestins are likely “preloaded” with their interaction partners when they bind the receptor. Robust interaction of free arrestins with JNK3 and Mdm2 and their ability to regulate subcellular localization of these proteins may play an important role in the survival of photoreceptors and other neurons, as well as in retinal and neuronal degeneration. Arrestins specifically bind agonist-activated phosphorylated G protein-coupled receptors (GPCRs), 3The abbreviations used are: GPCR, G protein-coupled receptor; ERK, extracellular signal-regulated kinase; E3, ubiquitin-protein isopeptide ligase; NES, nuclear export signal; GFP, green fluorescent protein; HEK, human embryonic kidney; LMB, leptomycin B; WT, wild type; JNK, c-Jun N-terminal kinase. terminating further G protein activation and often redirecting signaling to alternative pathways (1Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (240) Google Scholar, 2Lefkowitz R.J. Shenoy S.K. Science. 2005; 308: 512-517Crossref PubMed Scopus (1439) Google Scholar). Visual arrestin plays a key role in the regulation of rhodopsin signaling in rod photoreceptors. Non-visual arrestins 2 and 3 are expressed in most cells and regulate the signaling of a wide variety of GPCRs. Arrestin2 is the most abundant subtype in mature neurons (3Gurevich E.V. Benovic J.L. Gurevich V.V. Neuroscience. 2002; 109: 421-436Crossref PubMed Scopus (90) Google Scholar, 4Gurevich E.V. Benovic J.L. Gurevich V.V. J. Neurochem. 2004; 91: 1404-1416Crossref PubMed Scopus (79) Google Scholar). Receptor-bound non-visual arrestins link GPCRs to the activation of c-Src (5Luttrell 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 (1264) Google Scholar), serve as scaffolds for receptor activation-dependent phosphorylation of ERK1/2 (6Luttrell 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 (704) Google Scholar) and JNK3 (7McDonald 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: 1515-1518Crossref PubMed Google Scholar), and mobilize the E3 ubiquitin ligase Mdm2 to the arrestin-receptor complex (8Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (718) Google Scholar), etc. Binding to the receptor is accompanied by a global conformational change in the arrestin molecule (4Gurevich E.V. Benovic J.L. Gurevich V.V. J. Neurochem. 2004; 91: 1404-1416Crossref PubMed Scopus (79) Google Scholar), which is widely believed to underlie preferential interaction of numerous nonreceptor partners with receptor-bound, rather than with free, arrestin (2Lefkowitz R.J. Shenoy S.K. Science. 2005; 308: 512-517Crossref PubMed Scopus (1439) Google Scholar, 9Gurevich V.V. Gurevich E.V. Structure. 2003; 11: 1037-1042Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Non-visual arrestins 2 and 3 were recently shown to shuttle between the nucleus and the cytoplasm (10Scott 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 (169) Google Scholar, 11Wang P. Wu Y. Ge X. Ma L. Pei G. J. Biol. Chem. 2003; 278: 11648-11653Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Arrestin3 with its native nuclear export signal (NES) removes some of its interaction partners, such as JNK3 (10Scott 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 (169) Google Scholar) and Mdm2 (11Wang P. Wu Y. Ge X. Ma L. Pei G. J. Biol. Chem. 2003; 278: 11648-11653Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), from the nucleus. This must be a function of free arrestin, because membrane-imbedded GPCRs are not transported through the aqueous nuclear pore. Here we used the ability of arrestin proteins to bring their binding partners out of the nucleus as a readout to study the interactions of free arrestins 2 and 3 and visual arrestin with JNK3 and Mdm2, two proteins that play a pivotal role in the regulation of cell death and survival. We found that all three arrestins interact with JNK3 and Mdm2 and dramatically change their subcellular localization. Comparison of and mutants in the basal state that both JNK3 and Mdm2 bind arrestins in their basal state and Mdm2 the of visual arrestin G. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), Benovic J.L. J. Caron M.G. Lefkowitz R.J. Science. PubMed Scopus Google Scholar), and arrestin3 Gurevich V.V. P. Benovic J.L. J. Biol. Chem. 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Benovic Arrestin2 and arrestin3 were the by were by for GFP-JNK3 and the human of were from Luttrell of and Pei for and were in with and in a with cells were and the cells were and with of for and with for in with the of for the and of and by the and of and the of were and subcellular localization of visual arrestin in as A. Gurevich E.V. S.A. J. Gurevich V.V. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). were with and with were in the in and of the were with for (3Gurevich E.V. Benovic J.L. Gurevich V.V. Neuroscience. 2002; 109: 421-436Crossref PubMed Scopus (90) Google Scholar, 4Gurevich E.V. Benovic J.L. Gurevich V.V. J. Neurochem. 2004; 91: 1404-1416Crossref PubMed Scopus (79) Google Scholar). were used in by and were a fluorescent equipped with a cells were in for cells were with and with in for and arrestins were with L. S. V. Curr. PubMed Scopus Google Scholar) and by the and the human of were in with the cells were with of leptomycin for to of and the were in for were and with the to the GFP-JNK3 and GFP-Mdm2 were an equipped with a were three to In the of Mdm2, arrestin in cells nucleus nucleus the were by of with arrestin and as the of both and the interaction between were of with for an of cells and in protein by by and to Arrestin with L. S. V. Curr. PubMed Scopus Google Scholar). of the proteins were to the for of Arrestins in of arrestins in native not we the localization of the two visual A. Gurevich E.V. S.A. J. Gurevich V.V. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) and (4Gurevich E.V. Benovic J.L. Gurevich V.V. J. Neurochem. 2004; 91: 1404-1416Crossref PubMed Scopus (79) Google Scholar), for which for are Non-visual arrestins 2 and 3 are expressed in all (3Gurevich E.V. Benovic J.L. Gurevich V.V. Neuroscience. 2002; 109: 421-436Crossref PubMed Scopus (90) Google Scholar). Arrestin2 in mature neurons, its that of arrestin3 (4Gurevich E.V. Benovic J.L. Gurevich V.V. J. Neurochem. 2004; 91: 1404-1416Crossref PubMed Scopus (79) Google Scholar). is in both the and cytoplasm in all of neurons its between these two in neurons, in and the of in the nucleus that in the cytoplasm and in neurons is predominantly and In other of neuronal cells nucleus of the and a of other is between the nucleus and the cytoplasm protein is to through the nuclear pore. its preferential localization in the nucleus the cytoplasm that are cell that in of these compartments. localization of visual arrestin in rod photoreceptors is A. Gurevich E.V. S.A. J. Gurevich V.V. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In the in the whereas in the is in the and cell Visual arrestin is from the is in the cytoplasm and the of visual arrestin is not to its the nuclear K. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), must be from the nucleus. of Visual and Arrestins 2 and 3 in we the localization of the three arrestins expressed in arrestin3 is exclusively in the whereas visual arrestin and predominantly localization with a in the nucleus Thus, in cells is to its in neurons and localization of arrestins 2 and 3 that have a not change their the localization of the native proteins Thus, the of arrestins between the nucleus and the cytoplasm of cells their in neurons that these of a the nuclear of visual arrestin and arrestin3 not change the of protein be from the nucleus for two not because is to the nuclear and not have a nuclear localization signal to a may and be is equipped with a nuclear export signal of the native and arrestins is not to their whereas arrestins are than that they and the nuclear K. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the for the subcellular of arrestin we used LMB, an of a nuclear export F. Science. 278: PubMed Scopus Google Scholar, S. E. PubMed Scopus Google Scholar). of cells with of the of visual arrestin and arrestin3 in the nucleus, whereas change in in the when are visual arrestin and arrestin3 dramatically to the nucleus, and the nuclear of that is predominantly by an whereas and the other two arrestin an to a the of all three arrestins that the export of visual arrestin and arrestin3 out of the nucleus is when these proteins are with a that out of the nucleus the a S. E. PubMed Scopus Google Scholar), a of a J.L. S.S. Full Text PDF PubMed Scopus Google Scholar). arrestin3 shown to have a native in its which be by a (10Scott 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 (169) Google Scholar, 11Wang P. Wu Y. Ge X. Ma L. Pei G. J. Biol. 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This is because the of the visual mutant than that of the and ability of the three of to Mdm2 the all three proteins expressed the mutant Mdm2 to the cytoplasm and the of whereas to redistribute Mdm2 Arrestin3 also to Mdm2 to the of Mdm2 in the of cells in the of the mutant to of arrestin3 we not have and cells to the arrestin3 is in Mdm2 than suggesting that its for Mdm2 is that a of in the nucleus its of and binding Mdm2. of the of visual arrestin and that all bind Mdm2 the of the interaction arrestin Thus, all three arrestins interact with Mdm2, and in all the Mdm2 binding of mutants in the basal conformation is that free arrestin in the cytoplasm may be with Mdm2 binding to its receptor and that receptor binding is likely to the Mdm2 from the Arrestins are of signaling that bind the phosphorylated of their receptors with further G protein activation Gurevich V.V. Benovic J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the of receptor binding arrestin a global conformational that the of free and arrestin are V.V. Gurevich E.V. Sci. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, V.V. Gurevich E.V. PubMed Scopus Google Scholar). This conformational change is believed to the interaction for binding partners preferential binding of signaling to the complex (2Lefkowitz R.J. Shenoy S.K. Science. 2005; 308: 512-517Crossref PubMed Scopus (1439) Google Scholar, 9Gurevich V.V. Gurevich E.V. Structure. 2003; 11: 1037-1042Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). rhodopsin signaling is widely the function of visual arrestin, whereas non-visual arrestins 2 and 3 also interact with F. Gurevich V.V. Benovic J.L. PubMed Scopus Google Scholar) and S.A. J. Ferguson S.S. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar), the complex for In the complex and c-Src (5Luttrell 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 (1264) Google Scholar), scaffolds kinase to the activation of ERK1/2 (6Luttrell 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 (704) Google Scholar) and JNK3 (7McDonald 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: 1515-1518Crossref PubMed Google Scholar), and the ubiquitin ligase Mdm2 to the receptor (8Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (718) Google Scholar). activation-dependent JNK3 phosphorylation as a function of arrestin, arrestin3 (7McDonald 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: 1515-1518Crossref PubMed Google Scholar). in arrestin3 as a W.E. McDonald P.H. Field M.E. Davis R.J. Lefkowitz R.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), is not in other Gurevich V.V. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the of free other than receptor is to non-visual arrestins were shown to the nucleus J. Y. G. F. X. F. X. Pei G. Ma L. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), nuclear localization signal were found in their Arrestin3 a in its that its shuttling between the nucleus and the Mdm2 (11Wang P. Wu Y. Ge X. Ma L. Pei G. J. Biol. Chem. 2003; 278: 11648-11653Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and JNK3 (10Scott 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 (169) Google Scholar) in the GPCRs are not transported to the nucleus, these that free arrestin3 in its basal conformation with these two the ability to Mdm2 (11Wang P. Wu Y. Ge X. Ma L. Pei G. J. Biol. Chem. 2003; 278: 11648-11653Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and JNK3 from the nucleus, suggesting that also both Mdm2 and JNK3 in its free In recently shown to regulate of suggesting that J. Y. G. F. X. F. X. Pei G. Ma L. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). all of the the subcellular localization of arrestins from the of (10Scott 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 (169) Google Scholar, 11Wang P. Wu Y. Ge X. Ma L. Pei G. J. Biol. Chem. 2003; 278: 11648-11653Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). of the localization of native visual arrestin and in neurons that that visual arrestin is from the nucleus, whereas in of neurons in cells arrestins and arrestins that a whereas the of the subcellular localization of visual arrestin and arrestin3 and in to the protein the of the of the native arrestins that arrestins are to through the nuclear that in their their to and from the nucleus to of the In of the of arrestins with JNK3 and Mdm2 of in the of arrestin3 and both in visual arrestin the localization of these proteins that arrestins have some of which are not by that they be alternative pathways. that all three arrestins both and pathways to the nucleus, whereas in complex with interaction partners, arrestins 2 and 3 an of visual arrestin is most the ability of protein to JNK3 from the nucleus is suggesting the of in the visual arrestin with in both JNK3 as as the protein to in H. S. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). of of function have is with the binding of the to H. S. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). may the of a complex the and of the interaction P. J. L. J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). two the of all in visual arrestin have the as JNK3 of the of visual arrestin that were not by the be to wild redistribute JNK3 in an Thus, be in the of the visual arrestin with two is by and in the nucleus with its interaction whereas mutants are not in the complex with free to an alternative nuclear export that is not by We used the ability of arrestins to their binding partners from the nucleus to the cytoplasm as a to study their interactions with JNK3 and Mdm2. In with (11Wang P. Wu Y. Ge X. Ma L. Pei G. J. Biol. 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PubMed Scopus Google Scholar) that than signaling proteins interact with the that binding partners likely for the In the binding of signaling proteins to free arrestin may which signaling be arrestin to the receptor. of active arrestin mutants Gurevich V.V. J. Biol. 2005; PubMed Scopus Google Scholar) and mutants in their basal state S.A. Gurevich Gurevich V.V. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (86) Google Scholar) to the conformational of arrestin interactions with JNK3 and Mdm2. is important to that the used the arrestin by its interactions with the of the in some of the of S.A. Gurevich Gurevich V.V. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the likely free arrestin to a conformation to that of arrestin to the which is from the active conformation of arrestin to the S.A. Gurevich V.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). not arrestin the active state to that of arrestin Gurevich V.V. 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In of an of arrestin for Mdm2 when a conformation is to that Mdm2 by arrestin to the receptor to ubiquitin to arrestin active that the of receptor localization of signaling is for their between the nucleus and the cytoplasm is important for proteins that regulate This which and the a of the complex in photoreceptors A. E. P. Y. A. J. 2002; PubMed Scopus Google Scholar), and Mdm2, which and for G. 2001; PubMed Scopus Google Scholar). Thus, the ability of visual arrestin expressed in rod photoreceptors J. PubMed Scopus (126) Google Scholar) and and 3 expressed in (4Gurevich E.V. Benovic J.L. Gurevich V.V. J. Neurochem. 2004; 91: 1404-1416Crossref PubMed Scopus (79) Google Scholar) to dramatically change the of JNK3 and Mdm2 between the nucleus and the cytoplasm may play a role in neuronal survival. that the of between the nucleus and cytoplasm in neuronal likely in localization of signaling with which We L. and G. Pei for and GFP-Mdm2 and for of the