β-Arrestin-mediated Signaling Regulates Protein Synthesis
2008; Elsevier BV; Volume: 283; Issue: 16 Linguagem: Inglês
10.1074/jbc.m710515200
ISSN1083-351X
AutoresScott M. DeWire, Jihee Kim, Erin J. Whalen, Seungkirl Ahn, Minyong Chen, Robert J. Lefkowitz,
Tópico(s)Computational Drug Discovery Methods
ResumoSeven transmembrane receptors (7TMRs) exert strong regulatory influences on virtually all physiological processes. Although it is historically assumed that heterotrimeric G proteins mediate these actions, there is a newer appreciation that β-arrestins, originally thought only to desensitize G protein signaling, also serve as independent receptor signal transducers. Recently, we found that activation of ERK1/2 by the angiotensin receptor occurs via both of these distinct pathways. In this work, we explore the physiological consequences of β-arrestin ERK1/2 signaling and delineate a pathway that regulates mRNA translation and protein synthesis via Mnk1, a protein that both physically interacts with and is activated by β-arrestins. We show that β-arrestin-dependent activation of ERK1/2, Mnk1, and eIF4E are responsible for increasing translation rates in both human embryonic kidney 293 and rat vascular smooth muscle cells. This novel demonstration that β-arrestins regulate protein synthesis reveals that the spectrum of β-arrestin-mediated signaling events is broader than previously imagined. Seven transmembrane receptors (7TMRs) exert strong regulatory influences on virtually all physiological processes. Although it is historically assumed that heterotrimeric G proteins mediate these actions, there is a newer appreciation that β-arrestins, originally thought only to desensitize G protein signaling, also serve as independent receptor signal transducers. Recently, we found that activation of ERK1/2 by the angiotensin receptor occurs via both of these distinct pathways. In this work, we explore the physiological consequences of β-arrestin ERK1/2 signaling and delineate a pathway that regulates mRNA translation and protein synthesis via Mnk1, a protein that both physically interacts with and is activated by β-arrestins. We show that β-arrestin-dependent activation of ERK1/2, Mnk1, and eIF4E are responsible for increasing translation rates in both human embryonic kidney 293 and rat vascular smooth muscle cells. This novel demonstration that β-arrestins regulate protein synthesis reveals that the spectrum of β-arrestin-mediated signaling events is broader than previously imagined. Seven transmembrane-spanning receptors (7TMRs 3The abbreviations used are: 7TMR, seven transmembrane receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; AngII, angiotensin II; VSMC, vascular smooth muscle cell; rVSMC, rat VSMC; HEK, human embryonic kidney; siRNA, small interfering RNA; IP, immunoprecipitation; HA, hemagglutinin; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ANOVA, analysis of variance; JNK, c-Jun N-terminal kinase. /G protein-coupled receptors), the largest class of cell surface receptors, regulate nearly every known physiologic process in mammals. Canonical signaling by 7TMRs involves an agonist-induced change in receptor conformation that causes the activation of heterotrimeric G proteins, leading to second messenger generation and downstream signaling. The termination of this cascade, a process known as desensitization, occurs when G protein-coupled receptor kinases phosphorylate the activated receptor and promote the subsequent binding of β-arrestins, which act sterically to block further G protein activation. However, in recent years a previously unappreciated role of β-arrestins has become evident as distinct transducers of 7TMR signals independent of G proteins. By serving as multiprotein binding scaffolds, β-arrestins facilitate the activation of numerous signaling pathways, including the mitogen-activated protein kinases (MAPKs), c-Src, and Akt (1DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1166) Google Scholar, 2Lefkowitz R.J. Rajagopal K. Whalen E.J. Mol. Cell. 2006; 24: 643-652Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 3Lefkowitz R.J. Shenoy S.K. Science. 2005; 308: 512-517Crossref PubMed Scopus (1439) Google Scholar). The diverse array of cellular outcomes that involve β-arrestin-mediated signaling are just beginning to be appreciated and thus far include chemotaxis (4DeFea K.A. Annu. Rev. Physiol. 2007; 69: 535-560Crossref PubMed Scopus (92) Google Scholar), cardiomyocyte contractility (5Rajagopal K. Whalen E.J. Violin J.D. Stiber J.A. Rosenberg P.B. Premont R.T. Coffman T.M. Rockman H.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16284-16289Crossref PubMed Scopus (188) Google Scholar), and the prevention of apoptosis (6Povsic T.J. Kohout T.A. Lefkowitz R.J. J. Biol. Chem. 2003; 278: 51334-51339Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), among others (2Lefkowitz R.J. Rajagopal K. Whalen E.J. Mol. Cell. 2006; 24: 643-652Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). One of the most well studied targets of β-arrestin-mediated signaling from 7TMRs is the MAPK extracellular-regulated kinase 1/2 (ERK1/2) (7DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (690) 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 (704) Google Scholar). Classically, G protein stimulation via second messenger effectors like protein kinase C (PKC) and protein kinase A promotes ERK1/2 phosphorylation and nuclear translocation to modulate transcription. However, our laboratory and others have found that after β-arrestin-dependent ERK1/2 activation, the kinase remains solely in the cytoplasm (9Ahn S. Shenoy S.K. Wei H. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 35518-35525Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 10Tohgo A. Choy E.W. Gesty-Palmer D. Pierce K.L. Laporte S. Oakley R.H. Caron M.G. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2003; 278: 6258-6267Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar), where it presumably phosphorylates a distinct subset of targets. The elucidation of these cytosolic β-arrestin-ERK1/2 targets has become a focus of recent study. In reviewing the literature on such cytosolic substrates of ERK1/2, we found a number of proteins involved in the modulation of protein synthesis, including MAP kinase-interacting kinase 1 (Mnk1). Because Mnk1 can be activated by a 7TMR (11Ishida M. Ishida T. Nakashima H. Miho N. Miyagawa K. Chayama K. Oshima T. Kambe M. Yoshizumi M. Circ. Res. 2003; 93: 1218-1224Crossref PubMed Scopus (25) Google Scholar), the angiotensin II receptor, we reasoned it might well be a β-arrestin ERK1/2 substrate and that β-arrestins might be involved in signaling to protein synthesis. Protein synthetic regulation is critical for cellular growth and development, and its dysregulation is implicated in numerous diseases such as Alzheimer disease, tumorigenesis, and various proliferative conditions such as neointimal hyperplasia (12Proud C.G. Biochem. J. 2007; 403: 217-234Crossref PubMed Scopus (403) Google Scholar). Multiple cell signaling molecules, including ERK1/2, can elicit changes in the rate of mRNA translation. These situations arise when environmental conditions demand heightened protein synthesis, and cells can respond either by increasing the rate of general translation or that of only a specific subset of mRNAs, the latter typically occurring through sequence-specific factors in the 5′-non-translated region of the message. On the other hand, because nearly all mammalian mRNAs contain a 5′-methylguanosine cap, increasing the assembly of the multiprotein cap binding complex is one way that cells can elicit more general increases in message translation. The major rate-limiting factors in cap-dependent translation are the availability of and mRNA affinity for the cap binding complex member protein eukaryotic translation initiation factor 4E (eIF4E). The amount of free eIF4E available for cap binding is determined by 4E-binding proteins (4EBPs). Only when the 4EBPs are dephosphorylated are they capable of binding eIF4E (12Proud C.G. Biochem. J. 2007; 403: 217-234Crossref PubMed Scopus (403) Google Scholar). The 4EBP kinase that allows for eIF4E release is the mammalian target of rapamycin (mTOR), a protein also implicated in other mRNA translation steps (13Wang X. Proud C.G. Physiol. (Bethesda). 2006; 21: 362-369Crossref PubMed Scopus (542) Google Scholar). The affinity of eIF4E for mRNA is determined by its phosphorylation state: when phosphorylated at serine 209, affinity decreases (14Scheper G.C. van Kollenburg B. Hu J. Luo Y. Goss D.J. Proud C.G. J. Biol. Chem. 2002; 277: 3303-3309Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), a process carried out by Mnk1 (15Fukunaga R. Hunter T. EMBO J. 1997; 16: 1921-1933Crossref PubMed Scopus (559) Google Scholar, 16Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (793) Google Scholar). This decreased affinity for mRNA cap structure seems counterintuitive for a pathway that seeks to increase mRNA translation. However, the link between eIF4E phosphorylation and protein synthesis is well established, leading to the hypothesis that cap affinity needs to be reduced to allow the ribosome to move along the untranslated mRNA (17Scheper G.C. Proud C.G. Eur. J. Biochem. 2002; 269: 5350-5359Crossref PubMed Scopus (264) Google Scholar). Thus, Mnk1, a serine/threonine kinase and substrate of ERK1/2 and p38 (15Fukunaga R. Hunter T. EMBO J. 1997; 16: 1921-1933Crossref PubMed Scopus (559) Google Scholar, 16Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (793) Google Scholar), serves as a key point of convergence for signal transduction pathways regulating protein synthesis. Recently, the potential targeting of Mnk1 for proliferative diseases was described, based on the observation that HER2-positive breast cancer lines show heightened Mnk1 activity (18Chrestensen C.A. Shuman J.K. Eschenroeder A. Worthington M. Gram H. Sturgill T.W. J. Biol. Chem. 2007; 282: 4243-4252Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Using rat vascular smooth muscle cells, Ishida et al. (19Knauf U. Tschopp C. Gram H. Mol. Cell Biol. 2001; 21: 5500-5511Crossref PubMed Scopus (221) Google Scholar) measured the protein synthetic response to angiotensin II (AngII) (11Ishida M. Ishida T. Nakashima H. Miho N. Miyagawa K. Chayama K. Oshima T. Kambe M. Yoshizumi M. Circ. Res. 2003; 93: 1218-1224Crossref PubMed Scopus (25) Google Scholar). By using a chemical inhibitor of Mnk1 kinase activity, the authors found that Mnk1 activity was absolutely required for AngII stimulation of protein synthesis. Also, Mnk1 phosphorylation in response to AngII in these cells was entirely mediated by ERK1/2, and not by p38 (11Ishida M. Ishida T. Nakashima H. Miho N. Miyagawa K. Chayama K. Oshima T. Kambe M. Yoshizumi M. Circ. Res. 2003; 93: 1218-1224Crossref PubMed Scopus (25) Google Scholar). The upstream mediators of this signaling, however, remain unknown. Based on the observations that Mnk1 is a target of cytosolic ERK1/2 (15Fukunaga R. Hunter T. EMBO J. 1997; 16: 1921-1933Crossref PubMed Scopus (559) Google Scholar, 16Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (793) Google Scholar) and can be activated by the AT1AR (11Ishida M. Ishida T. Nakashima H. Miho N. Miyagawa K. Chayama K. Oshima T. Kambe M. Yoshizumi M. Circ. Res. 2003; 93: 1218-1224Crossref PubMed Scopus (25) Google Scholar), we hypothesized that Mnk1 might be a potential downstream target of β-arrestin-mediated ERK1/2 signaling. Thus, we sought to investigate the potential role of β-arrestins in 7TMR signaling to Mnk1 activation and the resulting increase in protein synthesis. Plasmids and Cloning—Mnk1 cDNA was amplified from pEBG3X-Mnk1 (gift from Jonathan Cooper, Fred Hutchinson Cancer Center) with an N-terminal FLAG epitope and cloned into pcDNA3.1. For bacterial expression, pGEX-Mnk1 was a gift from Tony Hunter (Salk Institute). The plasmid encoding HA-AT1AR has been previously described (20Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (555) Google Scholar); AT1ARDRY-AAY mutant was made by site-directed mutagenesis (QuikChange; Stratagene) of the wild type plasmid. For the COS7 studies in supplemental Fig. S2, β-arrestin overexpression plasmids were described previously (21McDonald 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). Reagents—(Sar1, Ile4, Ile8)-AngII was synthesized as described (20Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (555) Google Scholar, 22Miura S. Zhang J. Matsuo Y. Saku K. Karnik S.S. Hypertens. Res. 2004; 27: 765-770Crossref PubMed Scopus (48) Google Scholar) and used at 10 μm in all figures, in accordance with its KD being ∼200-fold less than AngII (22Miura S. Zhang J. Matsuo Y. Saku K. Karnik S.S. Hypertens. Res. 2004; 27: 765-770Crossref PubMed Scopus (48) Google Scholar). AngII (Sigma A9525) was used at 100 nm in all figures. Ro31–8425 (1 μm), U0126 (5 μm), and SB203580 (20 μm) were purchased from Calbiochem. Phospho-Mnk1 and phospho-eIF4E antibodies were purchased from Cell Signaling (2111 and 9741). Total Mnk1 and eIF4E antibodies were purchased from Santa Cruz Biotechnology (SC-6962 and SC-6968). CGP57380 was a gift from Hermann Gram, Novartis (Basel, Switzerland) and was used at 30 μm. Cell Culture—Vascular smooth muscle cells (VSMCs) were prepared from 10–12-week-old male Sprague-Dawley rats (∼250 g) as previously described (11Ishida M. Ishida T. Nakashima H. Miho N. Miyagawa K. Chayama K. Oshima T. Kambe M. Yoshizumi M. Circ. Res. 2003; 93: 1218-1224Crossref PubMed Scopus (25) Google Scholar). VSMCs were cultured in M199 medium with 10% fetal bovine serum and penicillin/streptomycin. rVSMCs were used for signaling and translation assays prior to four passages, due to loss of angiotensin receptor expression and responsiveness over time. Rat vascular smooth muscle cells were used because, unlike mouse VSMCs, they express a tractable amount of AT1AR. HEK-293 and COS7 cells were cultured in minimal essential medium with 10% fetal bovine serum and penicillin/streptomycin as described previously (23Violin J.D. Dewire S.M. Barnes W.G. Lefkowitz R.J. J. Biol. Chem. 2006; 281: 36411-36419Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Transfection of siRNAs in HEK-293s was carried out using Gene Silencer (Gene Therapy Systems) as described previously (24Ahn S. Nelson C.D. Garrison T.R. Miller W.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1740-1744Crossref PubMed Scopus (192) Google Scholar). Silencing achieved for β-arrestins was 70–80%, consistent with previous reports (24Ahn S. Nelson C.D. Garrison T.R. Miller W.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1740-1744Crossref PubMed Scopus (192) Google Scholar, 25Wisler, J. W., Dewire, S. M., Whalen, E. J., Violin, J. D., Drake, M. T., Ahn, S., Shenoy, S. K., and Lefkowitz, R. J. (2007) Proc. Natl. Acad. Sci. U. S. A.Google Scholar). Sequences of human siRNAs used were the following: β-arrestin 1 (5′-AAAGCCUUCUGCGCGGAGAAU-3′), β-arrestin2 (5′-AAGGACCGCAAAGUGUUUGUG-3′), and for supplemental Fig. S2 the second β-arrestin2 siRNA was (5′-CCAACCUCAUUGAAUUUGA-3′). Pooled human Mnk1 siRNA was purchased from Santa Cruz Biotechnology (sc-39106). Transfection of plasmids for all other experiments in HEK-293 cells was done using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. For siRNA experiments in rat cells, siRNAs were the following: β-arrestin2-1 (5′-AAGGACCGGAAAGTGTTTGTG-3′) and β-arrestin2-2 (5′-ACCAACCTCATTGAATTCGA-3′). Pooled murine Mnk1 siRNA was purchased from Santa Cruz Biotechnology (sc-39107). Transfection was achieved using 60 μl of Lipofectamine/10-cm dish of rVSMCs at 90% confluence according to the manufacturer's protocol. Immunoprecipitations and Western Blots—For IPs, HeLa cells (500 μg of total protein/sample) or mouse tissue homogenate (1 mg/sample) in radioimmune precipitation lysis buffer was pre-cleared with protein A beads and then incubated with A1CT antibody (26Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar) or an equivalent volume of preimmune serum from the same rabbit. 24 h later, bound proteins were precipitated with protein A-agarose beads and washed several times with radioimmune precipitation buffer. Western blots were carried out according to standard protocols. β-Arrestins were detected with a monoclonal antibody (BD 610550). For the antigen-blocking experiment, A1CT-coated beads were preincubated with 100 ng of recombinant ratβ-arr1 (or equivalent volume of buffer) and washed thoroughly before proceeding with IP. For epitope-tagged proteins, HA antibody was Covance 12CA5 and FLAG antibody was M2 (Sigma F9291). For agonist-stimulated IPs the time point was 30 min. Detection of β-arr2 in rVSMCs was performed with A2CT antibody (26Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar). Protein Synthesis Measurements—Rat VSMCs or HEK-293 cells were seeded in 12-well plates. After 24 h the medium was replaced with serum-free medium. 24 h later, 2 uCi/ml of [3H]leucine (NET460005MC; Amersham Biosciences) and either phosphate-buffered saline (for nonstimulated conditions), 100 nm AngII, or 10 μm SII were added to the medium. 24 h later, medium was removed and newly generated protein was quantitated as previously described (11Ishida M. Ishida T. Nakashima H. Miho N. Miyagawa K. Chayama K. Oshima T. Kambe M. Yoshizumi M. Circ. Res. 2003; 93: 1218-1224Crossref PubMed Scopus (25) Google Scholar). Mnk1 Activation Occurs in an ERK-dependent but PKC-independent Manner—In HEK-293 cells, AT1AR activation leads to ERK1/2 phosphorylation by both G protein/PKC and β-arrestin-dependent mechanisms (9Ahn S. Shenoy S.K. Wei H. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 35518-35525Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 20Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (555) Google Scholar, 27Ahn S. Wei H. Garrison T.R. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 7807-7811Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In the presence of a PKC inhibitor, all AngII-induced ERK1/2 phosphorylation is carried out by the β-arrestin2-specific signaling arm (27Ahn S. Wei H. Garrison T.R. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 7807-7811Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Thus, if AngII stimulation leads to phosphorylation of a protein that is resistant to PKC inhibition, but is sensitive to ERK inhibition, it is a candidate β-arrestin signaling target. We used this rationale as a screening strategy to identify potential β-arrestin-ERK1/2 signaling targets among a panel of known cytosolic ERK1/2 substrates. One protein, Mnk1, showed the expected pattern of a β-arrestin target but was very difficult to detect at endogenous levels in HEK-293 cells. Thus, we generated stable cells that express both the AT1AR and Mnk1 to further investigate this protein. These cells were pretreated with vehicle (Me2SO) or chemical inhibitors of PKC (Ro31–8425), MEK (U0126), p38 (SB203580), or a combination of inhibitors and then stimulated with AngII as shown in Fig. 1. AngII-induced Mnk1 phosphorylation was primarily dependent on ERK1/2 rather than p38, especially at later time points, consistent with previously published data for Mnk1 activation in rat vascular smooth muscle cells (11Ishida M. Ishida T. Nakashima H. Miho N. Miyagawa K. Chayama K. Oshima T. Kambe M. Yoshizumi M. Circ. Res. 2003; 93: 1218-1224Crossref PubMed Scopus (25) Google Scholar). In the early portion of the curve, there was a slight effect of p38 inhibition, PKC inhibition, and MEK inhibition, suggesting that all three effectors can contribute to Mnk1 phosphorylation at these shorter time points. The virtual superimposition of the p38 and PKC inhibition curves suggests that p38 and PKC may lie in the same pathway, which is parallel to and independent of the ERK1/2 pathway. Further support for this idea is provided by the additive nature of these curves. As can be seen in Fig. 1, C and D, the Mnk1 phosphorylation that is resistant to MEK inhibition (U0126) added to either the PKC-inhibited curve (Ro31) or p38-inhibited curve (SB) accounts for the total Mnk1 phosphorylation activity, drawn in blue lines. Similarly, the subtraction of the MEK-inhibited curve from the total activity curve results in a curve that perfectly overlies either the PKC- or p38-inhibited curve (Fig. 1D). At later times, Mnk1 phosphorylation is largely resistant to p38 and PKC inhibition, which strongly suggests G protein independence. These kinase inhibitor studies indicate that in HEK-293 cells activation of Mnk1 occurs through two pathways, one of which relies upon G protein activation, PKC, and p38 and the other which is likely mediated by β-arrestin2-dependent ERK1/2 signaling. Mnk1 and eIF4E Activation Depends upon Expression of β-Arrestin2—We used siRNA to directly test the involvement of β-arrestins in the AngII-induced Mnk1 activation in the same cell system used for Fig. 1. β-arrestin1 silencing had a small effect at early time points compared with control siRNA but had no effect at 30 and 60 min, times when Mnk1 was fully activated (Fig. 2A). However, β-arrestin2 siRNA inhibited Mnk1 phosphorylation at all time points tested by ∼50%. The pMnk1 signal that persisted after β-arrestin2 siRNA was largely the result of PKC-dependent, MEK-independent signaling, as predicted from the kinase inhibitor studies in Fig. 1 (data not shown). Western blotting for β-arrestins demonstrates that siRNA silencing was >70%, consistent with the amount of silencing previously reported (24Ahn S. Nelson C.D. Garrison T.R. Miller W.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1740-1744Crossref PubMed Scopus (192) Google Scholar). A second siRNA sequence targeting a different portion of the β-arrestin2 mRNA was also tested and a similar result was obtained (supplemental Fig. S1A), thus confirming the necessity of β-arrestin2 expression for full Mnk1 activation. Similarly, overexpression of β-arrestin2, but not β-arrestin1, in Cos7 cells, which have low levels of endogenous β-arrestins, resulted in an increase in Mnk1 phosphorylation (supplemental Fig. S1B). To confirm that β-arrestin-mediated Mnk1 activation indeed continues down the pathway to eIF4E, we used a similar approach (Fig. 2B). We found that eIF4E phosphorylation in HEK-293 cells stimulated by AngII was significantly inhibited by β-arrestin2 siRNA (Fig. 2B), indicating that β-arrestins are capable of signaling to eIF4E, the rate-limiting step in cap-dependent translation. β-Arrestins Physically Interact with Mnk1—β-Arrestins are known to serve as scaffolds for the activation of numerous signaling molecules (1DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1166) Google Scholar), including ERK in complex with Raf-1 and Mek-1. Based on the above observations, we speculated that similar to other MAPK pathway members, Mnk1 might be found in a complex with β-arrestins. Thus, we performed immunoprecipitations using an antibody that recognizes both β-arrestin1 and -2 from either HeLa cells (Fig. 3A) or mouse spleen (Fig. 3B) and immunoblotted for Mnk1. We found that Mnk1 was ∼10-fold more abundant in samples immunoprecipitated with the β-arrestin antibody versus an equivalent amount of pre-immune serum from the same rabbit. These results were consistent across all mouse tissues tested, including skeletal muscle, heart, kidney, and liver (data not shown). Further, when the β-arrestin antibody-coated beads were preincubated with the immunizing antigen, the Mnk1-β-arrestin co-immunoprecipitation was blocked (data not shown). These results confirm a specific physical interaction of these proteins at endogenous expression levels in cultured cells as well as in vivo. To determine whether there was β-arrestin isoform specificity for the physical interaction with Mnk1, we transfected HEK-293 cells with Mnk1 and epitope-tagged β-arrestin1 or -2 (Fig. 3C). We then immunoprecipitated the β-arrestins with epitope antibody and compared the levels of Mnk1. Although both β-arrestins bound more Mnk1 than the control condition, β-arrestin2 bound much more robustly than β-arrestin1. A reciprocal immunoprecipitation of Mnk1 followed by β-arrestin Western blotting was also performed and confirmed the interaction (Fig. 3D). In other systems, such as ERK1/2 and JNK3 (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 (704) Google Scholar, 21McDonald 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), the functional significance of β-arrestin scaffolding is to activate the target kinase by giving it access to its upstream activator. Thus, we explored whether agonist stimulation could modulate the interaction of βarrestin-2 and Mnk1 using a co-immunoprecipitation assay. We observed a moderate, yet significant, increase in the amount of Mnk1 immunoprecipitated by β-arrestin2 after stimulation of the AT1AR with either AngII or SII for 30 min (Fig. 3E). Also, the cellular stores of Mnk1 bound by β-arrestin2 were phosphorylated upon receptor stimulation, as confirmed by immunoblotting for phospho-Mnk1 in the β-arrestin2 immunoprecipitation (supplemental Fig. S3). These data suggest a dynamic interaction of β-arrestin2 and Mnk1 that can be regulated by the receptor and that may facilitate Mnk1 phosphorylation. Alternatively, the basal interaction of these two proteins alone may be important for facilitating Mnk1 phosphorylation. β-Arrestin-mediated Signaling to Mnk1—As mentioned above, when AngII binds the extracellular surface of the AT1AR it stimulates the independent coupling of receptor to both G protein- and β-arrestin-mediated signaling pathways (20Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (555) Google Scholar). To circumvent the issues of discerning which pathway is eliciting a particular response, we utilized two reagents that specifically activate only the β-arrestin signaling arm. S1I4I8-AngII (SII) is a mutated peptide analog of the natural ligand that induces a receptor conformation that is unable to activate Gαq yet maintains the capacity to activate ERK1/2 in a β-arrestin-dependent manner (20Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (555) Google Scholar, 22Miura S. Zhang J. Matsuo Y. Saku K. Karnik S.S. Hypertens. Res. 2004; 27: 765-770Crossref PubMed Scopus (48) Google Scholar). Similarly, the AT1AR-DRY/AAY mutated receptor, when treated with AngII, only activates β-arrestin signaling and does not couple to G proteins (20Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (555) Google Scholar, 28Gaborik Z. Jagadeesh G. Zhang M. Spat A. Catt K.J. Hunyady L. Endocrinology. 2003; 144: 2220-2228Crossref PubMed Scopus (104) Google Scholar). Hence, these reagents allow for clearer investigation of the role of β-arrestin in Mnk1 activation. In HEK-293 cells, SII stimulated significant Mnk1 phosphorylation, ∼22% as well as AngII. We believe that this lower response, though statistically significant, is likely because the SII peptide leads to exclusive, albeit less efficient, coupling of the receptor to the β-arrestin signaling pathway. The SII mechanism for Mnk1 activation was dependent upon ERK1/2, but not PKC, activity (Fig. 4A). Similarly, the AT1AR-DRY/AAY, which is uncoupled from G proteins yet still recruits β-arrestins and leads to ERK1/2 activation, also stimulated Mnk1 activation (Fig. 4B). We also observed that eIF4E phosphorylation could be stimulated via SII-AngII treatment of the wild type AT1AR or by AngII stimulation of the AT1AR-DRY/AAY mutated receptor (data not shown). β-Arrestin2 and Mnk1 Dependence of AngII-induced Protein Synthesis—The terminal event in the Mnk1 signaling pathway is the stimulation of protein synthesis. As has been observed in other systems, AngII treatment led to an increase in protein synthesis by ∼50% in AT1AR-stable HEK-293 cells. Interestingly, SII stimulation also resulted in significant increases in protein synthesis over basal rates, with a magnitude of about half that observed with AngII (Fig. 5A). We confirmed that this pathway required β-arrestin2 expression by using siRNA transfection and found that AngII-induced translation was reduced and SII-induced translation was completely inhibited in the presence of a β-arrestin2-silencing RNA. Importantly, the basal rate of protein synthesis did not change upon transfection of β-arrestin2 siRNA. We also perfor
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