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The Extracellular Regulated Kinase-1 (ERK1) Controls Regulated α-Secretase-mediated Processing, Promoter Transactivation, and mRNA Levels of the Cellular Prion Protein

2011; Elsevier BV; Volume: 286; Issue: 33 Linguagem: Inglês

10.1074/jbc.m110.208249

ISSN

1083-351X

Autores

Moustapha Cissé, Eric Duplan, Marie‐Victoire Guillot‐Sestier, Joaquim Rumigny, Charlotte Bauer, Gilles Pagès, Hans‐Dieter Orzechowski, Barbara E. Slack, Frédéric Checler, Bruno Vincent,

Tópico(s)

Neurological diseases and metabolism

Resumo

The α-secretases A disintegrin and metalloprotease 10 (ADAM10) and ADAM17 trigger constitutive and regulated processing of the cellular prion protein (PrPc) yielding N1 fragment. The latter depends on protein kinase C (PKC)-coupled M1/M3 muscarinic receptor activation and subsequent phosphorylation of ADAM17 on its intracytoplasmic threonine 735. Here we show that regulated PrPc processing and ADAM17 phosphorylation and activation are controlled by the extracellular-regulated kinase-1/MAP-ERK kinase (ERK1/MEK) cascade. Thus, reductions of ERK1 or MEK activities by dominant-negative analogs, pharmacological inhibition, or genetic ablation all impair N1 secretion, whereas constitutively active proteins increase N1 recovery in the conditioned medium. Interestingly, we also observed an ERK1-mediated enhanced expression of PrPc. We demonstrate that the ERK1-associated increase in PrPc promoter transactivation and mRNA levels involve transcription factor AP-1 as a downstream effector. Altogether, our data identify ERK1 as an important regulator of PrPc cellular homeostasis and indicate that this kinase exerts a dual control of PrPc levels through transcriptional and post-transcriptional mechanisms. The α-secretases A disintegrin and metalloprotease 10 (ADAM10) and ADAM17 trigger constitutive and regulated processing of the cellular prion protein (PrPc) yielding N1 fragment. The latter depends on protein kinase C (PKC)-coupled M1/M3 muscarinic receptor activation and subsequent phosphorylation of ADAM17 on its intracytoplasmic threonine 735. Here we show that regulated PrPc processing and ADAM17 phosphorylation and activation are controlled by the extracellular-regulated kinase-1/MAP-ERK kinase (ERK1/MEK) cascade. Thus, reductions of ERK1 or MEK activities by dominant-negative analogs, pharmacological inhibition, or genetic ablation all impair N1 secretion, whereas constitutively active proteins increase N1 recovery in the conditioned medium. Interestingly, we also observed an ERK1-mediated enhanced expression of PrPc. We demonstrate that the ERK1-associated increase in PrPc promoter transactivation and mRNA levels involve transcription factor AP-1 as a downstream effector. Altogether, our data identify ERK1 as an important regulator of PrPc cellular homeostasis and indicate that this kinase exerts a dual control of PrPc levels through transcriptional and post-transcriptional mechanisms. IntroductionThe cellular prion protein is responsible for transmissible spongiform encephalopathies (TSE) 4The abbreviations used are: TSEtransmissible spongiform encephalopathiesPrPccellular prion proteinCAconstitutively activeMEFmouse embryonic fibroblastsDNdominant-negativePDBuphorbol ester 12,13-dibutyrateTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. that can affect several mammals including humans (1Aguzzi A. Calella A.M. Physiol. Rev. 2009; 89: 1105-1152Crossref PubMed Scopus (377) Google Scholar). The common central event in prion pathologies is the conversion of the host-encoded cellular prion protein (PrPc) into a pathogenic, insoluble, and partially protease-resistant isoform (PrPsc) that aggregates and accumulates in specific brain areas, triggers neuronal degeneration, and ultimately leads to dementia and death (2Prusiner S.B. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13363-13383Crossref PubMed Scopus (5088) Google Scholar).Besides its implication in the development of TSE, it was postulated that PrPc could fulfill physiological functions. Indeed, it has been suggested that PrPc could participate in lymphocyte activation, cellular adhesion processes, neuronal growth, synaptogenesis, cellular signaling, and cell survival/apoptosis (for review, see Ref. 3Linden R. Martins V.R. Prado M.A. Cammarota M. Izquierdo I. Brentani R.R. Physiol. Rev. 2008; 88: 673-728Crossref PubMed Scopus (474) Google Scholar).The cellular prion protein is physiologically cleaved at the 111/112 peptidyl bond, thereby generating the so-called N1 amino-terminal fragment and its carboxyl-terminal membrane tethered counterpart named C1 (4Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Interestingly, an additional cleavage occurring at the 90/91 peptide bond in Creutzfeldt-Jakob disease-affected brains yielding fragments referred to as N2 and C2 (4Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar) preserves the 106–126 PrPc domain. This peptide has been shown to be neurotoxic in vitro (5Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (893) Google Scholar) and in vivo (6Ettaiche M. Pichot R. Vincent J.P. Chabry J. J. Biol. Chem. 2000; 275: 36487-36490Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Therefore, understanding the mechanisms underlying PrPc processing could provide a means to interfere with PrPc-dependent effects in both physiological and pathological conditions.We and others previously established that PrPc metabolism could be either constitutive or regulated by protein kinase C (PKC) (7Vincent B. Paitel E. Frobert Y. Lehmann S. Grassi J. Checler F. J. Biol. Chem. 2000; 275: 35612-35616Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and that the disintegrins ADAM10 and ADAM17 were directly responsible for the constitutive and PKC-regulated processing of PrPc, respectively (8Vincent B. Paitel E. Saftig P. Frobert Y. Hartmann D. de Strooper B. Grassi J. Lopez-Perez E. Checler F. J. Biol. Chem. 2001; 276: 37743-37746Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Laffont-Proust I. Faucheux B.A. Hässig R. Sazdovitch V. Simon S. Grassi J. Hauw J.J. Moya K.L. Haïk S. FEBS Lett. 2005; 579: 6333-6337Crossref PubMed Scopus (46) Google Scholar). Moreover, we demonstrated that ADAM9 acted as an upstream activator of ADAM10 activity (10Cissé M.A. Sunyach C. Lefranc-Jullien S. Postina R. Vincent B. Checler F. J. Biol. Chem. 2005; 280: 40624-40631Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). We very recently showed that stimulation of the M1/M3 muscarinic receptors with several classical or more receptor-specific agonists promotes isoform-specific PKC-dependent processing of the cellular prion protein via catalytic activation of ADAM17 upon phosphorylation on its threonine 735 (11Alfa Cissé M. Sunyach C. Slack B.E. Fisher A. Vincent B. Checler F. J. Neurosci. 2007; 27: 4083-4092Crossref PubMed Scopus (46) Google Scholar, 12Alfa Cissé M. Louis K. Braun U. Mari B. Leitges M. Slack B.E. Fisher A. Auberger P. Checler F. Vincent B. Mol. Cell. Neurosci. 2008; 39: 400-410Crossref PubMed Scopus (18) Google Scholar). Moreover, we demonstrated that the conventional PKCα, the novel PKCδ and PKCϵ, but not the atypical PKCζ isoforms participate in the PDBu- or carbachol-stimulated N1 production (12Alfa Cissé M. Louis K. Braun U. Mari B. Leitges M. Slack B.E. Fisher A. Auberger P. Checler F. Vincent B. Mol. Cell. Neurosci. 2008; 39: 400-410Crossref PubMed Scopus (18) Google Scholar). Analysis of the amino acid sequence encompassing the intracytoplasmic Thr-735 of ADAM17 indicated that this residue is not part of the canonical (K/R)R(K/R/Q)GT(F/L/V)X consensus sequence that is required for phosphorylation by PKCα, -δ, or -ϵ isoforms, suggesting that PKC indirectly mediated phosphorylation of ADAM17 and thus, that N1 production required an additional kinase. Cautious analysis of mouse and human ADAM17 sequences revealed that the Thr-735 of ADAM17 was located in an APQTPG sequence corresponding to a canonical ERK1-targeted motif (XPXTPX).We show here that ERK1 is absolutely required for PDBU- and carbachol-induced processing of PrPc and that the inhibition of the ERK1 pathway totally impairs phosphorylation of ADAM17 on its Thr-735 and thereby, N1 production. In addition, we establish that, besides its crucial involvement in PrPc-regulated processing, ERK1 modulates PrPc protein and mRNA levels by a mechanism implying AP-1-dependent transcriptional control.DISCUSSIONAlthough the importance of the cellular prion protein in the development of TSEs has been extensively documented (see Ref. 1Aguzzi A. Calella A.M. Physiol. Rev. 2009; 89: 1105-1152Crossref PubMed Scopus (377) Google Scholar for review), less is known concerning the physiological roles fulfilled by this protein. Because PrPc knock-out mice are viable and fertile with no apparent dysfunction (32Büeler H. Fischer M. Lang Y. Bluethmann H. Lipp H.P. DeArmond S.J. Prusiner S.B. Aguet M. Weissmann C. Nature. 1992; 356: 577-582Crossref PubMed Scopus (1432) Google Scholar), it has long been thought that PrPc does not participate in any vital physiological process. However, subsequent studies showed that PrPc could contribute to various biological functions such as lymphocyte activation, cell adhesion, synaptic transmission, and apoptosis (see Ref. 3Linden R. Martins V.R. Prado M.A. Cammarota M. Izquierdo I. Brentani R.R. Physiol. Rev. 2008; 88: 673-728Crossref PubMed Scopus (474) Google Scholar for review). Noteworthy, PrPc is subjected to a physiological proteolytic breakdown at its 111/112 peptide bond (4Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). There exist strong evidence that disintegrins ADAM10 and ADAM17 are responsible for the constitutive and PKC-regulated pathway (8Vincent B. Paitel E. Saftig P. Frobert Y. Hartmann D. de Strooper B. Grassi J. Lopez-Perez E. Checler F. J. Biol. Chem. 2001; 276: 37743-37746Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 14Cissé M.A. Gandreuil C. Hernandez J.F. Martinez J. Checler F. Vincent B. Biochem. Biophys. Res. Commun. 2006; 347: 254-260Crossref PubMed Scopus (22) Google Scholar), respectively. It should be noted, however, that a few studies failed to find evidence for an involvement of ADAM proteases on PrPc processing but this discrepancy may be likely explained by distinct experimental conditions in which recombinant soluble enzyme were used or because only constitutive processing was examined (33Taylor D.R. Parkin E.T. Cocklin S.L. Ault J.R. Ashcroft A.E. Turner A.J. Hooper N.M. J. Biol. Chem. 2009; 284: 22590-22600Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 34Endres K. Mitteregger G. Kojro E. Kretzschmar H. Fahrenholz F. Neurobiol. Dis. 2009; 36: 233-241Crossref PubMed Scopus (40) Google Scholar).This proteolytic event further complicates our understanding of PrPc-related biological effects and raises the question whether this proteolytic attack represents a degradation process aimed at clearing full-length PrPc or indeed illustrates a maturation step yielding biologically active metabolites. Concerning the C1 fragment, we previously showed that its overexpression potentiates staurosporine-induced apoptosis in vitro (23Sunyach C. Cisse M.A. da Costa C.A. Vincent B. Checler F. J. Biol. Chem. 2007; 282: 1956-1963Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) as PrPc does (35Paitel E. Alves da Costa C. Vilette D. Grassi J. Checler F. J. Neurochem. 2002; 83: 1208-1214Crossref PubMed Scopus (62) Google Scholar, 36Paitel E. Fahraeus R. Checler F. J. Biol. Chem. 2003; 278: 10061-10066Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 37Paitel E. Sunyach C. Alves da Costa C. Bourdon J.C. Vincent B. Checler F. J. Biol. Chem. 2004; 279: 612-618Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). As far as the N1 fragment is concerned, the first indirect demonstration of an N1-related function was first brought by in vivo studies showing that transgenic mice expressing N-terminal-truncated PrPc constructs displayed exacerbated neurodegeneration and that this phenotype strictly requires the depletion of the 32-121 N-terminal sequence, thereby suggesting a putative N1-associated neuroprotective effect (38Shmerling D. Hegyi I. Fischer M. Blättler T. Brandner S. Götz J. Rülicke T. Flechsig E. Cozzio A. von Mering C. Hangartner C. Aguzzi A. Weissmann C. Cell. 1998; 93: 203-214Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 39Radovanovic I. Braun N. Giger O.T. Mertz K. Miele G. Prinz M. Navarro B. Aguzzi A. J. Neurosci. 2005; 25: 4879-4888Crossref PubMed Scopus (74) Google Scholar, 40Li A. Barmada S.J. Roth K.A. Harris D.A. J. Neurosci. 2007; 27: 852-859Crossref PubMed Scopus (40) Google Scholar). We brought the definitive and direct proof that N1 indeed conveys neuroprotection by showing that treatment with the recombinant N1 peptide or stimulation of N1 production by muscarinic agonists invariably protect cells from hypoxia-induced p53-dependent apoptosis and ischemia in vitro and in vivo (41Guillot-Sestier M.V. Sunyach C. Druon C. Scarzello S. Checler F. J. Biol. Chem. 2009; 284: 35973-35986Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Noteworthy, it has been very recently established that ablation of neuronal PrPc triggers a chronic demyelinating polyneuropathy due to impaired peripheral myelin maintenance (42Bremer J. Baumann F. Tiberi C. Wessig C. Fischer H. Schwarz P. Steele A.D. Toyka K.V. Nave K.A. Weis J. Aguzzi A. Nat. Neurosci. 2010; 13: 310-318Crossref PubMed Scopus (306) Google Scholar). This was an additional demonstration of a PrPc-associated pathology of the peripheral nervous system unrelated to TSEs. Interestingly, chronic demyelinating polyneuropathy can be rescued by PrPc variants that undergo disintegrin-mediated proteolytic processing at the 111/112 site but not by cleavage-resistant variants. Therefore, the N1 and/or C1 fragments derived from the physiological processing of PrPc are essential for myelin maintenance (42Bremer J. Baumann F. Tiberi C. Wessig C. Fischer H. Schwarz P. Steele A.D. Toyka K.V. Nave K.A. Weis J. Aguzzi A. Nat. Neurosci. 2010; 13: 310-318Crossref PubMed Scopus (306) Google Scholar). Altogether, and because we established that the N1-associated neuroprotective function was dominant over the C1-mediated toxic effect (41Guillot-Sestier M.V. Sunyach C. Druon C. Scarzello S. Checler F. J. Biol. Chem. 2009; 284: 35973-35986Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), these data suggests that up-regulating α-secretase processing of PrPc could convey beneficial effects in normal conditions as well as in pathological conditions unrelated to prion diseases. Therefore, a possible track would be to activate the PKC-regulated α-secretase hydrolysis of PrPc at the 111/112 peptide bond. Phosphorylation of ADAM17 on its intracytoplasmic threonine residue at position 735 is necessary to activate this pathway (11Alfa Cissé M. Sunyach C. Slack B.E. Fisher A. Vincent B. Checler F. J. Neurosci. 2007; 27: 4083-4092Crossref PubMed Scopus (46) Google Scholar). This amino acid is embedded in a consensus phosphorylation site targeted by ERK1/2. We therefore examined whether this kinase was directly responsible for ADAM17 activation through Thr-735 phosphorylation and, thereby, could act as a direct and functional activator of PKC-regulated α-secretase processing of PrPc. Four lines of data indicate that it is indeed the case. First, inhibition of MEK, a kinase occurring upstream in the MEK/ERK signaling cascade reduces α-secretase JMV2770-hydrolyzing activity and drastically impairs N1 secretion and ADAM17 phosphorylation. Second, transient overexpression of wild-type or constitutively active forms of these two kinases in both human cells and murine primary neurons activate PDBu- and carbachol-dependent N1 production, whereas dominant-negative kinases reduce the recovery of N1 in the conditioned medium. Third, ERK1 deletion impairs PKC-regulated α-secretase activity and abolishes PDBu- and carbachol-dependent N1 secretion. Fourth, ERK1 cDNA transfection in ERK1−/− cells rescues carbachol-dependent secretion of N1. Overall, we propose that the M1/M3 muscarinic receptors activation triggers ADAM17-dependent processing of PrPc at the 111/112 site after initiating the MEK/ERK pathway depicted in green in Fig. 11. ERK1-mediated control of PrPc processing is not the only example of such an ERK1-controlled proteolytic conversion. Thus, the neurotrophin receptor TrkA shedding is also stimulated through an ERK1-dependent phosphorylation of ADAM17 at its Thr-735 in both in vitro assay and cultured cells (43Díaz-Rodríguez E. Montero J.C. Esparís-Ogando A. Yuste L. Pandiella A. Mol. Biol. Cell. 2002; 13: 2031-2044Crossref PubMed Scopus (256) Google Scholar). Moreover, this molecular event induces maturation and trafficking of ADAM17 to the plasma membrane thereby explaining the gain of activity of the ERK1-dependent threonine 735-phosphorylated ADAM17 form (44Soond S.M. Everson B. Riches D.W. Murphy G. J. Cell Sci. 2005; 118: 2371-2380Crossref PubMed Scopus (200) Google Scholar). However, this cascade is not ubiquitous because we recently demonstrated that α-secretase-regulated processing of β-amyloid precursor protein indeed involved ADAM17 and PKCs but in an ERK-1-independent manner (45Cissé M. Braun U. Leitges M. Fisher A. Pagès G. Checler F. Vincent B. Mol. Cell. Neurosci. 2011; 47: 223-232Crossref PubMed Scopus (29) Google Scholar).Interestingly, PrPc-like immunoreactivity was significantly reduced in ERK1−/− MEFs. This led us to examine the putative involvement of ERK1 in the regulation of PrPc expression. We established that the observed ERK1-dependent reduction in PrPc immunoreactivity results from a direct effect on PrPc transcription. First, ERK1 depletion lowers PrPc promoter transactivation and mRNA levels. Second, transient expression of dominant-negative forms of ERK1 or MEK1 significantly reduced PrPc promoter activation and mRNA levels in HEK293 cells. Third, constitutively active MEK1 triggers the opposite effect. How does ERK signaling modulate PrPc promoter transactivation? Several clues came from molecular cloning and characterization of the human, bovine, and mouse PRNP gene (25Funke-Kaiser H. Theis S. Behrouzi T. Thomas A. Scheuch K. Zollmann F.S. Paterka M. Paul M. Orzechowski H.D. J. Mol. Med. 2001; 79: 529-535Crossref PubMed Scopus (15) Google Scholar, 26Mahal S.P. Asante E.A. Antoniou M. Collinge J. Gene. 2001; 268: 105-114Crossref PubMed Scopus (41) Google Scholar, 46Westaway D. Cooper C. Turner S. Da Costa M. Carlson G.A. Prusiner S.B. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 6418-6422Crossref PubMed Scopus (91) Google Scholar, 47Inoue S. Tanaka M. Horiuchi M. Ishiguro N. Shinagawa M. J. Vet. Med. Sci. 1997; 59: 175-183Crossref PubMed Scopus (53) Google Scholar) that revealed consensus binding sequences for several transcription factors, namely Sp1, AP-1, AP-2, c-Rel, Nkx2–5, Ets-1, and NF-AT. Several of these putative effectors proved to be functional for modulating PrPc transcription in pathophysiological situations such as Sp1-dependent control of copper homeostasis (48Bellingham S.A. Coleman L.A. Masters C.L. Camakaris J. Hill A.F. J. Biol. Chem. 2009; 284: 1291-1301Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) or presenilin-dependent p53-mediated PrPc apoptosis (49Vincent B. Sunyach C. Orzechowski H.D. St. Georges-Hyslop P. Checler F. J. Neurosci. 2009; 29: 6752-6760Crossref PubMed Scopus (46) Google Scholar).We clearly showed that transcription factor AP-1, but not Sp1, contributes, at least partly, to the ERK1-dependent control of PrPc. Thus, cDNA constructs harboring various 5′ deletions indicate that the ablation of the promoter region containing the AP-1 binding site, but not Sp1-related GC box elements, strongly impaired PrPc promoter transactivation. Accordingly, inactivating mutations of the AP-1 binding sequence in the full-length promoter significantly reduce its promoter transactivation in two different cell lines. However, the fact that the extent of inhibition triggered by the −284/−131 deletion was higher than that resulting from the AP-1 site mutation (55–70% compared with 30% inhibition, Fig. 10C), suggests that additional transcription factors, the binding sites of which would be located within position −284/−131, may also modulate PrPc expression. It is noteworthy that binding sites for Ets-1, NF-AT, AP-2, YY1, and E4BP4 have been delineated in such a region (25Funke-Kaiser H. Theis S. Behrouzi T. Thomas A. Scheuch K. Zollmann F.S. Paterka M. Paul M. Orzechowski H.D. J. Mol. Med. 2001; 79: 529-535Crossref PubMed Scopus (15) Google Scholar, 50Burgess S.T. Shen C. Ferguson L.A. O'Neill G.T. Docherty K. Hunter N. Goldmann W. J. Biol. Chem. 2009; 284: 6716-6724Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Altogether, these data clearly established an important functional contribution of ERK1 and AP-1 in the positive control of PrPc transcription (Fig. 11, orange arrowheads).Because ERK increases both PrPc expression and the production of its α-secretase-derived catabolite N1, one can question whether the increase of N1 is just the consequence of an upstream elevation of its precursor PrPc or if ERK triggers dual and fully independent phenotypes. Close examination of some of the data indicates that the latter hypothesis is more likely. Thus, PDBu- and carbachol-stimulated N1 production is abolished in ERK-deficient cells, whereas PrPc expression remains similar in treated and untreated null fibroblasts. This shows that agonist-dependent phosphorylation of ADAM17 and subsequent N1 production is impaired by ERK1 ablation, whereas reduction of PrPc still stands. Second, ERK-1 rescues the carbachol-induced increase in N1 production without affecting PrPc expression levels. This set of data reflects a clear discrimination between the two events that could be likely accounted for by distinct time frames for the two processes, with an early phase involving ADAM17 phosphorylation and enhanced PrPc catabolism and a later phase needing a necessary delay to activate the transcriptional machinery.Another interesting aspect of this work concerns the putative functional cross-talk between PrPc and the MEK/ERK signaling pathway. Thus, PrPc cross-linking or overexpression triggers ERK phosphorylation in neuronal and non-neuronal cells (51Schneider B. Mutel V. Pietri M. Ermonval M. Mouillet-Richard S. Kellermann O. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13326-13331Crossref PubMed Scopus (155) Google Scholar, 52Monnet C. Gavard J. Mège R.M. Sobel A. FEBS Lett. 2004; 576: 114-118Crossref PubMed Scopus (47) Google Scholar, 53Stuermer C.A. Langhorst M.F. Wiechers M.F. Legler D.F. von Hanwehr S.H. Guse A.H. Plattner H. FASEB J. 2004; 18: 1731-1733Crossref PubMed Scopus (123) Google Scholar, 54Shyu W.C. Lin S.Z. Chiang M.F. Ding D.C. Li K.W. Chen S.F. Yang H.I. Li H. J. Neurosci. 2005; 25: 8967-8977Crossref PubMed Scopus (101) Google Scholar, 55Pantera B. Bini C. Cirri P. Paoli P. Camici G. Manao G. Caselli A. J. Neurochem. 2009; 110: 194-207Crossref PubMed Scopus (55) Google Scholar). Other studies established that the specific PrPc ligand stress-inducible protein 1 (STI1) (56Zanata S.M. Lopes M.H. Mercadante A.F. Hajj G.N. Chiarini L.B. Nomizo R. Freitas A.R. Cabral A.L. Lee K.S. Juliano M.A. de Oliveira E. Jachieri S.G. Burlingame A. Huang L. Linden R. Brentani R.R. Martins V.R. EMBO J. 2002; 21: 3307-3316Crossref PubMed Scopus (370) Google Scholar) likely accounts for PrPc-dependent ERK phosphorylation in neurons and astrocytes (57Lopes M.H. Hajj G.N. Muras A.G. Mancini G.L. Castro R.M. Ribeiro K.C. Brentani R.R. Linden R. Martins V.R. J. Neurosci. 2005; 25: 11330-11339Crossref PubMed Scopus (213) Google Scholar, 58Caetano F.A. Lopes M.H. Hajj G.N. Machado C.F. Arantes C.P. Magalhães A.C. Vieira Mde P. Américo T.A. Massensini A.R. Priola S.A. Vorberg I. Gomez M.V. Linden R. Prado V.F. Martins V.R. Prado M.A. J. Neurosci. 2008; 28: 6691-6702Crossref PubMed Scopus (79) Google Scholar, 59Arantes C. Nomizo R. Lopes M.H. Hajj G.N. Lima F.R. Martins V.R. Glia. 2009; 57: 1439-1449Crossref PubMed Scopus (53) Google Scholar). Therefore, the MEK/ERK cascade could self-stimulate its own signaling via the increase of PrPc expression and thereby explain both ERK and PrPc-related protective phenotypes documented in certain cell systems (3Linden R. Martins V.R. Prado M.A. Cammarota M. Izquierdo I. Brentani R.R. Physiol. Rev. 2008; 88: 673-728Crossref PubMed Scopus (474) Google Scholar, 60Han B.H. Holtzman D.M. J. Neurosci. 2000; 20: 5775-5781Crossref PubMed Google Scholar, 61Cavanaugh J.E. Jaumotte J.D. Lakoski J.M. Zigmond M.J. J. Neurosci. Res. 2006; 84: 1367-1375Crossref PubMed Scopus (75) Google Scholar). Along with this hypothesis, one could envision that part of an ERK1-dependent PrPc-associated antiapoptotic function could be mediated by an ERK-1-mediated increase in N1 because we recently characterized this catabolite as a neuroprotective factor both in vitro and in vivo (41Guillot-Sestier M.V. Sunyach C. Druon C. Scarzello S. Checler F. J. Biol. Chem. 2009; 284: 35973-35986Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar).In other cases, PrPc has been shown to be toxic and trigger p53-dependent cell death (35Paitel E. Alves da Costa C. Vilette D. Grassi J. Checler F. J. Neurochem. 2002; 83: 1208-1214Crossref PubMed Scopus (62) Google Scholar, 36Paitel E. Fahraeus R. Checler F. J. Biol. Chem. 2003; 278: 10061-10066Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 37Paitel E. Sunyach C. Alves da Costa C. Bourdon J.C. Vincent B. Checler F. J. Biol. Chem. 2004; 279: 612-618Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In this case, the ERK-1-mediated production of N1 could be seen as a cellular response to counterbalance PrPc-mediated toxicity. This hypothesis seems to be supported by the observation that in cells overexpressing a PrPc construct lacking the 32–134 amino-terminal sequence (that encompasses most of the N1 domain), ERK phosphorylation is still stimulated but leads to cell death, oxidative injury, and neurodegeneration (62Gavín R. Braun N. Nicolas O. Parra B. Ureña J.M. Mingorance A. Soriano E. Torres J.M. Aguzzi A. del Río J.A. FEBS Lett. 2005; 579: 4099-4106Crossref PubMed Scopus (27) Google Scholar, 63Pietri M. Caprini A. Mouillet-Richard S. Pradines E. Ermonval M. Grassi J. Kellermann O. Schneider B. J. Biol. Chem. 2006; 281: 28470-28479Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 64Nicolas O. Gavín R. Braun N. Ureña J.M. Fontana X. Soriano E. Aguzzi A. del Río J.A. FASEB J. 2007; 21: 3107-3117Crossref PubMed Scopus (33) Google Scholar). This could be due to the fact that whereas N-terminal-truncated PrPc is increased, its neuroprotective counterpart N1 is lacking.Overall, our study suggests that under normal conditions, ERK1 contributes to cell survival and neuroprotection through the augmentation of N1 secretion (41Guillot-Sestier M.V. Sunyach C. Druon C. Scarzello S. Checler F. J. Biol. Chem. 2009; 284: 35973-35986Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) via an increase in ADAM17 activity and PrPc expression. It is likely that in pathological situations, N1 could temporarily compensate for the PrP scrapie-associated toxicity and cell death and thereby partly could explain the long asymptomatic time course of TSEs.Note Added in ProofWe want to make clear that in some of the gels appearing in the article, lanes have been reorganized for purposes of clarity. All initial gels have been seen and reviewed by referees. All quantifications have been performed on initial gels before any rearrangements. As explained in the text, quantifications of data are means of several independent experiments, and gels are only representative of one of these experiments. IntroductionThe cellular prion protein is responsible for transmissible spongiform encephalopathies (TSE) 4The abbreviations used are: TSEtransmissible spongiform encephalopathiesPrPccellular prion proteinCAconstitutively activeMEFmouse embryonic fibroblastsDNdominant-negativePDBuphorbol ester 12,13-dibutyrateTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. that can affect several mammals including humans (1Aguzzi A. Calella A.M. Physiol. Rev. 2009; 89: 1105-1152Crossref PubMed Scopus (377) Google Scholar). The common central event in prion pathologies is the conversion of the host-encoded cellular prion protein (PrPc) into a pathogenic, insoluble, and partially protease-resistant isoform (PrPsc) that aggregates and accumulates in specific brain areas, triggers neuronal degeneration, and ultimately leads to dementia and death (2Prusiner S.B. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13363-13383Crossref PubMed Scopus (5088) Google Scholar).Besides its implication in the development of TSE, it was postulated that PrPc could fulfill physiological functions. Indeed, it has been suggested that PrPc could participate in lymphocyte activation, cellular adhesion processes, neuronal growth, synaptogenesis, cellular signaling, and cell survival/apoptosis (for review, see Ref. 3Linden R. Martins V.R. Prado M.A. Cammarota M. Izquierdo I. Brentani R.R. Physiol. Rev. 2008; 88: 673-728Crossref PubMed Scopus (474) Google Scholar).The cellular prion protein is physiologically cleaved at the 111/112 peptidyl bond, thereby generating the so-called N1 amino-terminal fragment and its carboxyl-terminal membrane tethered counterpart named C1 (4Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Interestingly, an additional cleavage occurring at the 90/91 peptide bond in Creutzfeldt-Jakob disease-affected brains yielding fragments referred to as N2 and C2 (4Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar) preserves the 106–126 PrPc domain. This peptide has been shown to be neurotoxic in vitro (5Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (893) Google Scholar) and in vivo (6Ettaiche M. Pichot R. Vincent J.P. Chabry J. J. Biol. Chem. 2000; 275: 36487-36490Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Therefore, understanding the mechanisms underlying PrPc processing could provide a means to interfere with PrPc-dependent effects in both physiological and pathological conditions.We and others previously established that PrPc metabolism could be either constitutive or regulated by protein kinase C (PKC) (7Vincent B. Paitel E. Frobert Y. Lehmann S. Grassi J. Checler F. J. Biol. Chem. 2000; 275: 35612-35616Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and that the disintegrins ADAM10 and ADAM17 were directly responsible for the constitutive and PKC-regulated processing of PrPc, respectively (8Vincent B. Paitel E. Saftig P. Frobert Y. Hartmann D. de Strooper B. Grassi J. Lopez-Perez E. Checler F. J. Biol. Chem. 2001; 276: 37743-37746Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Laffont-Proust I. Faucheux B.A. Hässig R. Sazdovitch V. Simon S. Grassi J. Hauw J.J. Moya K.L. Haïk S. FEBS Lett. 2005; 579: 6333-6337Crossref PubMed Scopus (46) Google Scholar). Moreover, we demonstrated that ADAM9 acted as an upstream activator of ADAM10 activity (10Cissé M.A. Sunyach C. Lefranc-Jullien S. Postina R. Vincent B. Checler F. J. Biol. Chem. 2005; 280: 40624-40631Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). We very recently showed that stimulation of the M1/M3 muscarinic receptors with several classical or more receptor-specific agonists promotes isoform-specific PKC-dependent processing of the cellular prion protein via catalytic activation of ADAM17 upon phosphorylation on its threonine 735 (11Alfa Cissé M. Sunyach C. Slack B.E. Fisher A. Vincent B. Checler F. J. Neurosci. 2007; 27: 4083-4092Crossref PubMed Scopus (46) Google Scholar, 12Alfa Cissé M. Louis K. Braun U. Mari B. Leitges M. Slack B.E. Fisher A. Auberger P. Checler F. Vincent B. Mol. Cell. Neurosci. 2008; 39: 400-410Crossref PubMed Scopus (18) Google Scholar). Moreover, we demonstrated that the conventional PKCα, the novel PKCδ and PKCϵ, but not the atypical PKCζ isoforms participate in the PDBu- or carbachol-stimulated N1 production (12Alfa Cissé M. Louis K. Braun U. Mari B. Leitges M. Slack B.E. Fisher A. Auberger P. Checler F. Vincent B. Mol. Cell. Neurosci. 2008; 39: 400-410Crossref PubMed Scopus (18) Google Scholar). Analysis of the amino acid sequence encompassing the intracytoplasmic Thr-735 of ADAM17 indicated that this residue is not part of the canonical (K/R)R(K/R/Q)GT(F/L/V)X consensus sequence that is required for phosphorylation by PKCα, -δ, or -ϵ isoforms, suggesting that PKC indirectly mediated phosphorylation of ADAM17 and thus, that N1 production required an additional kinase. Cautious analysis of mouse and human ADAM17 sequences revealed that the Thr-735 of ADAM17 was located in an APQTPG sequence corresponding to a canonical ERK1-targeted motif (XPXTPX).We show here that ERK1 is absolutely required for PDBU- and carbachol-induced processing of PrPc and that the inhibition of the ERK1 pathway totally impairs phosphorylation of ADAM17 on its Thr-735 and thereby, N1 production. In addition, we establish that, besides its crucial involvement in PrPc-regulated processing, ERK1 modulates PrPc protein and mRNA levels by a mechanism implying AP-1-dependent transcriptional control.

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