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• W2028097766 abstract "Huntington disease (HD) is caused by an abnormal expanded polyglutamine repeat in the huntingtin protein. Insulin-like growth factor-1 is of particular interest in HD because it strongly inhibits polyQ-huntingtin-induced neurotoxicity. This neuroprotective effect involves the phosphorylation of huntingtin at Ser421 by the prosurvival kinase Akt (Humbert, S., Bryson, E. A., Cordelières, F. P., Connors, N. C., Datta, S. R., Finkbeiner, S., Greenberg, M. E., and Saudou, F. (2002) Dev. Cell 2, 831–837). Here, we report that Akt inhibits polyQ-huntingtin-induced toxicity in the absence of phosphorylation of huntingtin at Ser421, suggesting that Akt also acts on other downstream effector(s) to prevent neuronal death in HD. We show that this survival effect involves the ADP-ribosylation factor-interacting protein arfaptin 2, the levels of which are increased in HD patients. Akt phosphorylated arfaptin 2 at Ser260. Lack of phosphorylation of arfaptin 2 at this site substantially modified its subcellular distribution and increased neuronal death and intranuclear inclusions caused by polyQ-huntingtin. In contrast, arfaptin 2 had a neuroprotective effect on striatal neurons when phosphorylated by Akt. This effect is mediated through the proteasome, as phosphorylated arfaptin 2 inhibited the blockade of the proteasome induced by polyQ-huntingtin. This study points out a new mechanism by which Akt promotes neuroprotection in HD, emphasizing the potential therapeutic interest of this pathway in the disease. Huntington disease (HD) is caused by an abnormal expanded polyglutamine repeat in the huntingtin protein. Insulin-like growth factor-1 is of particular interest in HD because it strongly inhibits polyQ-huntingtin-induced neurotoxicity. This neuroprotective effect involves the phosphorylation of huntingtin at Ser421 by the prosurvival kinase Akt (Humbert, S., Bryson, E. A., Cordelières, F. P., Connors, N. C., Datta, S. R., Finkbeiner, S., Greenberg, M. E., and Saudou, F. (2002) Dev. Cell 2, 831–837). Here, we report that Akt inhibits polyQ-huntingtin-induced toxicity in the absence of phosphorylation of huntingtin at Ser421, suggesting that Akt also acts on other downstream effector(s) to prevent neuronal death in HD. We show that this survival effect involves the ADP-ribosylation factor-interacting protein arfaptin 2, the levels of which are increased in HD patients. Akt phosphorylated arfaptin 2 at Ser260. Lack of phosphorylation of arfaptin 2 at this site substantially modified its subcellular distribution and increased neuronal death and intranuclear inclusions caused by polyQ-huntingtin. In contrast, arfaptin 2 had a neuroprotective effect on striatal neurons when phosphorylated by Akt. This effect is mediated through the proteasome, as phosphorylated arfaptin 2 inhibited the blockade of the proteasome induced by polyQ-huntingtin. This study points out a new mechanism by which Akt promotes neuroprotection in HD, emphasizing the potential therapeutic interest of this pathway in the disease. Huntington disease (HD) 1The abbreviations used are: HD, Huntington disease; IGF-1, insulin-like growth factor-1; GST, glutathione S-transferase; Akt-ca, constitutively activated Akt; SGK, serum- and glucocorticoid-induced kinase; GFP, green fluorescent protein; NIIs, neuronal intranuclear inclusions; MT, microtubule; UPS, ubiquitin/proteasome system; ANOVA, analysis of variance. is a fatal neurodegenerative disorder characterized by involuntary movements, personality changes, and dementia (1Young A.B. J. Clin. Investig. 2003; 111: 299-302Crossref PubMed Scopus (92) Google Scholar). The dominantly inherited causal gene encodes the huntingtin protein, which contains an abnormal polyglutamine expansion (polyQ) in HD patients. HD develops when this expansion exceeds 35 glutamine residues. There is a strong inverse correlation between the number of glutamines and the age at onset of the disease. HD leads to the selective dysfunction and death of striatal neurons in the brain. The presence of neuritic and intranuclear inclusions in neurons is also characteristic of the disease. There is currently no effective treatment to prevent or to delay disease progression, and death usually occurs 10–20 years after the appearance of the first clinical symptoms. We recently reported that the insulin-like growth factor-1 (IGF-1)/Akt signaling pathway could have a beneficial effect in HD (2Humbert S. Bryson E.A. Cordelières F.P. Connors N.C. Datta S.R. Finkbeiner S. Greenberg M.E. Saudou F. Dev. Cell. 2002; 2: 831-837Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). In striatal neurons, the most vulnerable neurons in HD, IGF-1 completely blocks polyQ-huntingtin-induced toxicity (2Humbert S. Bryson E.A. Cordelières F.P. Connors N.C. Datta S.R. Finkbeiner S. Greenberg M.E. Saudou F. Dev. Cell. 2002; 2: 831-837Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). This effect is mediated by the serine/threonine kinase Akt, which directly phosphorylates polyQ-huntingtin at Ser421, inhibiting the toxic properties of polyQ-huntingtin. These findings reveal that the IGF-1/Akt signaling pathway is of particular interest in HD, as it directly affects the protein that causes the disease. We observed that Akt is altered in the brains of HD patients and particularly in the striatum, showing that this kinase plays an important role in HD (2Humbert S. Bryson E.A. Cordelières F.P. Connors N.C. Datta S.R. Finkbeiner S. Greenberg M.E. Saudou F. Dev. Cell. 2002; 2: 831-837Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Akt is a potent prosurvival kinase that exerts its effects by phosphorylating key substrates such as components of the cell death machinery (3Brazil D.P. Yang Z.Z. Hemmings B.A. Trends Biochem. Sci. 2004; 29: 233-242Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar). Akt could be particularly important in HD, as it abrogates the toxicity of polyQ-huntingtin by phosphorylating Ser421. It also acts on other substrates that promote survival by general mechanisms or by mechanisms relevant to HD. The phosphorylation by Akt of substrates other than huntingtin may be important in the latter stages of the disease. During the pathological process in HD, huntingtin is cleaved by several proteases, including caspases, calpains, and other unidentified proteases (4Wellington C.L. Singaraja R. Ellerby L. Savill J. Roy S. Leavitt B. Cattaneo E. Hackam A. Sharp A. Thornberry N. Nicholson D.W. Bredesen D.E. Hayden M.R. J. Biol. Chem. 2000; 275: 19831-19838Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 5Kim Y.J. Yi Y. Sapp E. Wang Y. Cuiffo B. Kegel K.B. Qin Z.H. Aronin N. DiFiglia M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12784-12789Crossref PubMed Scopus (327) Google Scholar, 6Gafni J. Ellerby L.M. J. Neurosci. 2002; 22: 4842-4849Crossref PubMed Google Scholar, 7Goffredo D. Rigamonti D. Tartari M. De Micheli A. Verderio C. Matteoli M. Zuccato C. Cattaneo E. J. Biol. Chem. 2002; 277: 39594-39598Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8Lunkes A. Lindenberg K.S. Ben-Haiem L. Weber C. Devys D. Landwehrmeyer G.B. Mandel J.L. Trottier Y. Mol. Cell. 2002; 10: 259-269Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). This cleavage generates short N-terminal amino acid fragments that contain the pathological polyglutamine expansion and that are more toxic to neurons than full-length huntingtin (4Wellington C.L. Singaraja R. Ellerby L. Savill J. Roy S. Leavitt B. Cattaneo E. Hackam A. Sharp A. Thornberry N. Nicholson D.W. Bredesen D.E. Hayden M.R. J. Biol. Chem. 2000; 275: 19831-19838Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 9Gafni J. Hermel E. Young J.E. Wellington C.L. Hayden M.R. Ellerby L.M. J. Biol. Chem. 2004; 279: 20211-20220Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Some of these short N-terminal fragments do not contain the Ser421 phosphorylation site, suggesting that the direct neuroprotective effect of Akt on huntingtin is lost during proteolysis (10Li S.H. Li X.J. Trends Genet. 2004; 20: 146-154Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). We report here that Akt blocks the polyglutamine-dependent neuronal death and inclusion formation that are induced by an N-terminal fragment of huntingtin that does not contain Ser421. We demonstrate that this Ser421 huntingtin-independent mechanism involves the ADP-ribosylation factor-interacting protein arfaptin 2. Phosphorylation of arfaptin 2 at Ser260 by Akt promoted neuronal survival and decreased intranuclear inclusion formation in a neuronal model of HD. This demonstrates that arfaptin 2 is neuroprotective in HD. Finally, we show that phosphorylated arfaptin 2 inhibits the polyQ-huntingtin-induced blockade of the proteasome. Constructs—The vectors encoding β-galactosidase, hemagglutinin-tagged huntingtin N-terminal fragments 171-17 and 171-68, (exon 1 of huntingtin with 17 glutamines (exon 1-17) and 68 glutamines (exon 1-68), glutathione S-transferase (GST)-fused human huntingtin-(384–467), constitutively activated Akt (Akt-ca), inactivated Akt, and hemagglutinin-tagged Akt have been described previously (2Humbert S. Bryson E.A. Cordelières F.P. Connors N.C. Datta S.R. Finkbeiner S. Greenberg M.E. Saudou F. Dev. Cell. 2002; 2: 831-837Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 11Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4946) Google Scholar, 12Saudou F. Finkbeiner S. Devys D. Greenberg M.E. Cell. 1998; 95: 55-66Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar). A bacterial expression plasmid encoding GST-fused arfaptin 2 (13Peters P.J. Ning K. Palacios F. Boshans R.L. Kazantsev A. Thompson L.M. Woodman B. Bates G.P. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 240-245Crossref PubMed Scopus (43) Google Scholar) was used as a template to generate plasmid encoding the C-terminal fragment of arfaptin 2. A mammalian expression plasmid (pcDNA3.1/GS) encoding full-length human arfaptin 2 fused to a His6 tag was purchased from Invitrogen (GeneStorm). The arfaptin 2 mutants for Akt phosphorylation sites were generated by QuikChange site-directed mutagenesis (Stratagene) using the mammalian or bacterial plasmid of arfaptin 2 as a non-mutated parental template and complementary oligonucleotides containing the desired mutation for S260A arfaptin 2 (5′-CGTCGACTTGAGGCTGCCCAGGCCACTTTC-3′) and for S260D arfaptin 2 (5′-CGTGGTCGACTTGAGGATGCCCAGGCCACTTTC-3′). Cell Culture and Transfection—Primary cultures of striatal neurons were prepared from embryonic day 17 Sprague-Dawley rats and transfected at 4 days in vitro by a modified calcium phosphate technique (12Saudou F. Finkbeiner S. Devys D. Greenberg M.E. Cell. 1998; 95: 55-66Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar). The mouse neuroblastoma NG108-15, human embryonic kidney 293, and the monkey fibroblast-like kidney COS-7 cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum (Invitrogen) (or serum-starved when indicated) and antibiotics (50 units/ml penicillin and 50 μg/ml streptomycin) and transfected by the calcium phosphate technique. Kinase Assays—Kinase assays were performed as described (2Humbert S. Bryson E.A. Cordelières F.P. Connors N.C. Datta S.R. Finkbeiner S. Greenberg M.E. Saudou F. Dev. Cell. 2002; 2: 831-837Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 14Rangone H. Poizat G. Troncoso J. Ross C.A. MacDonald M.E. Saudou F. Humbert S. Eur. J. Neurosci. 2004; 19: 273-279Crossref PubMed Scopus (112) Google Scholar). 293T cells were transfected with hemagglutinin-Akt vectors; soluble protein extracts were recovered; and hemagglutinin-Akt was immunoprecipitated. Recombinant SGK (Upstate Biotechnology, Inc.) was used. GST-fused proteins were produced in BL21 competent strains and purified with glutathione-Sepharose-coupled beads. Kinases and substrates were incubated for 30 min at 30 °C with 5 μCi of [γ-32P]ATP. The reaction products were resolved by 12% SDS-PAGE and stained with Coomassie Blue, and 32P-labeled proteins were visualized by autoradiography. Two-dimensional Phosphopeptide Mapping—After in vitro phosphorylation by Akt in the presence of [γ-32P]ATP, two-dimensional phosphopeptide mapping of wild-type or S260A arfaptin 2 was performed as described previously (15Girault J-A. Hemmings Jr., H.C. Williams K.R. Nairn A.C. Greengard P. J. Biol. Chem. 1989; 264: 21748-21759Abstract Full Text PDF PubMed Google Scholar). GST-fused wild-type and S620A arfaptin 2 were incubated with thrombin protease (Amersham Biosciences) following the manufacturer's instructions to remove the GST tag. After in vitro phosphorylation by Akt, two-dimensional phosphopeptide mapping of wild-type or S260A arfaptin 2 was performed (15Girault J-A. Hemmings Jr., H.C. Williams K.R. Nairn A.C. Greengard P. J. Biol. Chem. 1989; 264: 21748-21759Abstract Full Text PDF PubMed Google Scholar). Briefly, gel pieces containing phosphorylated proteins were excised, destained, and dried. Arfaptin 2 was then digested with tosylphenylalanyl chloromethyl ketone-treated trypsin (75 μg/ml; Worthington) in 25 mm ammonium carbonate buffer (pH 8) at 37 °C for 12–16 h. Samples were dried, and peptides were collected in 10 μl of electrophoresis buffer (10% glacial acetic acid and 1% pyridine (pH 3.5)). Samples were spotted 4 cm from the bottom and 5 cm from the right side of a thin-layer chromatography sheet (Analtech Inc.) together with basic fuschin dye. First-dimension electrophoresis was carried out at pH 3.5 for 3 h at 350 V, and second-dimension chromatography was performed on dried sheets in chromatography buffer (37.5% pyridine, 25% butanol, and 7.5% acetic acid) for 4 h. Phosphopeptides were visualized with a PhosphorImager (Storm 820, Amersham Biosciences). Antibody against Arfaptin 2 Phosphorylated at Ser260—Phosphopeptide CRDAGTRGRLEpSAQA was synthesized, coupled to keyhole limpet hemocyanin (Neosystem), and injected into rabbits. Polyclonal antibody to the keyhole limpet hemocyanin-coupled peptide was obtained and affinity-purified by two chromatography steps (Neosystem). Briefly, the serum was filtered (0.22-μm filter), and following an addition of 1 m Tris (pH 8.0) up to a final concentration of 100 mm, it was applied to a SulfoLink column (Pierce) coupled to the non-phosphorylated peptide. The flow-through was collected, concentrated, and applied to the phosphopeptide column. Elution was performed with 100 mm glycine buffer (pH 2.7). Cell and Tissue Extracts—Cells were lysed 2 days post-transfection with Nonidet P-40 lysis buffer (20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 2 mm EGTA, 1% Nonidet P-40, 10 mm β-glycerophosphate, 5 mm NaF, 1 mm sodium inorganic pyrophosphate, 2 mm dithiothreitol, 1 mm sodium vanadate, and 100 μm phenylmethylsulfonyl fluoride) for 20 min at 4 °C. Soluble and insoluble Nonidet P-40 fractions were separated after centrifugation at 10,000 rpm for 15 min at 4 °C. Human tissues were from the Harvard Brain Tissue Resource Center (Belmont, MA). Brain samples were homogenized in Nonidet P-40 lysis buffer and cleared by centrifugation at 6000 × g for 15 min at 4 °C. 50 μg of homogenates were subjected to Western blot analysis. Samples 1–5 correspond to brain numbers 4741, 4744, 4751, 4797, and 4740, respectively, as numbered by the Harvard Brain Tissue Resource Center. Quantification of Western blots was performed and is expressed relative to actin levels. Human biopsies were procured following the guidelines recommended by the National Institutes of Health. Proteins were loaded onto 10% SDS-PAGE; transferred to polyvinylidene difluoride membrane; and immunoblotted with anti-arfaptin 2 (1:1000) (13Peters P.J. Ning K. Palacios F. Boshans R.L. Kazantsev A. Thompson L.M. Woodman B. Bates G.P. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 240-245Crossref PubMed Scopus (43) Google Scholar), anti-arfaptin 2/POR1 (1:100; N19, Santa Cruz Biotechnology, Inc.), and anti-β-actin (1:5000; AC15, Sigma) antibodies. Immunofluorescence—Transfected cells were grown on laminin- and/or poly-d-lysine-coated glass coverslip, fixed with 4% paraformaldehyde for 20 min, and incubated with the following primary antibodies: anti-arfaptin 2 (1:200) (13Peters P.J. Ning K. Palacios F. Boshans R.L. Kazantsev A. Thompson L.M. Woodman B. Bates G.P. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 240-245Crossref PubMed Scopus (43) Google Scholar), anti-His tag (1:500; Cell Signaling Technology), anti-GM130 (1:100, BD Biosciences), and fluorescein isothiocyanate-conjugated anti-α-tubulin (1:100, Sigma). Pictures of fixed cells were captured with a three-dimensional deconvolution imaging system. Analysis of Arfaptin 2 Effect on Proteasome Activity in Cells—GFPu-1 cells (16Bence N.F. Sampat R.M. Kopito R.R. Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1821) Google Scholar) were transfected with constructs of interest. DsRed2-C1 DNA (Clontech) was included in the transfection mixture at a 1:5 ratio. After 72 h, cells were trypsinized and analyzed in a BD Biosciences FACS-Calibur flow cytometer. A minimum of 104 Discosoma sp. red fluorescent protein-transfected cells were analyzed for green fluorescent protein (GFP) fluorescence under each condition using CellQuest software. The levels of fluorescence were standardized to the exon 1-17 condition. The results from three independent experiments are presented as median values of green fluorescence histogram plots. Measurement of Neuronal Survival and Intranuclear Inclusions— Four days post-plating, primary cultures of striatal neurons were transfected with wild-type huntingtin or polyQ-huntingtin and GFP to identify the transfected cells. To be certain that each neuron synthesizing GFP also expressed the huntingtin construct, transfections were performed by the modified calcium phosphate technique with a high ratio of DNA to GFP DNA (10:1) (2Humbert S. Bryson E.A. Cordelières F.P. Connors N.C. Datta S.R. Finkbeiner S. Greenberg M.E. Saudou F. Dev. Cell. 2002; 2: 831-837Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Under these circumstances, >95% of the GFP-positive neurons also expressed the huntingtin construct (data not shown). GFP-positive neurons were scored by fluorescence microscopy in a blinded manner 16 and 36 h post-transfection. Cell death occurring within the GFP-positive cells was determined as the difference in the number of surviving neurons between the two time points and is expressed as a percentage of cell survival. For intranuclear inclusion scoring, striatal neurons were transfected with vectors of interest and a plasmid encoding β-galactosidase (10:1 ratio). Neurons were fixed 5 days post-transfection, immunostained, and analyzed for the presence of ubiquitin-positive intranuclear inclusions (anti-β-galactosidase antibody (1:300), 5 Prime → 3 Prime, Inc.; and anti-ubiquitin antibody (1:100), Dako Corp.). Each graph represents at least two to three independent experiments performed in duplicate or triplicate. Each bar in a given graph corresponds to the scoring of ∼2000 neurons in neuronal survival experiments and to 500 neurons for inclusion scoring. Data were submitted to complete statistical analyses (see figure legends). Akt Exerts a Neuroprotective Effect Independently of Ser421 Phosphorylation in Huntingtin—We tested whether Akt could promote neuroprotection independently of Ser421 phosphorylation. We used an in vitro neuronal model of HD to determine whether Akt could block the neuronal toxicity induced by an N-terminal fragment of huntingtin not containing Ser421. In primary cultures of striatal neurons, fragments of huntingtin containing the abnormal polyglutamine expansion induce neuronal death and the formation of neuronal intranuclear inclusions (12Saudou F. Finkbeiner S. Devys D. Greenberg M.E. Cell. 1998; 95: 55-66Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar). We transfected striatal neurons with constructs encoding the first 171 amino acids of huntingtin containing 17 glutamine residues (wild-type, construct 171-17) or 68 glutamine residues (mutant, construct 171-68) and with a construct encoding Akt-ca and analyzed neuronal survival (Fig. 1A). As expected, construct 171-68 induced a statistically significant decrease in neuronal survival compared with construct 171-17. However, Akt completely inhibited neuronal death induced by huntingtin fragment 171-68 (Fig. 1A). We then analyzed the effect of Akt on the formation of neuronal intranuclear inclusions (NIIs). We found that Akt significantly decreased the percentage of neurons containing NIIs (Fig. 1B). In vitro phosphorylation assays showed that neither fragment 171-17 nor fragment 171-68 was phosphorylated by Akt-ca (data not shown). This suggests that the protective effects of Akt are probably not due to phosphorylation between amino acids 1 and 171 of huntingtin. We have previously demonstrated that part of the neuroprotective effect of IGF-1 is mediated through the phosphorylation of huntingtin at Ser421 (2Humbert S. Bryson E.A. Cordelières F.P. Connors N.C. Datta S.R. Finkbeiner S. Greenberg M.E. Saudou F. Dev. Cell. 2002; 2: 831-837Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). We show here that Akt protected striatal neurons from polyQ-huntingtin-induced toxicity not only by directly phosphorylating huntingtin, but also by phosphorylating other substrates that in turn modulate polyQ-huntingtin-induced toxicity. This indirect effect of Akt led to inhibition of both the death of striatal neurons and the formation of intranuclear inclusions (Fig. 1). Therefore, Akt may act on a substrate that directly controls polyQ-huntingtin-induced toxicity. To identify such an Akt substrate, we screened data bases for proteins containing an Akt consensus phosphorylation site (17Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1399) Google Scholar). We found, among many others, arfaptin 2, which has been shown to regulate polyQ-huntingtin aggregation (13Peters P.J. Ning K. Palacios F. Boshans R.L. Kazantsev A. Thompson L.M. Woodman B. Bates G.P. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 240-245Crossref PubMed Scopus (43) Google Scholar). Arfaptin 2 Is Up-regulated in HD—The striatum is the most affected region in HD. To determine whether arfaptin 2 is modified in the pathological situation, we analyzed the levels of arfaptin 2 in human striatal samples from control (CT), grade 3 HD (HD3), and grade 4 HD (HD4) patients (Fig. 2A). Brain extracts were analyzed using anti-arfaptin 2/POR1 antibody (upper panel) and anti-β-actin antibody as a control for protein levels (lower panel). Arfaptin 2 levels were higher in brain extracts from HD patients than in those from control individuals (Fig. 2B). This agrees with previous work on HD transgenic mouse brains showing up-regulation of arfaptin 2 (13Peters P.J. Ning K. Palacios F. Boshans R.L. Kazantsev A. Thompson L.M. Woodman B. Bates G.P. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 240-245Crossref PubMed Scopus (43) Google Scholar) and further suggests its involvement in HD. Arfaptin 2 Is a Substrate of Akt—Arfaptin 2 has a putative Akt phosphorylation site at Ser260 (Fig. 3A). To test whether Akt phosphorylates arfaptin 2 in vitro, we incubated GST-fused arfaptin 2 with Akt-ca or inactivated Akt in the presence of [γ-32P]ATP. Akt-ca phosphorylated arfaptin 2, whereas inactivated Akt did not (Fig. 3B). To demonstrate that Ser260 is phosphorylated by Akt-ca, we generated a GST-fused C-terminal fragment of arfaptin 2 (amino acids 249–341) with either Ser260 or a Ser-to-Ala mutation at this position (S260A). The C-terminal fragment of arfaptin 2 was no longer phosphorylated by Akt-ca when Ser260 was replaced with Ala (Fig. 3B), even though the amount of the GST-fused protein was similar. We carried out two-dimensional phosphopeptide mapping of full-length wild-type and S260A arfaptin 2 to confirm Ser260 as a site of phosphorylation of arfaptin 2 (Fig. 3C). The major phosphopeptide observed in wild-type arfaptin 2 (see arrows) was not seen when Ser260 was mutated to Ala, suggesting Ser260 as the main site of phosphorylation of arfaptin 2 by Akt. To unequivocally identify Ser260 as an Akt phosphorylation site, we raised a polyclonal antibody that specifically recognizes arfaptin 2 phosphorylated at Ser260. As shown in Fig. 3D, the antibody against arfaptin 2 phosphorylated at Ser260 recognized arfaptin 2 only when coexpressed with Akt-ca, but failed to recognize the same protein with the S260A mutation. These results show that Akt phosphorylates arfaptin 2 at Ser260 in cells and that arfaptin 2 is a substrate of Akt. We investigated whether SGK phosphorylates arfaptin 2 because this kinase has a protective effect by phosphorylating huntingtin at Ser421 after IGF-1 activation (14Rangone H. Poizat G. Troncoso J. Ross C.A. MacDonald M.E. Saudou F. Humbert S. Eur. J. Neurosci. 2004; 19: 273-279Crossref PubMed Scopus (112) Google Scholar). Unlike huntingtin, arfaptin 2 was not phosphorylated by SGK, indicating that Akt specifically phosphorylates arfaptin 2 at Ser260 (Fig. 3E). Phosphorylation of Arfaptin 2 Modifies Its Cellular Distribution—Arfaptin 2 is located mainly in the perinuclear region of Chinese hamster ovary cells, in particular around the microtubule (MT)-organizing center and in cytoplasmic vesicular structures (13Peters P.J. Ning K. Palacios F. Boshans R.L. Kazantsev A. Thompson L.M. Woodman B. Bates G.P. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 240-245Crossref PubMed Scopus (43) Google Scholar). To investigate whether phosphorylation alters the distribution of arfaptin 2, we transfected NG108-15 neuroblastoma cells with His-tagged wild-type or S260A arfaptin 2. We used immunofluorescence to compare the distribution of arfaptin 2 and the Golgi marker protein GM130 (Fig. 4A). The transfected wild-type and endogenous arfaptin 2 proteins appeared in the cytoplasm as diffuse punctate staining and partially co-localized with GM130 in the perinuclear region. However, the distribution of S260A arfaptin 2 was very different. The protein was redistributed to bundle structures in the cytoplasm and was no longer detected in Golgi bodies or vesicles. To ensure that this effect was due to loss of phosphorylation at Ser260, we assessed the effect of constitutive phosphorylation of Ser260 by replacing it with Asp (S260D). S260D arfaptin 2 had the same subcellular localization as arfaptin 2 in neuroblastoma cells (Fig. 4A) and in striatal neurons (data not shown). S260A arfaptin 2 also re-localized into bundle structures in neuritic extensions of striatal neurons (Fig. 4B). Quantification of bundle formation in neuroblastoma cells and neurons demonstrated that it was specifically associated with loss of phosphorylation at Ser260, as wild-type and S260D arfaptin 2 never formed bundles. In contrast, bundles were observed in 35–45% of NG108-15 neuroblastoma cells and neurons expressing S260A arfaptin 2 (Fig. 4C). Arfaptin 2 Subcellular Localization Depends on the MT Network—Proteins that associate with MTs often induce bundles when overexpressed in cells (18Ligon L.A. Shelly S.S. Tokito M. Holzbaur E.L. Mol. Biol. Cell. 2003; 14: 1405-1417Crossref PubMed Scopus (152) Google Scholar). Bundle formation induced by loss of phosphorylation of arfaptin 2 at Ser260 together with the localization of arfaptin 2 around the MT-organizing center led us to investigate the effect of phosphorylation on the distribution of arfaptin 2 in relation to the MT network (Fig. 5A). Unfortunately, the paraformaldehyde fixation step, which is essential for the detection of the anti-histidine antibody that localizes transfected arfaptin 2, slightly disrupted the MT network. Nevertheless, wild-type arfaptin 2 was located mainly around the MT-organizing center in a punctate staining pattern as described previously (13Peters P.J. Ning K. Palacios F. Boshans R.L. Kazantsev A. Thompson L.M. Woodman B. Bates G.P. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 240-245Crossref PubMed Scopus (43) Google Scholar), whereas in cells expressing S260A arfaptin 2, the MT network was disrupted (Fig. 5A). To ensure that the localization of arfaptin 2 and its regulation by phosphorylation depend on MTs, we treated cells with nocodazole, an MT-depolymerizing agent. We found that both wild-type and S260A arfaptin 2 distributions were disrupted, with the proteins being concentrated in dense clusters throughout the cytoplasm (Fig. 5B). These results show that arfaptin 2 localization depends on the integrity of the MT network and that loss of phosphorylation of arfaptin 2 leads to an alteration of the MT organization. We next studied the effect of phosphorylation on arfaptin 2 distribution in cells. We analyzed arfaptin 2 in whole cell extracts and in soluble and insoluble Nonidet P-40 fractions from COS-7 cells transfected with various arfaptin 2 constructs (Fig. 5C). Wild-type and S260D arfaptin 2 were concentrated in the soluble Nonidet P-40 protein fraction, whereas S260A arfaptin 2 was strongly enriched in the insoluble Nonidet P-40 protein fraction. Thus, we have demonstrated by immunostaining and biochemical experiments that loss of phosphorylation of arfaptin 2 at Ser260 dramatically alters the cellular distribution of arfaptin 2. This results in its accumulation into insoluble bundle structures and to the partial disruption of the MT network. Arfaptin 2 Modulates PolyQ-huntingtin-induced Toxicity: Effect of Phosphorylation by Akt—We studied the physiological consequences of arfaptin 2 expression and phosphorylation of Ser260 on polyQ-huntingtin-induced to" @default.
• W2028097766 created "2016-06-24" @default.
• W2028097766 creator A5020007462 @default.
• W2028097766 creator A5045098548 @default.
• W2028097766 creator A5065550629 @default.
• W2028097766 creator A5070112095 @default.
• W2028097766 creator A5088515419 @default.
• W2028097766 date "2005-06-01" @default.
• W2028097766 modified "2023-10-18" @default.
• W2028097766 title "Phosphorylation of Arfaptin 2 at Ser260 by Akt Inhibits PolyQ-huntingtin-induced Toxicity by Rescuing Proteasome Impairment" @default.
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