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• W1983103334 abstract "Activation of Akt-mediated signaling pathways is crucial for survival and regeneration of injured neurons. In this study, we attempted to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt. PC12 cells that overexpressed constitutively active Akt were used. Using two-dimensional PAGE, we identified protein spots that exhibited increased immunostaining of the antibody. Mass spectrometry revealed several major spots as the neuronal intermediate filament protein, peripherin. Using several peripherin fragments, the phosphorylation site was determined as Ser66 in its head domain in vitro. Furthermore, a co-immunoprecipitation experiment revealed that Akt interacted with the head domain of peripherin in HEK 293T cells. An antibody against phosphorylated peripherin was raised, and induction of phosphorylated peripherin was observed not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. Activation of Akt-mediated signaling pathways is crucial for survival and regeneration of injured neurons. In this study, we attempted to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt. PC12 cells that overexpressed constitutively active Akt were used. Using two-dimensional PAGE, we identified protein spots that exhibited increased immunostaining of the antibody. Mass spectrometry revealed several major spots as the neuronal intermediate filament protein, peripherin. Using several peripherin fragments, the phosphorylation site was determined as Ser66 in its head domain in vitro. Furthermore, a co-immunoprecipitation experiment revealed that Akt interacted with the head domain of peripherin in HEK 293T cells. An antibody against phosphorylated peripherin was raised, and induction of phosphorylated peripherin was observed not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. Akt (also known as protein kinase B) is a Ser/Thr kinase that plays essential roles in various cellular processes such as cell survival, proliferation, and differentiation (1Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar). In the nervous system, Akt is suggested to be involved in neurogenesis (2Sinor A.D. Lillien L. J. Neurosci. 2004; 24: 8531-8541Crossref PubMed Scopus (99) Google Scholar, 3Peng Y. Jiang B.H. Yang P.H. Cao Z. Shi X. Lin M.C. He M.L. Kung H.F. J. Biol. Chem. 2004; 279: 28509-28514Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), neuronal survival (4Brunet A. Datta S.R. Greenberg M.E. Curr. Opin. Neurobiol. 2001; 11: 297-305Crossref PubMed Scopus (1011) Google Scholar), axon or dendrite formation (5Markus A. Zhong J. Snider W.D. Neuron. 2002; 35: 65-76Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 6Yoshimura T. Kawano Y. Arimura N. Kawabata S. Kikuchi A. Kaibuchi K. Cell. 2005; 120: 137-149Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar), synaptogenesis (7Akama K.T. McEwen B.S. J. Neurosci. 2003; 23: 2333-2339Crossref PubMed Google Scholar, 8Znamensky V. Akama K.T. McEwen B.S. Milner T.A. J. Neurosci. 2003; 23: 2340-2347Crossref PubMed Google Scholar), and synaptic transmission (9Wang Q. Liu L. Pei L. Ju W. Ahmadian G. Lu J. Wang Y. Liu F. Wang Y.T. Neuron. 2003; 38: 915-928Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The most evident role of all may be its neuroprotective action. For instance, several previous papers have demonstrated a strong protective effect of Akt on damaged neurons in vivo (10Namikawa K. Honma M. Abe K. Takeda M. Mansur K. Obata T. Miwa A. Okado H. Kiyama H. J. Neurosci. 2000; 20: 2875-2886Crossref PubMed Google Scholar, 11Manabe Y. Nagano I. Gazi M.S. Murakami T. Shiote M. Shoji M. Kitagawa H. Setoguchi Y. Abe K. Apoptosis. 2002; 7: 329-334Crossref PubMed Scopus (60) Google Scholar, 12Ohba N. Kiryu-Seo S. Maeda M. Muraoka M. Ishii M. Kiyama H. Neurosci. Lett. 2004; 359: 159-162Crossref PubMed Scopus (34) Google Scholar, 13Endo H. Nito C. Kamada H. Yu F. Chan P.H. Stroke. 2006; 37: 2140-2146Crossref PubMed Scopus (102) Google Scholar). Of particular interest, Akt was proven to have a crucial role in neuronal survival after peripheral nerve injury (10Namikawa K. Honma M. Abe K. Takeda M. Mansur K. Obata T. Miwa A. Okado H. Kiyama H. J. Neurosci. 2000; 20: 2875-2886Crossref PubMed Google Scholar). In the peripheral nervous system, in which most neurons can survive and regenerate after injury, glial cells secrete various trophic factors to promote survival and regeneration of nerve-injured neurons. Astrocytes and microglia, which are located around the neuronal cell bodies, are thought to secrete various factors toward injured neurons (14Polazzi E. Contestabile A. Rev. Neurosci. 2002; 13: 221-242Crossref PubMed Scopus (188) Google Scholar, 15Liberto C.M. Albrecht P.J. Herx L.M. Yong V.W. Levison S.W. J. Neurochem. 2004; 89: 1092-1100Crossref PubMed Scopus (389) Google Scholar). Furthermore, in the distal stump of axons far from neuronal cell bodies, Schwann cells also secrete trophic factors (16Frostick S.P. Yin Q. Kemp G.J. Microsurgery. 1998; 18: 397-405Crossref PubMed Scopus (416) Google Scholar). Such factors released from those glial cells include a wide range of growth factors such as nerve growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and fibroblast growth factor-2 (17Terenghi G. J. Anat. 1999; 194: 1-14Crossref PubMed Google Scholar, 18Grothe C. Nikkhah G. Anat. Embryol. (Berl.). 2001; 204: 171-177Crossref PubMed Scopus (134) Google Scholar, 19Boyd J.G. Gordon T. Mol. Neurobiol. 2003; 27: 277-324Crossref PubMed Scopus (377) Google Scholar). They are known to activate the phosphatidylinositol 3-kinase-Akt pathway in injured neurons via their respective receptors (20Patapoutian A. Reichardt L.F. Curr. Opin. Neurobiol. 2001; 11: 272-280Crossref PubMed Scopus (921) Google Scholar, 21Besset V. Scott R.P. Ibanez C.F. J. Biol. Chem. 2000; 275: 39159-39166Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 22Karajannis M.A. Vincent L. Direnzo R. Shmelkov S.V. Zhang F. Feldman E.J. Bohlen P. Zhu Z. Sun H. Kussie P. Rafii S. Leukemia. 2006; 20: 979-986Crossref PubMed Scopus (45) Google Scholar). In fact, our previous study showed that Akt activity was markedly induced in motor neurons after nerve injury (10Namikawa K. Honma M. Abe K. Takeda M. Mansur K. Obata T. Miwa A. Okado H. Kiyama H. J. Neurosci. 2000; 20: 2875-2886Crossref PubMed Google Scholar). We also revealed that activated Akt accelerated axonal elongation, as well as neuronal survival. It is well established that activated Akt exerts its function by phosphorylating its substrates; however, the substrates that specifically exist in neurons are largely unidentified. Thus, identification of novel neuronal substrates is pivotal to gain further insight into the function of Akt in neuronal regeneration. In this study, we attempted to identify novel Akt substrates in neurons by a proteomic approach, using a unique antibody that recognizes the consensus motif phosphorylated by Akt. Here we demonstrate that peripherin, which is a peripheral nervous system neuron-specific intermediate filament protein, is a novel Akt substrate, and that Ser66 of peripherin is the phosphorylation site. Peripherin phosphorylation is apparently induced in motor neurons after nerve injury, suggesting that the Akt-mediated peripherin phosphorylation may play a role in motor nerve regeneration. Materials—Anti-phospho-Akt substrate antibody (antibody 9611; Cell Signaling Technology, Danvers, MA), anti-phospho-Akt antibody (antibody 4051; Cell Signaling Technology), anti-peripherin antibody (antibody MAB1527 for Western blotting; antibody AB1530 for immunohistochemistry; Chemicon, Temecula, CA), anti-glutathione S-transferase (GST) 2The abbreviations used are: GST, glutathione S-transferase; HA, hemagglutinin; HEK, human embryonic kidney; WT, wild type; CA, constitutively active; DN, dominant negative; MOI, multiplicity of infection; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; p70S6K, p70 S6 kinase; NF, neurofilament; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. antibody (antibody sc-138; Santa Cruz Biotechnology, Santa Cruz, CA), anti-His antibody (antibody 1922416; Roche Applied Science), anti-hemagglutinin (HA) antibody (antibody 1583816; Roche Applied Science; and antibody sc-138; Santa Cruz Biotechnology), anti-FLAG antibody (antibody F3166; Sigma), and anti-glyceraldehydes-3-phosphate dehydrogenase (antibody 4300; Ambion, Huntington, UK) were used as primary antibodies. As secondary antibodies, horseradish peroxidase-conjugated antibodies (Amersham Biosciences) and Alexa Fluor-conjugated antibodies (Molecular Probes, Eugene, OR) were used for Western blotting and immunohistochemistry, respectively. All of the inhibitors were obtained from Calbiochem (La Jolla, CA). Cell Culture—Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Invitrogen) and 0.05 mg/ml penicillin/streptomycin (Invitrogen). PC12 cells were maintained on cell culture dishes coated with collagen in RPMI 1640 containing 5% fetal bovine serum, 10% horse serum, and 0.05 mg/ml penicillin/streptomycin. Both cell types were cultured at 37 °C under 5% CO2. Adenoviral Vectors—The detailed procedure for constructing recombinant adenoviral vectors was described previously (10Namikawa K. Honma M. Abe K. Takeda M. Mansur K. Obata T. Miwa A. Okado H. Kiyama H. J. Neurosci. 2000; 20: 2875-2886Crossref PubMed Google Scholar). Briefly, HA-tagged wild type Akt (HA-WT-Akt), constitutively active Akt (HA-CA-Akt), which lacks its pleckstrin homology domain but has a Src myristoylation signal sequence, and dominant negative Akt (T308A/S473A; HA-DN-Akt; kindly provided by Drs. M. Kasuga and W. Ogawa) were subcloned into pAxCALNLw Cre-lox P system-mediated expression cassette (23Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 24Sato Y. Tanaka K. Lee G. Kanegae Y. Sakai Y. Kaneko S. Nakabayashi H. Tamaoki T. Saito I. Biochem. Biophys. Res. Commun. 1998; 244: 455-462Crossref PubMed Scopus (94) Google Scholar, 25Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar). The adenoviral vectors AxCALNLHA-WT-Akt, AxCALNLHA-CA-Akt, and AxCALNLHA-DN-Akt were then constructed by the COS-terminal protein complex method (26Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (787) Google Scholar). AxCANCre and AxCALNLLacZ were kindly provided by Drs. I. Saito and Y. Kanegae (27Kanegae Y. Lee G. Sato Y. Tanaka M. Nakai M. Sakaki T. Sugano S. Saito I. Nucleic Acids Res. 1995; 23: 3816-3821Crossref PubMed Scopus (598) Google Scholar). Two-dimensional PAGE—PC12 cells grown on 10-cm cell culture dishes were infected with AxCALNLLacZ (multiplicity of infection (MOI) 100) or AxCALNLHA-CA-Akt (MOI 100) together with AxCANCre (MOI 30). The cells were collected 48 h after infection, washed once with PBS, and lysed in a buffer containing 40 mm Tris base, 8 m urea, and 2% CHAPS. After centrifugation at 10,000 × g for 20 min at 4 °C, the supernatants were aliquoted and stored at -80 °C until use. Two-dimensional PAGE was performed according to the previous report with slight modification (28Konishi H. Namikawa K. Kiyama H. Glia. 2006; 53: 723-732Crossref PubMed Scopus (36) Google Scholar). Immobiline DryStrips (pH 3–10, 7 cm; pH 4.5–5.5, 24 cm; Amersham Biosciences) were rehydrated with rehydration solution containing 60 μg (for 7 cm gel) or 240 μg (for 24 cm gel) of the supernatants, 8 m urea, 2% CHAPS, 0.5% IPG buffer (Amersham Biosciences), 20 mm dithiothreitol, and bromphenol blue for 12 h at 20 °C. Isoelectric focusing was then performed using the IPGphor Isoelectric Focusing System (Amersham Biosciences) (500 V for 1 h, 1000 V for 1 h, and 8000 V for 2–3 h at 20 °C). The strips were equilibrated with a buffer containing Tris-HCl, pH 6.8, 6 m urea, 30% glycerol, 2% SDS, and 65 mm dithiothreitol for 20 min at room temperature, fixed vertically on top of the SDS-polyacrylamide gel by 1.5% agarose in running buffer, and subjected to 25 mA/gel in a cold room. The gels were analyzed by Western blotting for immunostaining or SYPRO Ruby for protein staining according to the manufacturer’s protocol (Molecular Probes, Eugene, OR). In-gel Digestion—Protein spots were punched out from the gel, trimmed into small pieces, destained in a solution containing 50% acetonitrile and 25 mm NH4HCO3, and dehydrated. The gel pieces were then rehydrated in a solution containing 10 mm dithiothreitol and 25 mm NH4HCO3 and subsequently treated with 25 mm NH4HCO3 containing 55 mm iodoacetoamide. Following the dehydration step, gel pieces were rehydrated in trypsin solution containing 10 mg/ml trypsin and 25 mm NH4HCO3 overnight at 37 °C, and finally digested peptides were eluted with 50% acetonitrile containing 5% trifluoroacetic acid. Mass Spectrometry—The eluate containing digested peptides was desalted with ZipTip (Millipore, Bedford, MA). The solution was then mixed with an equal volume of saturated α-cyano-4-hydroxycinnamic acid solution dissolved in 30% acetonitrile and 0.1% trifluoroacetic acid and spotted onto a target plate. Mass spectrometry was performed on a matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometer Reflex III (Bruker Daltonics, Billerica, MA) with reflector mode. Obtained peptide mass fingerprinting data were searched against the NCBI data base using the MASCOT search engine (Matrix Science, Boston, MA). Western Blotting—Protein extracts were separated by SDS-PAGE, and blots were prepared on polyvinylidene difluoride membranes (Millipore). For two-dimensional gels, whole 7-cm gels or part of 24-cm gels were prepared on the membrane. The blots were probed with primary and subsequent secondary antibodies and visualized by using the chemiluminescence system (Western Lightning; PerkinElmer Life Sciences). If necessary, the membranes were stripped of antibodies by incubating in stripping buffer containing 62.5 mm Tris-HCl, pH 6.8, 2% SDS, and 100 mm 2-mercaptoethanol for 30 min at 50 °C and then probed with another antibody. Preparation of Recombinant Proteins—To generate GST fusion proteins, partial sequences for 1–60, 51–100, 181–251, and 301–350 amino acids of peripherin were amplified from full-length mouse peripherin cDNA (kindly provided by Dr. F. Landon) and subcloned into pGEX 5X-1 (Amersham Biosciences). Site-directed mutagenesis (Ser66 or Ser79 to Ala) was introduced by PCR primers carrying these mutations. BL21 bacteria transformed with these vectors were stimulated with 0.2 mm isopropyl-β-d-thiogalactopyranoside overnight at 20 °C, harvested by brief centrifugation, and lysed in PBS containing 1% Triton X-100 for 30 min at 4 °C. The supernatants were subsequently incubated with glutathione-Sepharose 4B (Amersham Biosciences) for 1 h at 4 °C, and the bound proteins were eluted by adding 10 mm reduced glutathione in 50 mm Tris-HCl, pH 8.0. After removal of glutathione by dialysis against PBS, the proteins were checked by SDS-PAGE followed by Coomassie Brilliant Blue R-250 staining and stored at -80 °C until use. In Vitro Kinase Assay—2.5 μg of GST fusion proteins were incubated with or without 100 ng of recombinant His-tagged CA-Akt (His-CA-Akt) (Upstate Biotechnology) in 20 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 20 μm ATP, and 30 kBq [γ-32P]ATP (PerkinElmer Life Sciences) for 30 min at 30 °C. The reaction mixtures were subjected to SDS-PAGE, and phosphorylation of the fragments was detected by autoradiography. For Western blot analysis, 1 μg of GST fusion proteins was reacted with 100 ng of His-CA-Akt, and one-tenth of the reaction mixtures was analyzed. Phosphorylation-specific Antibody—A rat monoclonal antibody that specifically recognized phosphorylated peripherin (anti-pPer antibody) was raised in accordance with the previous report (29Kishiro Y. Kagawa M. Naito I. Sado Y. Cell Struct. Funct. 1995; 20: 151-156Crossref PubMed Scopus (170) Google Scholar, 30Ushijima R. Sakaguchi N. Kano A. Maruyama A. Miyamoto Y. Sekimoto T. Yoneda Y. Ogino K. Tachibana T. Biochem. Biophys. Res. Commun. 2005; 330: 880-886Crossref PubMed Scopus (46) Google Scholar). Briefly, a 10-week-old female WKY/NCrj rat was immunized with a synthetic peptide containing phosphorylated Ser66 (ARLGpS66FRAPRC). Three weeks after immunization, lymph nodes obtained from the rat were dispersed, and lymphocytes were fused with mouse myeloma Sp2/0-Ag14 cells. The phosphorylation-specific antibody was screened by enzyme-linked immunosorbent assay using hybridoma supernatants, and clone 2C2 was selected. Finally, 2C2 hybridoma cells were injected into the abdominal cavity of nude mice, and prepared ascites were used for immunological assays. Detection of Peripherin Phosphorylation in Cultured Cells—HEK 293T cells seeded on 60-mm culture dishes were grown to ∼80% confluence and transfected with pcDNA3-peripherin and pcDNA3-HA-WT-Akt using Lipofectamin 2000 (Invitrogen). After 8 h, the cells were seeded into 12 well culture dishes and cultured for another 24 h. The cells were then serum-starved for 10 h, treated with insulin (Sigma), and subjected to Western blot analysis using the anti-pPer antibody. If necessary, inhibitors were added to the cultured medium 30 min before insulin treatment. As for the phosphorylation of endogenous peripherin, PC12 cells infected with AxCALNLLacZ (MOI 100), AxCALNLHA-WT-Akt (MOI 100), AxCALNLHA-CA-Akt (MOI 100), or AxCALNLHA-DN-Akt (MOI 100) together with AxCANCre (MOI 30) for 48 h were examined. Immunoprecipitation—HEK 293T cells seeded on 6-well culture dishes were transfected with pcDNA3-HA-WT-Akt together with pcDNA3 empty vector or FLAG-tagged head domain of peripherin (FLAG-Per 1–103) subcloned into pcDNA3 using Lipofectamin 2000. After 32 h, the cells were serum-starved for 10 h and treated with 100 nm insulin for 20 min. The cells were then washed in Tris-buffered saline briefly and lysed in radioimmunoprecipitation assay buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 0.25% sodium deoxycholate, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mm Na3VO4, and 10 mm NaF). After centrifugation at 10,000 × g for 10 min at 4 °C, the soluble fractions were collected and reacted with anti-FLAG antibody followed by precipitation using protein G-Sepharose 4B (Sigma). Immunoprecipitates were rinsed four times with lysis buffer and eluted by adding 2× SDS sample buffer. Immunohistochemistry—Adult male Wistar rats weighing ∼150 g were anesthetized with pentobarbital (40 mg/kg) and positioned supine, and their right hypoglossal nerves were crushed with forceps. The rats were perfused with 4% paraformaldehyde in 0.1 m phosphate buffer 5 days after surgery. The brains were quickly removed, post-fixed overnight at 4 °C in the fixative, and immersed in 0.1 m phosphate buffer containing 25% sucrose for an additional day. Sections were cut on a cryostat (18 μm in thickness), washed once in PBS, and treated with 10 μg/ml proteinase K for 10 min. After two washes in PBS, the sections were blocked with PBS containing 10% normal goat serum for 1 h and subsequently reacted with primary antibodies (anti-peripherin antibody; 1:1000, anti-pPer antibody; 1:1000) in PBS containing 1% normal goat serum overnight at 4 °C. After three washes in PBS, the sections were incubated with secondary antibodies for 1 h and finally washed three times in PBS. The sections were visualized by fluorescent microscopy (AX70; Olympus, Tokyo, Japan). Identification of Peripherin as an Akt Substrate in Neurons—To identify novel neuronal substrates for Akt, we utilized the anti-phospho-Akt substrate antibody. Akt preferentially phosphorylates Ser or Thr in the RXRXX(S/T) motif, and the antibody specifically recognizes this motif only when Ser or Thr is phosphorylated. PC12 cells infected with adenovirus expressing LacZ or CA-Akt were subjected to Western blot analysis using this antibody, and proteins exhibiting more intense signal in the CA-Akt-expressing preparation were searched. Our preliminary experiment using ordinary SDS-PAGE demonstrated stacked positive bands where isolation of the individual positive band was impossible (data not shown). We therefore performed two-dimensional PAGE to also separate proteins by their isoelectric points, and the two-dimensional gels were analyzed by Western blotting using the antibody. We initially used a wide pH range gel for the first dimension and found numerous spots were intensely stained in the CA-Akt-expressing preparation; in particular in the region in which the isoelectric point was 5.0–5.5 and molecular mass was ∼60 kDa (Fig. 1A). We therefore focused on this region and separated proteins more precisely by using narrow pH range gels for the first dimension (Fig. 1B). Six spots that exhibited the intense positive immunostaining were identical to the protein spots in the protein-stained gels (spots 1–4, 8, and 9 in Fig. 1C). Judging from their sequential spot patterns, we assumed that spots 1–4 were the same proteins, each of which might have different post-translational modifications. Similarly, the spots 8 and 9 were assumed to be the same protein. As representative samples, spots 1 and 9 were punched out from the gel and analyzed by MALDI-TOF mass spectrometry to identify the corresponding proteins. The subsequent data base search revealed that both spots were identical to peripherin. All spots (spots 1–4, 8, and 9) were confirmed as peripherin by Western blot analysis using the anti-peripherin antibody (Fig. 1D). Akt Phosphorylates Ser66 of Peripherin in Vitro—Peripherin, whose expression is mostly restricted to neurons in the peripheral nervous system, is a member of type III intermediate filament proteins (31Coulombe P.A. Ma L. Yamada S. Wawersik M. J. Cell Sci. 2001; 114: 4345-4347Crossref PubMed Google Scholar). Because peripherin has not been identified as an Akt substrate, we performed further analysis. First, we aimed to determine the phosphorylation site by Akt in vitro using recombinant proteins. Although no typical consensus sequence for the Akt substrate, RXRXX(S/T), was found in peripherin, five potent sequences existed (Fig. 2A). Because several previous papers indicated that Akt could possibly recognize some similar sequences as its target (details are described under “Discussion”), we examined the possibility that Akt was able to recognize and phosphorylate some similar sequences. Four types of GST fusion proteins that contained one or two potent sequences were generated and reacted with recombinant CA-Akt protein in the presence of [γ-32P]ATP (Fig. 2B). Autoradiography showed that one fragment containing 51–100 amino acids of peripherin (GST-Per 51–100) was exclusively phosphorylated by CA-Akt among four fragments. Because GST-Per 51–100 contained two potent sequences, SARLGS66 and ALRLPS79, we then introduced site-directed mutagenesis to GST-Per 51–100 to produce unphosphorylated mutants. These proteins were analyzed by an in vitro kinase assay (Fig. 2C). The S66A mutation entirely prevented Akt phosphorylation, whereas the S79A mutation did not cause any alterations. These results demonstrate that Akt phosphorylates Ser66 of peripherin in vitro. The sequence containing Ser66 is highly conserved among mammalian species (Fig. 2D). Ser66 of Peripherin Is Phosphorylated in Akt-activated Cultured Cells—To evaluate peripherin phosphorylation in vivo, a monoclonal antibody (anti-pPer antibody) was raised against the synthetic peptide ARLGpS66FRAPRC. Specificity of this antibody was tested by Western blot analysis using GST-Per 51–100 in vitro (Fig. 3A). The anti-pPer antibody could detect GST-Per 51–100 only when the fragment was reacted with CA-Akt, and the intense immunoreactivity entirely disappeared when the S66A mutant was used. Using this antibody, peripherin phosphorylation was examined in HEK 293T cells. HEK 293T cells were transfected with WT-Akt and peripherin, because they have no endogenous peripherin, subsequently stimulated with insulin to activate Akt, and peripherin phosphorylation was detected by Western blot analysis. First, HEK 293T cells were treated with increasing doses of insulin, and peripherin phosphorylation was examined (Fig. 3B). Both Akt activation, which was evaluated by the phosphorylation state of Akt (32Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar), and peripherin phosphorylation occurred in a dose-dependent manner. Next, we observed changes in peripherin phosphorylation over time after insulin treatment (Fig. 3C). Peripherin was phosphorylated in a time-dependent manner, which paralleled Akt activation. To further demonstrate that Akt kinase activity regulated peripherin phosphorylation, we used several inhibitors to modulate Akt activity. Both Akt activation and peripherin phosphorylation were almost prevented by pretreating cells with LY294002 (phosphatidylinositol 3-kinase inhibitor, which inhibited upstream signaling of Akt). In contrast, peripherin phosphorylation was not prevented by Me2SO (the vehicle for control), U0126 (MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor, which prevented mitogen-activated protein kinase signaling), or rapamycin (mTOR inhibitor, which prevented one of the downstream signaling of Akt). We also examined the phosphorylation of endogenous peripherin in PC12 cells by Western blot analysis (Fig. 3E). Peripherin phosphorylation was hardly detected in PC12 cells infected with adenovirus expressing LacZ, WT-Akt, or DN-Akt. In contrast, peripherin phosphorylation was clearly observed in cells expressing CA-Akt. Although some minor additional bands were observed at different molecular masses in this blotting using PC12 cells, we assumed those bands would be nonspecific because their intensity was not affected by WT-, CA-, or DN-Akt expression. Together with the results obtained by HEK 293T cells, these results demonstrate that Ser66 of peripherin is phosphorylated in an Akt-mediated pathway in cultured cells. Akt Interacts with the Head Domain of Peripherin in Vivo—It is likely that Akt may directly phosphorylate Ser66 of peripherin in vivo. To provide further support for this possibility, we examined whether these two proteins could interact in vivo using a co-immunoprecipitation experiment. Full-length peripherin, most of which may form intermediate filament in cells, is almost detergent-insoluble (33Giasson B.I. Mushynski W.E. J. Neurochem. 1998; 70: 1869-1875Crossref PubMed Scopus (22) Google Scholar), and we assumed that peripherin might not be solubilized in a typical lysis buffer for immunoprecipitation. Therefore, we used a deletion form of peripherin for the immunoprecipitation experiment. Because our preliminary experiment showed that the head domain of peripherin (1–103 amino acids), which contained Ser66, could be solubilized entirely in radioimmunoprecipitation assay buffer (data not shown), we used the head domain instead of full-length peripherin in this assay. HEK 293T cells transfected with FLAG-Per 1–103 and WT-Akt were treated with or without insulin and subjected to immunoprecipitation using the anti-FLAG antibody (Fig. 4). Akt was co-precipitated with FLAG-Per 1–103, indicating that they could interact in vivo.It was of note that this interaction was not dependent on Akt activity because insulin treatment did not enhance the interaction. A similar activity-independent binding has also been reported on several other Akt substrates (34Datta 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, 35Kim A.H. Khursigara G. Sun X. Franke T.F. Chao M.V. Mol. Cell. Biol. 2001; 21: 893-901Crossref PubMed Scopus (623) Google Scholar). Ser66 of Peripherin Is Phosphorylated in Regenerating Hypoglossal Motor Neurons—Previous reports have revealed that Akt was activated in response to neuronal injury (10Namikawa K. Honma M. Abe K. Takeda M. Mansur K. Obata T. Miwa A. Okado H. Kiyama H. J. Neurosci. 2000; 20: 2875-2886Crossref PubMed Google Scholar, 13Endo H. Nito C. Kamada H. Yu F. Chan P.H. Stroke. 2006; 37: 2140-2146Crossref PubMed Scopus (102) Google Scholar, 36Yu F. Sugawara T. Maier C.M. Hsieh L.B. Chan P.H. Neurobiol. Dis. 2005; 20: 491-499Crossref PubMed Scopus (53) Google Scholar). In particular, Akt activation is crucial for nerve-injured motor neurons to regenerate (10Namikawa K. Honma M. Abe K. Takeda M. Mansur K. Obata T. Miwa A. Okado H. Kiyama H. J. Neurosci. 2000; 20: 2875-2886Crossref PubMed Google Scholar). We therefore examined the phosphorylation of endogenous peripherin in nerve-injured hypoglossal motor neurons. We crushed the hypoglossal nerve, and then peripherin expression and phosphorylation were examined by immunohistochemistry 5 days after i" @default.
• W1983103334 created "2016-06-24" @default.
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• W1983103334 creator A5073257513 @default.
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• W1983103334 date "2007-08-01" @default.
• W1983103334 modified "2023-10-15" @default.
• W1983103334 title "Identification of Peripherin as a Akt Substrate in Neurons" @default.
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