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Pin1-dependent Prolyl Isomerization Modulates the Stress-induced Phosphorylation of High Molecular Weight Neurofilament Protein

2008; Elsevier BV; Volume: 283; Issue: 39 Linguagem: Inglês

10.1074/jbc.m801633200

1083-351X

Parvathi Rudrabhatla, Yali Zheng, Niranjana D. Amin, Sashi Kesavapany, Wayne Albers, Harish C. Pant,

Hereditary Neurological Disorders

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H2O2) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H2O2 and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases. Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H2O2) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H2O2 and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases. The reversible phosphorylation on Ser/Thr-Pro ((S/T)P) motifs governed by Pro-directed protein kinases and phosphatases is a major regulatory mechanism for the control of various cellular processes. The phosphorylation state of a protein reflects the balance between the activation of kinases and phosphatases, and also of partners, such as isomerases, able to support their activity through conformational changes. Neurofilament proteins are among the most highly phosphorylated proteins in the nervous system (1Nixon R.A. Sihag R.K. Trends Neurosci. 1991; 1411: 501-506Abstract Full Text PDF Scopus (287) Google Scholar, 2Elhanany E. Jaffe H. Link W.T. Sheeley D.M. Gainer H. Pant H.C. J. Neurochem. 1994; 63: 2324-2335Crossref PubMed Scopus (69) Google Scholar, 3Pant H.C. Veeranna Biochem. Cell Biol. 1995; 73: 575-592Crossref PubMed Scopus (171) Google Scholar). Extensive phosphorylation of (S/T)P repeats in the tail domain of high molecular weight neurofilament protein (NF-H) 2The abbreviations used are: NF-Hhigh molecular weight neurofilament proteinADAlzheimer diseaseCdk5cyclin-dependent kinase 5EGFepidermal growth factorErk1/2extracellular signal-regulated kinases 1 and 2JNKc-Jun N-terminal kinasePin1protein interacting with NIMA (never in mitosis A)-1p-NF-Hphosphorylated NF-HMAPmitogen-activated proteinMAPKmitogen-activated protein kinaseTUNELterminal deoxynucleotidyl transferase-mediated nick end labelingsiRNAshort interfering RNAALSamyotrophic lateral sclerosisSAPKstress-activated protein kinaseDNdominant-negativeGSTglutathione S-transferaseDICdays in cultureErkextracellular signal-regulated kinasePDParkinson disease. 2The abbreviations used are: NF-Hhigh molecular weight neurofilament proteinADAlzheimer diseaseCdk5cyclin-dependent kinase 5EGFepidermal growth factorErk1/2extracellular signal-regulated kinases 1 and 2JNKc-Jun N-terminal kinasePin1protein interacting with NIMA (never in mitosis A)-1p-NF-Hphosphorylated NF-HMAPmitogen-activated proteinMAPKmitogen-activated protein kinaseTUNELterminal deoxynucleotidyl transferase-mediated nick end labelingsiRNAshort interfering RNAALSamyotrophic lateral sclerosisSAPKstress-activated protein kinaseDNdominant-negativeGSTglutathione S-transferaseDICdays in cultureErkextracellular signal-regulated kinasePDParkinson disease. occurs primarily in axons (4Sternberger L.A. Sternberger N.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6126-6130Crossref PubMed Scopus (1034) Google Scholar, 5Lee V.M. Otvos Jr., L. Carden M.J. Hollosi M. Dietzschold B. Lazzarini R.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1998-2002Crossref PubMed Scopus (347) Google Scholar). The deregulation of NF-H tail domain phosphorylation is correlated with some pathology seen in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis (ALS) in which aberrant tail domain phosphorylation occurs in neuronal perikarya (6Manetto V. Sternberger N.H. Perry G. Sternberger L.A. Gambetti P. J. Neuropathol. Exp. Neurol. 1988; 47: 642-653Crossref PubMed Scopus (189) Google Scholar, 7Cleveland D.W. Rothstein J.D. Nat. Rev. Neurosci. 2001; 2: 806-819Crossref PubMed Scopus (1160) Google Scholar). high molecular weight neurofilament protein Alzheimer disease cyclin-dependent kinase 5 epidermal growth factor extracellular signal-regulated kinases 1 and 2 c-Jun N-terminal kinase protein interacting with NIMA (never in mitosis A)-1 phosphorylated NF-H mitogen-activated protein mitogen-activated protein kinase terminal deoxynucleotidyl transferase-mediated nick end labeling short interfering RNA amyotrophic lateral sclerosis stress-activated protein kinase dominant-negative glutathione S-transferase days in culture extracellular signal-regulated kinase Parkinson disease. high molecular weight neurofilament protein Alzheimer disease cyclin-dependent kinase 5 epidermal growth factor extracellular signal-regulated kinases 1 and 2 c-Jun N-terminal kinase protein interacting with NIMA (never in mitosis A)-1 phosphorylated NF-H mitogen-activated protein mitogen-activated protein kinase terminal deoxynucleotidyl transferase-mediated nick end labeling short interfering RNA amyotrophic lateral sclerosis stress-activated protein kinase dominant-negative glutathione S-transferase days in culture extracellular signal-regulated kinase Parkinson disease. Rat NF-H has 52 KSP repeats in the tail domain, and almost all of them are phosphorylated in vivo (8Jaffe H. Veeranna Pant H.C. Biochemistry. 1998; 37: 16211-16224Crossref PubMed Scopus (116) Google Scholar, 9Veeranna Amin N.D. Ahn N.G. Jaffe H. Winters C.A. Grant P. Pant H.C. J. Neurosci. 1998; 64: 4008-4021Crossref Google Scholar). The multiple repeats of the SP moiety suggest that reconfiguration of NF-H may involve additional factors to catalyze peptidylprolyl isomerization. One such candidate for this is the peptidylprolyl isomerase, Pin1 (protein interacting with NIMA (never in mitosis A)-1). Pin1 catalyzes the cis to trans isomerization of peptide bonds that link Ser(P) or Thr(P) residues to proline. Although Pin1 has been widely studied in relation to the cell cycle and cancer, little is known about its role in the brain. Recent data suggested that Pin1 might be involved in AD pathogenesis, because it is found in neurofibrillary tangles (10Lu P.J. Wulf G. Zhou X.Z. Davies P. Lu K.P. Nature. 1999; 399: 784-788Crossref PubMed Scopus (617) Google Scholar). Pin1 has also been shown to facilitate Tau dephosphorylation by protein phosphatase 2A (10Lu P.J. Wulf G. Zhou X.Z. Davies P. Lu K.P. Nature. 1999; 399: 784-788Crossref PubMed Scopus (617) Google Scholar). We have shown recently that Pin1 may also be implicated in post-phosphorylation modulation of pSer/pThr-Pro (KSP) repeats in NF-H, which, when deregulated, may lead to perikaryal motor-neuron inclusions typical of ALS (11Kesavapany S. Patel V. Zheng Y.L. Pareek T.K. Bjelogrlic M. Albers W. Amin N. Jaffe H. Gutkind J.S. Strong M.J. Grant P. Pant H.C. Mol. Biol. Cell. 2007; 9: 3645-3655Crossref Google Scholar). A growing number of phosphorylated proteins participating in important cellular processes such as RNA polymerase II (12Xu Y.-X. Hirose Y. Zhou X.Z. Lu K.P. Manley J.L. Genes Dev. 2003; 17: 2765-2776Crossref PubMed Scopus (135) Google Scholar) and CDC25 (13Crenshaw D.G. Yang J. Means A.R. Kornbluth S. EMBO J. 1998; 17: 1315-1327Crossref PubMed Scopus (166) Google Scholar) are regulated through prolyl isomerase-induced conformational changes. Pin1 activity has been reported to influence its target proteins in two completely opposite fashions: 1) It can facilitate the dephosphorylation of Tau by protein phosphatase 2A (10Lu P.J. Wulf G. Zhou X.Z. Davies P. Lu K.P. Nature. 1999; 399: 784-788Crossref PubMed Scopus (617) Google Scholar). 2) Pin1 is shown to facilitate hyperphosphorylation and inhibit dephosphorylation of RNA polymerase II (12Xu Y.-X. Hirose Y. Zhou X.Z. Lu K.P. Manley J.L. Genes Dev. 2003; 17: 2765-2776Crossref PubMed Scopus (135) Google Scholar). Several processes are up-regulated by Pin1: some of these involve its actions on Cyclin D1 (14Liou Y.C. Ryo A. Huang H.K. Lu P.J. Bronson R. Fujimori F. Uchida T. Hunter T. Lu K.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1335-1340Crossref PubMed Scopus (291) Google Scholar), CK2 (15Messenger M.M. Saulnier R.B. Gilchrist A.D. Diamond P. Gorbsky G.J. Litchfield D.W. J. Biol. Chem. 2002; 277: 23054-23064Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), c-Jun (16Wulf G.M. Ryo A. Wulf G.G. Lee S.W. Niu T. Petkova V. Lu K.P. EMBO J. 2001; 20: 3459-3472Crossref PubMed Scopus (475) Google Scholar), c-Fos (17Monje P. Hernandez-Losa J. Lyons R.J. Castellone M.D. Gutkind J.S. J. Biol. Chem. 2005; 280: 35081-35084Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), and Gephyrin (18Zita M.M. Marchionni I. Bottos E. Righi M. Del Sal G. Cherubini E. Zacchi P. EMBO J. 2007; 26: 1761-1771Crossref PubMed Scopus (73) Google Scholar). On the other hand, SRC3 (19Yi P. Wu R.C. Sandquist J. Wong J. Tsai S.Y. Tsai M.J. Means A.R. O'Malley B.W. Mol. Cell. Biol. 2005; 25: 9687-9699Crossref PubMed Scopus (81) Google Scholar), c-Myc (20Yeh E. Cunningham M. Arnold H. Chasse D. Monteith T. Ivaldi G. Hahn W.C. Stukenberg P.T. Shenolikar S. Uchida T. Counter C.M. Nevins J.R. Means A.R. Sears R. Nat. Cell Biol. 2004; 6: 308-318Crossref PubMed Scopus (612) Google Scholar), and Cyclin E (21van Drogen F. Sangfelt O. Malyukova A. Matskova L. Yeh E. Means A.R. Reed S.I. Mol. Cell. 2006; 23: 37-48Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) involve processes that are down-regulated by Pin1. Oxidative stress, an early event in AD that occurs prior to cytopathology, can induce rapid hyperphosphorylation of KSP epitopes of NF-H in cultured neurons. This is likely to be mediated by activation of proline-directed kinases or by protein phosphatase 2A inactivation (22Jaffe H. Veeranna Shetty K.T. Pant H.C. J. Neurochem. 1995; 64: 2681-2690PubMed Google Scholar). The role of stress-activated protein kinases such as JNK (SAPK) were reported to be involved in stress-induced NF-H phosphorylation (23Giasson B.I. Mushynski W.E. J. Biol. Chem. 1996; 271: 30404-30409Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 24Brownlees J. Yates A. Bajaj N.P. Davis D. Anderton B.H. Leigh P.N. Shaw C.E. Miller C.C.J. J. Cell Sci. 2000; 113: 401-407Crossref PubMed Google Scholar). Among the JNK isoforms, JNK3 (SAPK1β) is the major stress activated protein kinase widely expressed in the nervous system (25Martin J.H. Mohit A. Miller C.A. Mol. Brain. Res. 1996; 35: 47-57Crossref PubMed Google Scholar), specifically in neuronal cell bodies (26Merritt S.E. Matas M. Nihalani D. Zhu X. Holzman L.B. J. Biol. Chem. 1999; 274: 10195-10202Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). JNK3 is shown to phosphorylate neurofilament (NF-H) and is activated under glutamate stress (24Brownlees J. Yates A. Bajaj N.P. Davis D. Anderton B.H. Leigh P.N. Shaw C.E. Miller C.C.J. J. Cell Sci. 2000; 113: 401-407Crossref PubMed Google Scholar). Absence of excitotoxicity-induced apoptosis in the hippocampus of JNK3 knockout mice has been reported (27Davis R.J. Rakic J.P. Flavell R.A. Nature. 1997; 389: 865-870Crossref PubMed Scopus (1110) Google Scholar). In this study, we show that Pin1 elevates the NF-H phosphorylation by proline-directed kinases such as Erk1/2, Cdk5, and JNK3 in vitro. Both dominant-negative (DN) Pin1 and Pin1 siRNA reduced oxidative and heat stress-induced aberrant NF-H hyperphosphorylation. We show that Pin1 modulates the H2O2 and heat stress-induced perikaryal phosphorylation of NF-H via the activation of JNK3. Homology Modeling—Modeling of the structure of rat Pin1 was performed based on the x-ray structures of the proteins that produced the best E value when using BLAST against the protein in the protein data bank (PDB) data base using the Swiss PDB Viewer package (www.expasy.ch/swissmod/SWISS-MODEL.html) (28Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9399) Google Scholar). The molecular modeling method used was ProMod II (28Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9399) Google Scholar). Three-dimensional models of rat Pin1 were predicted using the coordinates of human Pin1 (PDB code 1nmv, determined at 1.45 Å) (29Bayer E. Goettsch S. Mueller J.W. Griewel B. Guiberman E. Mayr L.M. Bayer P. J. Biol. Chem. 2003; 278: 26183-26193Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The Pin1 protein sequence was aligned with the sequence of the homologous Pin1 using the advanced BLAST (www.ncbi.nlm.nih.gov/BLAST/). Refinement of side chains and terminal chains was done using the Molecular Operation Environment software package (Version 2001.01, Chemical Computing Group, Montreal, Canada). The generated model was then energy minimized in SYBYL (Tripos Associates, St. Louis, MO) using a three-stage protocol involving simplex, conjugate-gradient, and Powell minimization methods, by moving side chains alone, to relieve short contacts at the interprotomer interfaces. The quality of the three-dimensional model was evaluated using PROCHECK and Prosa II version 3.0 (30Sippl M.J. Proteins. 1993; 17: 355-362Crossref PubMed Scopus (1736) Google Scholar). Improvements in the model were obtained by an iterative sequence-structure alignment procedure, yielding the final sequence alignment between the Pin1 domain and homologous structures. The modeled structure was stable at room temperature during a 140-ps unconstrained full protein molecular dynamics simulation. Three-dimensional models were visualized by RasMol (31Sayle R. Milner-White E.J. Trends Biochem. Sci. 1995; 20: 374Abstract Full Text PDF PubMed Scopus (2298) Google Scholar), and calculations were performed on Silicon Graphics IRIS 4D/25 workstations. Primary Embryonic Neuronal Culture—Rat primary cortical neurons were prepared from E17-18-day-old Wistar rat embryos as follows. The brain and meninges were the first to be removed. Cortex was carefully dissected out and mechanically dissociated in culture medium by triturating with a polished Pasteur pipette. Once dissociated and after trypan blue counting, cells were plated at a density of 5 × 105 cells/cm2 in poly-d-lysine-coated 6-well plates (BD Biosciences). For dissociation, plating, and maintenance, we used Neurobasal medium supplemented with 2% B27 containing 200 μm glutamine and 1% antibiotic-antimycotic agent (Invitrogen). The medium was replenished every 3 days and 24 h before cell treatment. Pin1 and NF-H cDNAs—Dominant-negative Pin1 was produced by making a point mutation to produce an alanine at serine 16, using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Transfections of wild-type and dominant-negative Pin1 constructs were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Wild-type Pin1 was cloned into pGEX-5X-1 obtained from Amersham Bio-sciences for GST expression studies. Pin1 siRNA—Employing the HiPerformance Design Algorithm, synthetic 21-mer siRNA (short interfering RNA) was designed and synthesized (Qiagen). Pin1 siRNA (silencing) sense and antisense sequences were: 5′-r(GCUCAGGCCGAGUGUACUA)dTdT-3′ and 5′-r(UAGUACACUCGGCCUGAGC)dTdT-3′, respectively. All Stars negative control siRNA, a scrambled, non-silencing control siRNA, was obtained (Qiagen). Sense and antisense sequence for non silencing siRNA were: 5′-r(UUCUCCGAACGUGUCACGU)d(TT)-3′ and 5′-r(ACGUGACACGUUCGGAGAA)d(TT)-3′, respectively. To ensure the absence of possible complementary binding sites in mammalian genomes, the siRNA sequences were extensively checked against the GenBank™ data base using the Smith-Waterman algorithm. This strategy was used to exclude significant sequence homology within the genomes of mammalian species, which may interfere with the target specificity of the siRNA and contribute to unwanted off-target effects in later experiments. The sense and antisense strands were annealed to create the double-stranded siRNA at a 20 μm concentration. Control siRNA and Pin1 siRNA were dissolved in suspension buffer to obtain a 20 μm solution and then heated at 90 °C for 1 min. The final transfection step was preceded by a 60-min incubation at 37 °C. Delivery of siRNA was performed as described previously (11Kesavapany S. Patel V. Zheng Y.L. Pareek T.K. Bjelogrlic M. Albers W. Amin N. Jaffe H. Gutkind J.S. Strong M.J. Grant P. Pant H.C. Mol. Biol. Cell. 2007; 9: 3645-3655Crossref Google Scholar). Transfection efficiency of siRNA in HEK293 cells and cortical neurons was judged to be greater than 80% using fluorescent-labeled oligonucleotides. For experiments using cell lines, cells were transfected with siRNA or the indicated constructs using Lipofectamine 2000 (Invitrogen) in Opti-MEM I for 24 h, and then the medium was changed back to growth medium for additional incubation. For experiments using primary cultures, 5 DIC cortical neurons were transfected with siRNA using Lipofectamine 2000 in Opti-MEM I for 1.5 h. Subsequently, the medium was changed back to neurobasal medium (Invitrogen), and neurons were cultured for additional periods of time before further treatment. HEK293 Cell Culture and Transfection—HEK293 cells were obtained from the American Type Culture Collection, cultured in Dulbecco's modified Eagle's medium with 10% calf serum, and supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. The cells were transiently transfected using Lipofectamine (Invitrogen) according to the manufacturer's instructions. The human NF-H tail domain expression construct, wild-type Pin1, and dominant-negative Pin1 constructs were either transfected independently or co-transfected. Twenty-four hours after transfection, the cells were starved in the presence of 0.2% calf serum overnight (to reduce any back-ground stimulation by serum factors), and then the cells were induced with EGF (10 nm) or PD98059. Finally, the cells were fixed for immunocytochemistry analysis or lysed with lysis buffer for Western blot analysis. Cell Treatment—Cortical neurons 7 days in culture (7 DIC) or HEK293 cells transfected with NF-H for 2 days were treated with 1 mm H2O2 for 1 h, or exposed to 44 °C in a 5% CO2 incubator for 30 min. Thirty percent stock H2O2 (Sigma) was dissolved in 0.2 m phosphate-buffered saline (pH 7.4). JNK inhibitor (SP600125) was purchased from Calbiochem and used at 100 μm in HEK293 cells and cortical neurons. Antibodies—Pin1 antibodies were obtained from Cell Signaling Technologies (Beverly, MA) and Oncogene Research Products and used for Western blotting (1:1,000) and immunohistochemistry (1:100), respectively. RT-97 antibody that specifically stains p-NF-H was provided by Drs. Ralph Nixon and Veeranna (Nathan Kline Institute, Orangeburg, NY) and used at 1:500-1000 dilutions for immunofluorescence and 1:5000 dilutions for Western blotting. SMI31 was obtained from Covance (Princeton, NJ) and used at 1:500 for immunofluorescence and 1:2500 for Western blotting. Anti-tubulin antibody (clone DM1A), total NF-H (clone N52), and 4′, 6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO) were used at 1:10,000 for Western blotting and 1:1,000 for nuclear counter-staining, respectively. Anti-phospho-Jun, c-Jun, JNK, and actin antisera were purchased from Cell Signaling (Beverly, MA). Electrophoresis and Immunoblotting—Equal volumes of cells harvested in Nu-PAGE sample buffer (Invitrogen) were denaturated at 100 °C during 5 min, loaded onto 4-12% Nu-PAGE Novex gels (Invitrogen), and transferred to nitrocellulose. Membranes were blocked in Tris-buffered saline, pH 8, 0.05% Tween 20 with 5% skim milk or bovine serum albumin, and incubated with primary antibody. Membranes were incubated with horseradish peroxidase-labeled secondary antibody (goat anti-rabbit or anti-mouse IgGs, Sigma), and bands were visualized by chemiluminescence (ECL, Amersham Biosciences). Expression and Purification of the Fusion Proteins—The pGEX recombinant plasmid containing Pin1 was expressed and purified as described earlier (32Pant A.C. Veeranna Pant H.C. Amin N. Brain Res. 1997; 2: 259-266Crossref Scopus (59) Google Scholar). Rat NF-H was purified as described (22Jaffe H. Veeranna Shetty K.T. Pant H.C. J. Neurochem. 1995; 64: 2681-2690PubMed Google Scholar). Assay Conditions of Phosphorylation—Bacterially expressed and purified NF-H (2.5 μg) was incubated in an in vitro kinase assay reaction buffer (Tris-HCl, pH 7.4, 5 mm MgCl2, 1 mm each of vanadate, EGTA, EDTA, and dithiothreitol) 1 mm ATP, 1 mm dithiothreitol. Reaction was performed at 30 °C for 2 h with 1 unit of p42/44 MAPK or Cdk5 or JNK. In vitro JNK assay was performed using (GST)-cJun-(1-79) (Stratagene) as a substrate. Endogenous JNK from 7 DIC cortical neurons was immunoprecipitated using a polyclonal JNK3-selective antibody (Santa Cruz Biotechnology, Santa Cruz, CA). c-Jun fusion protein at a concentration of 2 μg/20 μl reaction was phosphorylated using JNK3 in an in vitro kinase assay with 1× Kinase Buffer (25 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 5 mm β-glycerophosphate, 0.1 mm Na3VO4, 2 mm dithiothreitol, 50 μm ATP) and 1 mm ATP. After a 30-min assay at 30 °C, the reaction was terminated by the addition of Laemmli sample buffer (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar), and phosphorylation was detected by Western blot with phospho-specific c-Jun antibodies (Santa Cruz Biotechnology). Immunocytochemical Staining of Neuronal Cultures—Primary rat cortical neurons were plated on polylysine-coated glass coverslips and processed for immunocytochemistry as previously described (34Kesavapany S. Amin N. Zheng Y.L. Nijhara R. Jaffe H. Sihag R. Gutkind J.S. Takahashi S. Kulkarni A. Grant P. Pant H.C. J. Neurosci. 2004; 24: 4421-4431Crossref PubMed Scopus (54) Google Scholar). Coverslips were mounted using Gel-Mount (Biomeda, Foster City, CA). Double immunostainings were carried out using anti-Pin1 and RT-97 antibodies. Pin1 staining was revealed with a goat anti-rabbit IgG (H+L) antibody coupled to Alexa Fluor®488 and RT-97 with a goat anti-mouse IgG (H+L) antibody coupled to Alexa Fluor®568 (Molecular Probes). In situ cytotoxicity kits were obtained from Roche Applied Science (Indianapolis, IN) and TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) staining was performed according to the manufacturer's instructions before immunocytochemistry was carried out. Images were captured with an oil immersion 63× objective on a Zeiss LSM510 using LSM Image Software. Statistics—Each experiment was repeated at least four times. The data were expressed as the means ± S.D. Student's t tests were used to compare the effects of all treatments. The differences were considered statistically significant as: *, p < 0.01; and **, p < 0.001. The presence of multiple SP repeats in NF-H protein suggests that Pin1 could play a direct role in NF-H function by influencing its phosphorylation pattern (Fig. 1A). Comparative modeling based on human Pin1 model (29Bayer E. Goettsch S. Mueller J.W. Griewel B. Guiberman E. Mayr L.M. Bayer P. J. Biol. Chem. 2003; 278: 26183-26193Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) revealed a conserved molecular model for mouse and rat Pin1 (Fig. 1B) with a WW domain on the N terminus and an isomerase catalytic domain on the C terminus (Fig. 1C). We expressed and purified human Pin1 (as a GST fusion protein in Escherichia coli) and rat NF-H (recombinant protein in E. coli) (Fig. 1, D and E). Pin1 Stimulates NF-H Phosphorylation by p42/44 MAPK and Cdk5/p35—Initially, we sought to test the effect of Pin1 on the ability of proline-directed kinases to phosphorylate NF-H. Several proline-directed kinases have been identified, and they function under different physiological conditions. Both, MAPKs (Erk1/2 and JNKs) and CdK5 play pivotal roles in NF phosphorylation and in the development of the nervous system by mediating both neurogenesis and neuronal differentiation. To examine the potential role of Pin1 in NF-H phosphorylation by MAPKs and CdK5, we first used purified NF-H as a substrate. Recombinant and purified NF-H was incubated with p42/44 MAPK in a kinase assay buffer containing ATP either without or with increasing concentrations of GST-Pin1. The phosphorylated NF-H was run on SDS-PAGE and detected by RT-97 antibody, a monoclonal antibody that detects the p-NF-H. Strikingly, addition of GST-Pin1 increased phosphorylation of NF-H in a dose-dependent manner (Fig. 2, A and B). Addition of GST alone to the reaction mixture did not alter the p-NF-H levels (data not shown). We next tested whether Pin1 might also stimulate phosphorylation of NF-H by Cdk5/p35. Bacterially expressed and purified NF-H was incubated with or without GST-Pin1 and CdK5/p35. NF-H phosphorylation was monitored by Western blotting with RT-97 antibody. GST-Pin1 significantly stimulated phosphorylation of NF-H by CdK5/p35 in a concentration-dependent manner (Fig. 2, C and D). In addition, GST-Pin1 elevated the phosphorylation of NF-H in a dose-dependent manner by JNK3 (Fig. 1, E and F). These results suggest that Pin1 facilitates the phosphorylation of NF-H by proline-directed kinases such as Erk1/2, Cdk5, and JNK3. Pin1 Modulates the NF-H Phosphorylation in HEK293 Cells—Because Pin1 has the potential to modulate the NF-H phosphorylation levels in vitro, it was important to determine if Pin1 directly modulates the NF-H phosphorylation levels in situ. HEK293 cells do not express endogenous NF. We initially examined the phosphorylation status of NF-H in NF-H-transfected HEK293 cells by Western blotting. EGF stimulation of HEK293 cells transiently transfected with NF-H increased the p-NF-H, suggesting that MAPKs phosphorylate NF-H in vivo. The EGF-mediated increase in p-NF-H is inhibited by the MAPK inhibitor, PD98059 (Fig. 3, A and B). If the function of the Pin1 is to increase the phosphorylation of NF-H, the cotransfection of NF-H and DN-Pin1 should reduce the p-NF-H levels. A DN-Pin1 construct, made by mutating Ser-16 to Ala, inhibits the isomerase activity of Pin1. Transfected DN-Pin1 migrated as a green fluorescence protein fusion protein at ∼50 kDa, whereas endogenous Pin1 migrated at ∼18 kDa. Expression of NF-H with transfected DN-Pin1 reduced NF-H phosphorylation in EGF-treated transfected cells (Fig. 3, A and B). Furthermore, in EGF-treated transfected cells, endogenous Pin1 colocalizes with p-NF-H (Fig. 3C). In addition, inhibition of Pin1 by Pin1 siRNA but not scrambled siRNA reduced the p-NF-H expression (Fig. 3, D and E). These results demonstrate that Pin1 modulates NF-H phosphorylation. Pin1 Modulates the Oxidative Stress-induced Phosphorylation of NF-H in Transfected HEK293 Cells—We next investigated possible functional consequences of Pin1-dependent hyperphosphorylation of NF-H. Experimental motor neuron disease models are characterized by oxidative stress leading to the accumulation of phospho-NFs within perikarya. Oxidative stress happens to be an early event in AD prior to cytopathology.