GSK-3 Phosphorylation of the Alzheimer Epitope within Collapsin Response Mediator Proteins Regulates Axon Elongation in Primary Neurons

• DOI: 10.1074/jbc.c400412200
• Publication Date: 2004-10-06T01:22:31Z
• Authors: Adam R. Cole; Axel Knebel; Nick A. Morrice; Laura A. Robertson; Andrew J. Irving; Chris N. Connolly; Calum Sutherland
• Keywords: Mediator; Elongation

Elevated glycogen synthase kinase-3 (GSK-3) activity is associated with Alzheimer disease. We have found that collapsin response mediator proteins (CRMP) 2 and 4 are physiological substrates of GSK-3. The amino acids targeted by GSK-3 comprise a hyperphosphorylated epitope first identified in plaques isolated from brain. Expression wild type CRMP2 primary hippocampal neurons or SH-SY5Y neuroblastoma cells promotes axon elongation. However, GSK-3-insensitive mutant has dramatically reduced ability to promote elongation, similar effect pharmacological inhibition Hence, we propose phosphorylation CRMP regulates This work provides direct connection between hyperphosphorylation these residues elevated activity, both which observed Glycogen 1The abbreviations used are: GSK-3, kinase-3; CRMP, protein; rCRMP, rat CRMP; hCRMP, human DYRK, dual tyrosine-regulated kinase; MS, mass spectrometry; GST, glutathione S-transferase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PBS, phosphate-buffered saline; GFP, green fluorescent IMAC, immobilized metal ion affinity chromatographyl; AD, Ser/Thr kinase evolutionarily conserved yeast humans. In mammals, there two closely related isoforms expressed separate genes, namely GSK-3α GSK-3β (1Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1154) Google Scholar). Both ubiquitously expressed, although highest expression the brain been shown phosphorylate more than 30 involved various cellular functions, including metabolism, signal transduction, apoptosis, gene transcription, microtubule dynamics, embryonic development, ubiquitin-mediated degradation, nuclear/cytoplasmic trafficking, only handful confirmed as targets for (for reviews, see Refs. 2Frame S. Cohen P. Biochem. 2001; 359: 1-16Crossref (1280) Scholar 3Doble B.W. Woodgett Cell Sci. 2003; 116: 1175-1186Crossref (1765) Most require prior another at serine threonine located C-terminal site phosphorylated (i.e. S/TXXXS/T(P), where X any acid) (2Frame downstream residue termed "priming" substrate can be regulated indirectly altering kinase. Meanwhile, per se directly distinct sites, Ser21/9 Tyr279/216 GSK-3α/β (4Sutherland C. Leighton I.A. 1993; 296: 15-19Crossref (757) Scholar, 5Cole A. Frame 2004; 377: 249-255Crossref (257) addition, interaction inhibitory proteins. For example, activation Wnt signaling pathway results possibly FRAT/GBP (6Thomas G.M. Goedert M. Nathke I. Polakis FEBS Lett. 1999; 458: 247-251Crossref (205) brain, throughout cell body processes post-mitotic (7Takahashi Tomizawa K. Kato R. Sato Uchida T. Fujita S.C. Imahori Neurochem. 1994; 63: 245-255Crossref (128) 8Leroy Brion J.P. Chem. Neuroanat. 16: 279-293Crossref Its widespread all regions developing adult it hippocampus, thalamus, cortex, Purkinje cerebellum adult, greatest neuronal plasticity 9Yao H.B. Shaw P.C. Wong C.C. Wan D.C. 2002; 23: 291-297Crossref (97) also late embryonic/early post-natal period 10Takahashi Ishiguro Brain Res. 2000; 857: 193-206Crossref (41) Overexpression transgenic mice causes relative reduction size bodies (11Lucas J.J. Hernandez F. Gomez-Ramos Moran M.A. Hen Avila 20: 27-39Crossref (813) 12Spittaels Van den H.C. Dorpe Terwel D. Vandezande Lasrado Bruynseels Irizarry Verhoye Lint Vandenheede Ashton Mercken Loos Hyman B. der L.A. Geerts H. Leuven Neuroscience. 113: 797-808Crossref (87) Conversely, lines reduces elongation rates but increases growth cones (13Owen Gordon-Weeks P.R. Mol. Cell. Neurosci. 626-637Crossref (86) implicated nerve factor control (14Zhou F.Q. Zhou Dedhar Wu Y.H. Snider W.D. Neuron. 42: 897-912Abstract Full Text PDF (461) These observations suggest an important regulator process extension synapse formation, little known about mechanisms regulating effectors Our data show specific on protein key part mechanism molecules combine induce identifies modulator Alzheimer-related epitope. Cloning, Mutagenesis, Protein Expression—The cDNA encoding full-length hCRMP2 was amplified PCR Image clone #6177866 using primers 5′-GGATCCGCCACCATGGACTACAAGGACGACGATGACAAGTCTTATCAGGGGAAGAAAAATATTCCACGC-3′ 5′-GAATTCTTAGCCCAGGCTGGTGATGTTGGC-3′. hCRMP4 #5725550 5′-GAATTCGCCACCATGGACTACAAGGACGACGATGACAAGTCCTACCAAGGCAAGAAGAACATCCCG-3′ 5′-GAATTCTTAACTCAGAGATGTGATATTAGAACGGCCG-3′. products were subcloned into pRK5 mammalian pGEX-6 bacterial expression. mutants hCRMP2/4[S522A/D] generated QuikChange mutagenesis kit (Stratagene). Recombinant DYRK2 Escherichia coli BL21 GST-tagged proteins, while His6-tagged Sf21 described previously (15Frame Biondi R.M. 7: 1321-1327Abstract (575) Purification Substrates Rat Brain—The identification novel (50 male Sprague-Dawley rats) KESTREL performed (16Knebel Morrice N. 4360-4369Crossref (210) Mass Spectrometry—Tryptic peptides analyzed Applied Biosystems 4700 Proteomics Analyser MALDI-TOF-TOF spectrometer 5 mg/ml α-cyanocinnamic acid matrix. spectra acquired reflector mode peptide sequences high energy tandem MS/MS selected precursors. precursor masses tryptic daughter ions experiments scanned against non-redundant databases (Celera) Mascot search software (Matrix Science). Phosphorylated enriched incubating digests 20 min μl PHOS-select™ chelate beads (Sigma) 0.25 m acetic acid/30% (v/v) acetonitrile. then packed pipette tip, washed same buffer, eluted 0.4 ammonium hydroxide. Solid phase sequencing 32P-labeled (17Campbell D.G. N.A. Biomol. Technol. 13: 119-130PubMed Phosphorylation Assays—Purified rCRMP2/4 incubated 1 milliunit milliunits His6-GSK-3β, 10 mm magnesium acetate, 0.1 [γ-32P]ATP buffer containing 50 Tris-HCl, pH 7.5, 0.03% Brij 35, 0.1% 2-mercaptoethanol concentrations times indicated. Reactions subjected SDS-PAGE, transferred nitrocellulose, autoradiographed. 32P-Labeled rCRMP bands Cerenkov counting. phosphatase PP1, without microcystin (1 μm), °C. Microcystin added tubes did not already contain it, followed addition h, °C). Relative amounts phosphate incorporated determined above. GST-hCRMP4 μm) 25 His6-GSK-3β GST-DYRK2, °C priming experiments, 2.5 milliunits/μl unlabeled ATP GST-DYRK2 removed reaction mixture Ni2+-agarose supernatant (2.5 milliunits/μl), Mg-[γ-32P]ATP (final concentration: ATP) Transfection HEK293 Cells—HEK293 transfected hCRMP2/4 vectors calcium method. DNA medium changed either Me2SO inhibitor CT-99021 (2 16 h. lysates 0.5 mg immunoprecipitated anti-FLAG-agarose overnight SDS-PAGE. Gels stained ProQ Diamond phospho-specific stain (Molecular Probes), CBR-250. LipofectAMINE 2000 (Invitrogen). Following incubation 37 60 fixed 4% (w/v) paraformaldehyde saline (PBS), permeabilized Triton X-100 blocked 10% fetal bovine serum, 0.5% serum albumin, anti-FLAG monoclonal antibody (Sigma), anti-mouse secondary pre-conjugated Alexa488 fluorophore. Hippocampal Neuron Culture, Transfection, Immunofluorescence Analysis—Hippocampi 2–3-day-old rats (18Rae M.G. Martin D.J. Collingridge G.L. Irving A.J. 8628-8636Crossref plating, neurobasal 2% B27 replacement l-glutamine 3–4 h 2000. After 36 fixed, permeabilized, sequentially anti-MAP2 (rabbit polyclonal; Chemicon) antibodies simultaneously anti-rabbit antibodies, conjugated Cy5 fluorophores, respectively. A scanning confocal imaging system (LSM 510 Carl Zeiss, Oberkochen, Germany) accompanying image acquisition analysis (cells analyzed; n = 35 > 80 hCRMP2[S522A]). Identification Novel Substrate GSK-3—The technique Scholar) identify lysate. brains homogenized fractionated heparin chromatography. An aliquot each fraction recombinant radiolabeled ATP. autophosphorylation band, several (potential substrates) varying molecular weight detected. Of particular interest group present Mr 60,000–70,000. fractions further purified exchange gel filtration (62,000–64,000 Mr) excised gel, digested trypsin, fingerprinting. 28 matched (rCRMP) (accession number gi 1351260), covering 62.8% protein, rCRMP4 25742568), 51.6% protein. Some detected common isoforms, many isoform. contained consensus No other detectable digest mixture. CRMPs family five microtubule-associated primarily during development (19Fukata Y. Kimura Kaibuchi 43: 305-315Crossref 20Charrier E. Reibel Rogemond V. Aguera Thomasset Honnorat Neurobiol. 28: 51-64Crossref (234) 21Arimura Menager Fukata 58: 34-47Crossref (150) screen mediate Semaphorin 3A (22Goshima Nakamura Strittmatter S.M. Nature. 1995; 376: 509-514Crossref (638) It formation polarization elongating axons hippocampus (23Inagaki Chihara Arimura Kawano Matsuo Nishimura Amano Nat. 4: 781-782Crossref (463) binds tubulin heterodimers preference polymerized thought play regulatory role polymerization microtubules (24Fukata Itoh T.J. Shiromizu Watanabe Inagaki Iwamatsu Hotani Biol. 583-591Crossref (635) Vitro—The rCRMP2 label roughly 62 64 kDa individually chromatography (IMAC) before being MS analysis. Three phosphopeptides band (T1–T3 Fig. 1A), four sites analyzing sequence (data lower 1B). They represented equivalent isoform; Ser522, Ser518, Thr514, Thr509 (Fig. example N-terminal Ser Thr residue. Thus, will modified include this variation. IMAC-enriched (T1–T3) solid γ-32P cycles corresponding Thr509, respectively shown). phosphopeptide phospho-Ser522 (T2 1B), suggesting Ser522 when Therefore, intact acts subsequent phosphorylation. To compare efficiency GSK-3β, up 1D). amount increased proportionally time Similar incorporation isoform observed. Preincubation PP1 (to remove endogenous phosphorylation) ablated 1E). confirms least portion Similarly, bacterially appreciable extent 2), demonstrating target Examination CRMP4 surrounding tyrosine (DYRK) likely Indeed, almost stoichiometrically 2A). major (and only) preincubated GSK-3β. Nearly 1.2 mol phosphate/mol following preincubation 2B). Ser518 phoshorylation sufficient prime vitro. Cells—It demonstrate vitro occurs vivo. FLAG-tagged presence absence 2C). compound most selective reported date (25Cohen Rev. Drug Discov. 3: 479-487Crossref (671) lysis, immunopurified gels Diamond, specifically detects phospho-proteins CBR-250 quantify loading. 2C) 2D) phosphorylated; however, completely inhibited co-incubation CT-99021. → Ala (S522A) Asp (S522D) prevented 2, C D). raised representing hCRMP2-(505–517) Thr514 2E). recognizes (509 514) 509 514. detect hCRMP2[S522D]. Together, three critical mutation negatively charged aspartate does substitute priming, non-consensus cells. Functional Consequences Neuronal transfection N1E-115 enhances neurite line, SH-SY5Y, hCRMP2, hCRMP2[S522A] (in differentiation factors (26Jamsa Hasslund Cowburn R.F. Backstrom Vasange Biophys. Commun. 319: 993-1000Crossref (142) Scholar)). Transfected immunoflourescence LSM software. induced 2–3-fold increase bearing neurites greater μm length 4-fold compared GFP 3, A–C). contrast, ∼1.5-fold over control, no significant difference experiment, cultures constructs D E). Axons dendrites differentially according their morphology staining somato-dendritic marker, MAP2. As Scholar), display (427.5 versus 280.11 average length, p < 0.0005) GFP-transfected D–F). There trend (but difference) toward (average length: 305.9 significantly (p 0.0002) those hCRMP2[S522A]. clear differences 200 400 outgrowth initiate neuroblastoma, greatly actions. polarity requires series complex intracellular initiated regulated. manipulation actin structure allows transport designate phenotype appropriate neurite. assembly required cone positioning Inhibition reduce protect apoptosis (27Pap Cooper 1998; 273: 19929-19932Abstract (955) 28Cross D.A. Culbert A.A. Chalmers K.A. Facci L. Skaper S.D. Reith A.D. 77: 94-102Crossref (343) regulate rate direction phosphorylates tau, promoting dissociation (29Wang J.Z. Q. Smith Grundke-Iqbal Iqbal 436: 28-34Crossref (174) 1B (MAP1B) presumably via tau way, proposed stability. now growth. considerable genetic biochemical evidence influences progression disease (AD). Patients AD develop insoluble deposits β-amyloid production aggregation promoted (30Phiel C.J. Wilson C.A. Lee V.M. Klein P.S. 423: 435-439Crossref (1091) tissue commonly contains neurofibrillary tangles, formed well characterized precise role(s) "tau-opathies" remains subject much study. Interestingly, abnormal linked tissue, exhibit match study (31Gu Hamajima Ihara Biochemistry. 39: 4267-4275Crossref (156) course, whether cause consequence affected up-regulation AD. summary, cluster residues, modification function CRMP2. inhibitors polarity, loss regulation and/or hence may explain association neurological disorders. thank James Hastie, Hilary Mclauchlan, Rudolfo Marquez, Sir Philip Cohen, School Life Sciences, University Dundee reagents Dervla O'Malley, Dave Finlay, Chris Lipina, Laidlaw, Laura Burgess technical advice assistance.