Key points are not available for this paper at this time.
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 Alzheimer brain. Expression of wild type CRMP2 in primary hippocampal neurons or SH-SY5Y neuroblastoma cells promotes axon elongation. However, a GSK-3-insensitive CRMP2 mutant has dramatically reduced ability to promote axon elongation, a similar effect to pharmacological inhibition of GSK-3. Hence, we propose that phosphorylation of CRMP proteins by GSK-3 regulates axon elongation. This work provides a direct connection between hyperphosphorylation of these residues and elevated GSK-3 activity, both of which are observed in Alzheimer brain. 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 Alzheimer brain. Expression of wild type CRMP2 in primary hippocampal neurons or SH-SY5Y neuroblastoma cells promotes axon elongation. However, a GSK-3-insensitive CRMP2 mutant has dramatically reduced ability to promote axon elongation, a similar effect to pharmacological inhibition of GSK-3. Hence, we propose that phosphorylation of CRMP proteins by GSK-3 regulates axon elongation. This work provides a direct connection between hyperphosphorylation of these residues and elevated GSK-3 activity, both of which are observed in Alzheimer brain. Glycogen synthase kinase-3 (GSK-3) 1The abbreviations used are: GSK-3, glycogen synthase kinase-3; CRMP, collapsin response mediator protein; rCRMP, rat CRMP; hCRMP, human CRMP; 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 protein; IMAC, immobilized metal ion affinity chromatographyl; AD, Alzheimer disease. is a Ser/Thr kinase evolutionarily conserved from yeast to humans. In mammals, there are two closely related isoforms expressed from separate genes, namely GSK-3α and GSK-3β (1Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1154) Google Scholar). Both are ubiquitously expressed, although highest expression is found in the brain (1Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1154) Google Scholar). GSK-3 has been shown to phosphorylate more than 30 proteins involved in various cellular functions, including glycogen metabolism, signal transduction, apoptosis, gene transcription, microtubule dynamics, embryonic development, ubiquitin-mediated degradation, and nuclear/cytoplasmic trafficking, although only a handful of these proteins have been confirmed as physiological targets for GSK-3 (for reviews, see Refs. 2Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1280) Google Scholar and 3Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1765) Google Scholar). Most GSK-3 substrates require prior phosphorylation by another kinase at a serine or threonine located 4 residues C-terminal to the site phosphorylated by GSK-3 (i.e. S/TXXXS/T(P), where X is any amino acid) (2Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1280) Google Scholar). This downstream residue is termed a “priming” site and phosphorylation of the substrate by GSK-3 can be regulated indirectly by altering the activity of the “priming” kinase. Meanwhile, GSK-3 activity per se is regulated directly by phosphorylation at two distinct sites, Ser21/9 and Tyr279/216 in GSK-3α/β (4Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (757) Google Scholar, 5Cole A. Frame S. Cohen P. Biochem. J. 2004; 377: 249-255Crossref PubMed Scopus (257) Google Scholar). In addition, GSK-3 activity is regulated by interaction with inhibitory proteins. For example, activation of the Wnt signaling pathway results in the inhibition of GSK-3 activity, possibly by interaction with FRAT/GBP (6Thomas G.M. Frame S. Goedert M. Nathke I. Polakis P. Cohen P. FEBS Lett. 1999; 458: 247-251Crossref PubMed Scopus (205) Google Scholar). In the brain, GSK-3 is expressed throughout the cell body and processes of post-mitotic neurons (7Takahashi M. Tomizawa K. Kato R. Sato K. Uchida T. Fujita S.C. Imahori K. J. Neurochem. 1994; 63: 245-255Crossref PubMed Scopus (128) Google Scholar, 8Leroy K. Brion J.P. J. Chem. Neuroanat. 1999; 16: 279-293Crossref PubMed Scopus (205) Google Scholar). Its expression is widespread throughout all regions of the developing and adult brain, although it is highest in the hippocampus, thalamus, cortex, and Purkinje cells of the cerebellum in the adult, which are regions of greatest neuronal plasticity (7Takahashi M. Tomizawa K. Kato R. Sato K. Uchida T. Fujita S.C. Imahori K. J. Neurochem. 1994; 63: 245-255Crossref PubMed Scopus (128) Google Scholar, 8Leroy K. Brion J.P. J. Chem. Neuroanat. 1999; 16: 279-293Crossref PubMed Scopus (205) Google Scholar, 9Yao H.B. Shaw P.C. Wong C.C. Wan D.C. J. Chem. Neuroanat. 2002; 23: 291-297Crossref PubMed Scopus (97) Google Scholar). Elevated expression is also observed at the late embryonic/early post-natal period (7Takahashi M. Tomizawa K. Kato R. Sato K. Uchida T. Fujita S.C. Imahori K. J. Neurochem. 1994; 63: 245-255Crossref PubMed Scopus (128) Google Scholar, 8Leroy K. Brion J.P. J. Chem. Neuroanat. 1999; 16: 279-293Crossref PubMed Scopus (205) Google Scholar, 10Takahashi M. Tomizawa K. Ishiguro K. Brain Res. 2000; 857: 193-206Crossref PubMed Scopus (41) Google Scholar). Overexpression of GSK-3β activity in transgenic adult mice causes a relative reduction in the size of neuronal cell bodies (11Lucas J.J. Hernandez F. Gomez-Ramos P. Moran M.A. Hen R. Avila J. EMBO J. 2001; 20: 27-39Crossref PubMed Scopus (813) Google Scholar, 12Spittaels K. Van den H.C. Van Dorpe J. Terwel D. Vandezande K. Lasrado R. Bruynseels K. Irizarry M. Verhoye M. Van Lint J. Vandenheede J.R. Ashton D. Mercken M. Loos R. Hyman B. Van der L.A. Geerts H. Van Leuven F. Neuroscience. 2002; 113: 797-808Crossref PubMed Scopus (87) Google Scholar). Conversely, inhibition of GSK-3 in neuronal cell lines reduces axon elongation rates but increases the size of axon growth cones (13Owen R. Gordon-Weeks P.R. Mol. Cell. Neurosci. 2003; 23: 626-637Crossref PubMed Scopus (86) Google Scholar). GSK-3 is also implicated in nerve growth factor control of axon growth (14Zhou F.Q. Zhou J. Dedhar S. Wu Y.H. Snider W.D. Neuron. 2004; 42: 897-912Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). These observations suggest that GSK-3 is an important regulator of neuronal process extension and synapse formation, although little is known about the mechanisms regulating the downstream effectors of GSK-3 in the brain. Our data show that phosphorylation of specific residues on collapsin response mediator protein (CRMP) 2 by GSK-3 is a key part of the mechanism by which these molecules combine to induce axon growth and identifies GSK-3 as the modulator of an Alzheimer-related epitope. Cloning, Mutagenesis, and Protein Expression—The cDNA encoding full-length hCRMP2 was amplified by PCR from Image clone #6177866 using the primers 5′-GGATCCGCCACCATGGACTACAAGGACGACGATGACAAGTCTTATCAGGGGAAGAAAAATATTCCACGC-3′ and 5′-GAATTCTTAGCCCAGGCTGGTGATGTTGGC-3′. The cDNA encoding full-length hCRMP4 was amplified by PCR from Image clone #5725550 using the primers 5′-GAATTCGCCACCATGGACTACAAGGACGACGATGACAAGTCCTACCAAGGCAAGAAGAACATCCCG-3′ and 5′-GAATTCTTAACTCAGAGATGTGATATTAGAACGGCCG-3′. The PCR products were subcloned into pRK5 for mammalian or pGEX-6 for bacterial expression. The mutants hCRMP2/4S522A/D were generated using the QuikChange mutagenesis kit (Stratagene). Recombinant DYRK2 were expressed in Escherichia coli BL21 cells as GST-tagged proteins, while GSK-3β was expressed as a His6-tagged protein in Sf21 cells as described previously (15Frame S. Cohen P. Biondi R.M. Mol. Cell. 2001; 7: 1321-1327Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). Purification of GSK-3 Substrates from Rat Brain—The identification of novel GSK-3 substrates from brain (50 male Sprague-Dawley rats) by KESTREL was performed as described previously (16Knebel A. Morrice N. Cohen P. EMBO J. 2001; 20: 4360-4369Crossref PubMed Scopus (210) Google Scholar). Mass Spectrometry—Tryptic peptides were analyzed using an Applied Biosystems 4700 Proteomics Analyser MALDI-TOF-TOF mass spectrometer with 5 mg/ml α-cyanocinnamic acid as the matrix. Mass spectra were acquired in the reflector mode and peptide sequences were confirmed by high energy tandem MS/MS of selected precursors. The precursor ion masses of tryptic peptides and the daughter ions from MS/MS experiments were scanned against non-redundant protein databases (Celera) using Mascot search software (Matrix Science). Phosphorylated tryptic peptides were enriched by incubating the tryptic digests for 20 min with 2 μl of PHOS-select™ metal chelate beads (Sigma) in 0.25 m acetic acid/30% (v/v) acetonitrile. The beads were then packed into a pipette tip, washed with the same buffer, and the peptides eluted with 20 μl of 0.4 m ammonium hydroxide. Solid phase sequencing of 32P-labeled peptides was performed as described previously (17Campbell D.G. Morrice N.A. J. Biomol. Technol. 2002; 13: 119-130PubMed Google Scholar). Phosphorylation Assays—Purified rCRMP2/4 was incubated with 1 milliunit or 5 milliunits of His6-GSK-3β, 10 mm magnesium acetate, and 0.1 mm γ-32PATP in buffer containing 50 mm Tris-HCl, pH 7.5, 0.03% (v/v) Brij 35, and 0.1% (v/v) 2-mercaptoethanol at the concentrations and times indicated. Reactions were subjected to SDS-PAGE, transferred to nitrocellulose, and autoradiographed. 32P-Labeled rCRMP bands were analyzed by Cerenkov counting. rCRMP2/4 was incubated with 10 milliunits of the phosphatase PP1, with or without microcystin (1 μm), for 30 min at 30 °C. Microcystin was then added to the tubes that did not already contain it, followed by addition of 5 milliunits of His6-GSK-3β, 10 mm magnesium acetate, and 0.1 mm γ-32PATP to all tubes (1 h, 30 °C). Relative amounts of phosphate incorporated into rCRMP2/4 were determined as described above. Recombinant GST-hCRMP4 (1 μm) was phosphorylated using 25 milliunits of His6-GSK-3β or GST-DYRK2, 10 mm magnesium acetate, and 0.1 mm γ-32PATP at 30 °C for the times indicated. For priming experiments, GST-hCRMP4 (1 μm) was phosphorylated using 2.5 milliunits/μl GST-DYRK2, 10 mm magnesium acetate, and 0.1 mm unlabeled ATP for 30 min at 30 °C. GST-DYRK2 was removed from the reaction mixture by addition of Ni2+-agarose and the supernatant incubated with His6-GSK-3β (2.5 milliunits/μl), Mg-γ-32PATP (final concentration: 10 mm magnesium acetate, 0.1 mm ATP) at 30 °C for the times indicated. Transfection of HEK293 and SH-SY5Y Cells—HEK293 cells were transfected with wild type and mutant hCRMP2/4 expression vectors using the calcium phosphate method. DNA was incubated with the cells for 4 h, then the medium changed and either Me2SO or the GSK-3β inhibitor CT-99021 (2 μm) was added for 16 h. Cell lysates containing 0.5 mg of protein were immunoprecipitated with anti-FLAG-agarose beads overnight at 4 °C and then subjected to SDS-PAGE. Gels were stained with ProQ Diamond phospho-specific stain (Molecular Probes), followed by CBR-250. SH-SY5Y neuroblastoma cells were transfected using LipofectAMINE 2000 (Invitrogen). Following incubation at 37 °C for 60 h, the cells were fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.1% (v/v) Triton X-100 in PBS, blocked in 10% (v/v) fetal bovine serum, 0.5% (w/v) bovine serum albumin, and 0.1% (v/v) Triton X-100 in PBS, then incubated with an anti-FLAG monoclonal antibody (Sigma), followed by an anti-mouse secondary antibody pre-conjugated to the Alexa488 fluorophore. Hippocampal Neuron Cell Culture, Transfection, and Immunofluorescence Analysis—Hippocampi isolated from 2–3-day-old Sprague-Dawley rats were isolated as described previously (18Rae M.G. Martin D.J. Collingridge G.L. Irving A.J. J. Neurosci. 2000; 20: 8628-8636Crossref PubMed Google Scholar). Following plating, cells were incubated in neurobasal medium containing 2% (v/v) B27 serum replacement (Sigma) and 2 mm l-glutamine for 3–4 h and then transfected using LipofectAMINE 2000. After 36 h, the cells were fixed, permeabilized, and blocked as described above. The cells were sequentially incubated with anti-FLAG and anti-MAP2 (rabbit polyclonal; Chemicon) antibodies and then incubated simultaneously with anti-mouse and anti-rabbit secondary antibodies, conjugated to Alexa488 and Cy5 fluorophores, respectively. A laser scanning confocal imaging system (LSM 510 Carl Zeiss, Oberkochen, Germany) and accompanying software was used for image acquisition and analysis (cells analyzed; n = 35 GFP, n > 80 hCRMP2 and n > 80 hCRMP2S522A). Identification of a Novel Substrate of GSK-3—The KESTREL technique (16Knebel A. Morrice N. Cohen P. EMBO J. 2001; 20: 4360-4369Crossref PubMed Scopus (210) Google Scholar) was used to identify novel GSK-3 substrates in rat brain lysate. Rat brains were homogenized and fractionated by heparin chromatography. An aliquot from each fraction was incubated with recombinant His6-GSK-3β and radiolabeled ATP. In addition to the GSK-3β autophosphorylation band, several 32P-labeled proteins (potential GSK-3 substrates) of varying molecular weight were detected. Of particular interest were a group of 32P-labeled bands present at Mr 60,000–70,000. The fractions containing these proteins were further purified by ion exchange and gel filtration chromatography. These substrates (62,000–64,000 Mr) were excised from a gel, digested with trypsin, and identified by peptide mass fingerprinting. The masses of 28 peptides were matched with rat collapsin response mediator protein (rCRMP) 2 (accession number gi 1351260), covering 62.8% of the protein, while the masses of 25 peptides were matched with rCRMP4 (accession number gi 25742568), covering 51.6% of the protein. Some of the peptides detected were common to both isoforms, although many were specific for either isoform. Both isoforms contained consensus sequences for phosphorylation by GSK-3. No other proteins were detectable in the tryptic digest mixture. CRMPs are a family of five microtubule-associated proteins primarily expressed during neuronal development (19Fukata Y. Kimura T. Kaibuchi K. Neurosci. Res. 2002; 43: 305-315Crossref PubMed Scopus (86) Google Scholar, 20Charrier E. Reibel S. Rogemond V. Aguera M. Thomasset N. Honnorat J. Mol. Neurobiol. 2003; 28: 51-64Crossref PubMed Scopus (234) Google Scholar, 21Arimura N. Menager C. Fukata Y. Kaibuchi K. J. Neurobiol. 2004; 58: 34-47Crossref PubMed Scopus (150) Google Scholar). CRMP2 was first identified in a screen for proteins that mediate Semaphorin 3A activity (22Goshima Y. Nakamura F. Strittmatter P. Strittmatter S.M. Nature. 1995; 376: 509-514Crossref PubMed Scopus (638) Google Scholar). It is key to axon formation during neuronal polarization and is enriched in elongating axons of the hippocampus (23Inagaki N. Chihara K. Arimura N. Menager C. Kawano Y. Matsuo N. Nishimura T. Amano M. Kaibuchi K. Nat. Neurosci. 2001; 4: 781-782Crossref PubMed Scopus (463) Google Scholar). CRMP2 binds to tubulin heterodimers in preference to polymerized tubulin and is thought to play a regulatory role in polymerization of microtubules (24Fukata Y. Itoh Kimura T. Menager C. Nishimura T. T. H. N. A. H. Kaibuchi K. Nat. Cell 2002; 4: PubMed Scopus Google Scholar). CRMP Phosphorylation by GSK-3 in mixture of purified and rCRMP4 was incubated with GSK-3β and γ-32PATP to the proteins and two 32P-labeled bands of and were observed by SDS-PAGE. The bands were digested and phosphorylated tryptic peptides enriched using immobilized metal ion affinity subjected to were detected in each in while phosphorylation were determined by the MS/MS data using Mascot software for the is shown in residues in each namely and Phosphorylation of is the first of a GSK-3 phosphorylation site located 5 residues to a or the consensus for phosphorylation by GSK-3 have to be to The were also subjected to phase was detected in to phosphorylation of and not was not detected in but a containing was detected by in that was already phosphorylated purified from rat brain. is phosphorylated in brain and as a priming site for GSK-3 the of phosphorylation by the purified rCRMP isoforms were incubated with GSK-3β and γ-32PATP for to 1 h The of incorporated into and rCRMP4 with rates of of into each were of purified and rCRMP4 with the protein phosphatase the ability of GSK-3β to phosphorylate rCRMP2/4 in a reaction This that at a of the purified rCRMP is phosphorylated at priming for phosphorylation by GSK-3. GSK-3β did not phosphorylate expressed GST-hCRMP4 to any that is not a for GSK-3. of the primary identified dual regulated kinase as a kinase. purified DYRK2 phosphorylated GST-hCRMP4 Meanwhile, analysis identified as the possibly residue phosphorylated by DYRK2 not GST-hCRMP4 was with DYRK2 to promote phosphorylation of prior to incubation with of of GST-hCRMP4 was incorporated into hCRMP4 by GSK-3β but only of hCRMP4 with DYRK2 The residues in GST-hCRMP4 phosphorylated by GSK-3 were identified by as and not is to for phosphorylation by GSK-3β in CRMP Phosphorylation by GSK-3 in is important to that phosphorylation observed in also in HEK293 cells were transfected with wild type hCRMP2 or hCRMP4 in the or of the GSK-3 inhibitor CT-99021 This is the GSK-3 inhibitor to P. Goedert M. Nat. 2004; PubMed Scopus Google Scholar). Following cell hCRMP2 and hCRMP4 were using anti-FLAG-agarose beads and subjected to SDS-PAGE. The were stained with ProQ which and then to Both hCRMP2 and hCRMP4 were was by with In addition, expression of the priming site mutants and phosphorylation of either and Phosphorylation of wild type hCRMP2 was confirmed using a phospho-specific antibody against a peptide phosphorylated at and This antibody dual phosphorylated peptide and but not peptide phosphorylated at only or This antibody did not these data that hCRMP2 and hCRMP4 are phosphorylated at residues by GSK-3 in cells and that phosphorylation of is for priming in In addition, of to the residue not for while the phosphorylation of by GSK-3 in of CRMP2 Phosphorylation by GSK-3 in has previously been that of neuroblastoma cells or primary neurons with hCRMP2 formation and growth (24Fukata Y. Itoh Kimura T. Menager C. Nishimura T. T. H. N. A. H. Kaibuchi K. Nat. Cell 2002; 4: PubMed Scopus Google Scholar). we transfected the human neuroblastoma cell with mammalian expression vectors containing either GFP, or and incubated for 60 h the of other A. K. A. M. Biochem. Res. 2004; PubMed Scopus Google cells were detected by and analyzed using 510 image Transfection with hCRMP2 a in the number of transfected cells than 20 in and a in cells than 50 in with cells transfected with In with only the number of cells than 20 in while there was in the number of cells with than 50 in between and control In a separate primary hippocampal were transfected with the same and and were identified to and for the previously (24Fukata Y. Itoh Kimura T. Menager C. Nishimura T. T. H. N. A. H. Kaibuchi K. Nat. Cell 2002; 4: PubMed Scopus Google cells transfected with wild type hCRMP2 a in axon elongation axon with neurons was a axon elongation in cells transfected with μm) with control However, the axon of transfected neurons containing wild type hCRMP2 was than containing In addition, in the number of cells with axons or were observed These data that CRMP2 expression can promote axon in primary neurons and extension in a while phosphorylation by GSK-3 these a of processes to be and For example, of of axon specific proteins that to the Meanwhile, of microtubules is for axon and growth (19Fukata Y. Kimura T. Kaibuchi K. Neurosci. Res. 2002; 43: 305-315Crossref PubMed Scopus (86) Google Scholar). of GSK-3 is known to axon (13Owen R. Gordon-Weeks P.R. Mol. Cell. Neurosci. 2003; 23: 626-637Crossref PubMed Scopus (86) Google Scholar) and also against neuronal M. G.M. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Neurochem. 2001; PubMed Scopus Google Scholar). many microtubule-associated proteins that the and of axon growth are known to be substrates for GSK-3. For example, GSK-3 of protein from microtubules Wu A. I. K. FEBS Lett. PubMed Scopus Google Scholar). In addition, phosphorylation of microtubule-associated protein regulates microtubule dynamics, similar mechanisms to (13Owen R. Gordon-Weeks P.R. Mol. Cell. Neurosci. 2003; 23: 626-637Crossref PubMed Scopus (86) Google Scholar). In GSK-3 is to microtubule We propose that phosphorylation of CRMP proteins is another mechanism by which GSK-3 regulates axon is and that GSK-3 the of Alzheimer with neuronal plaques that contain of protein. The and of the protein is by GSK-3α while reduced by pharmacological inhibition of GSK-3α Nature. 2003; PubMed Scopus Google Scholar). In addition, which are in part by hyperphosphorylation of the microtubule-associated protein another substrate of GSK-3. Hence, the of GSK-3 in the development of and other related the of CRMP2 expression has been to development, while hyperphosphorylated CRMP2 is found in and the residues that phosphorylation identified as targets for GSK-3 in Y. N. Y. 2000; PubMed Scopus Google Scholar). Of phosphorylation of CRMP2 by GSK-3 is a or a of require further We show that axon growth be by hyperphosphorylation of epitope of GSK-3 activity in In GSK-3 CRMP2 and on a of and regulates a of This interaction play a role in the observed effect of GSK-3 on axon growth and neuronal while of of GSK-3 the kinase CRMP the of both GSK-3 and CRMP2 with the development of a number of We and of of for and and for and
Cole et al. (Wed,) studied this question.