The unicellular, parasitic fungi of the phylum Sanchytriomycota (sanchytrids) were discovered a few years ago. These unusual chytrid-like fungi parasitize algae. The zoospores of the species of the phylum contain an extremely long kinetosome composed of microtubular singlets or doublets and a non-motile pseudocilium (i.e., a reduced posterior flagellum). Fungi provide an ideal opportunity to test and confirm the correlation between the occurrence of flagellar proteins (the ciliome) and that of the eukaryotic cilium/flagellum since the flagellum occurs in the early-branching phyla and not in terrestrial fungi. Tubulin polymerization promoting protein (TPPP)-like proteins, which contain a p25alpha domain, were also suggested to belong to the ciliome and are present in flagellated fungi. Although sanchytrids have lost many of the flagellar proteins, here it is shown that they possess a DNA sequence possibly encoding long (animal-type) TPPP, but not the fungal-type one characteristic of chytrid fungi. Phylogenetic analysis of p25alpha domains placed sanchytrids into a sister position to Blastocladiomycota, similarly to species phylogeny, with maximal support.
Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Δ3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 ± 0.3, 2.2 ± 0.3, and 0.9 ± 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr14, Ser18, Ser160 for Cdk5; Ser18, Ser160 for ERK2, and Ser32 for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (Kd = 2.5-2.7 μm) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr14 and/or Ser18 in its unfolded N-terminal tail. Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Δ3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 ± 0.3, 2.2 ± 0.3, and 0.9 ± 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr14, Ser18, Ser160 for Cdk5; Ser18, Ser160 for ERK2, and Ser32 for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (Kd = 2.5-2.7 μm) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr14 and/or Ser18 in its unfolded N-terminal tail. Previously we isolated a new, flexible, intrinsically unstructured protein from bovine brain, which was denoted tubulin polymerization-promoting protein (TPPP) 3The abbreviations used are:TPPPtubulin polymerization-promoting proteinMTmicrotubuleTEMtransmission electron microscopyERK2extracellular signal-regulated protein kinase 2PKAcAMP-dependent protein kinaseCdk5cyclin-dependent kinase 5MAPmicrotubule-associated proteinPSDpost source decayCIDcollision-induced dissociationDTTdithiothreitolMOPS4-morpholinepropanesulfonic acidMES4-morpholineethanesulfonic acidGAPDHglyceraldehyde-3-phosphate dehydrogenase.3The abbreviations used are:TPPPtubulin polymerization-promoting proteinMTmicrotubuleTEMtransmission electron microscopyERK2extracellular signal-regulated protein kinase 2PKAcAMP-dependent protein kinaseCdk5cyclin-dependent kinase 5MAPmicrotubule-associated proteinPSDpost source decayCIDcollision-induced dissociationDTTdithiothreitolMOPS4-morpholinepropanesulfonic acidMES4-morpholineethanesulfonic acidGAPDHglyceraldehyde-3-phosphate dehydrogenase./p25 accordingly to its in vitro function, tubulin polymerization-promoting protein, and its molecular mass (1Tirián L. Hlavanda E. Oláh J. Horváth I. Orosz F. Szabó B. Kovács J. Szabad J. Ovádi J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13976-13981Crossref PubMed Scopus (92) Google Scholar). In the HGNC data base this protein is assigned as TPPP, the first member of TPPP family. We have shown that TPPP can induce formation of double-walled microtubules (MTs) and aberrant tubulin aggregates (2Hlavanda E. Kovács J. Oláh J. Orosz F. Medzihradszky K.F. Ovádi J. Biochemistry. 2002; 41: 8657-8664Crossref PubMed Scopus (100) Google Scholar) as well as that it stabilizes MT network via its bundling activity in human cells (3Lehotzky A. Tirián L. Tőkési N. Lénárt P. Szabó B. Kovács J. Ovádi J. J. Cell Sci. 2004; 117: 6249-6259Crossref PubMed Scopus (56) Google Scholar). There are two TPPP homologues, p18 (TPPP2) and p20 (TPPP3) with distinct structural and functional features concerning their folding and tubulin binding properties (4Vincze O. Tőkési N. J. Oláh Hlavanda E. Zotter Á. Horváth I. Lehotzky A. Tirián L. Medzihradszky K.F. Kovács J. Orosz F. Ovádi J. Biochemistry. 2006; 45: 13818-13826Crossref PubMed Scopus (68) Google Scholar). The physiological functions of the TPPP protein family are unknown; however, TPPP has been suggested to take part in the stabilization of the MT network (3Lehotzky A. Tirián L. Tőkési N. Lénárt P. Szabó B. Kovács J. Ovádi J. J. Cell Sci. 2004; 117: 6249-6259Crossref PubMed Scopus (56) Google Scholar). tubulin polymerization-promoting protein microtubule transmission electron microscopy extracellular signal-regulated protein kinase 2 cAMP-dependent protein kinase cyclin-dependent kinase 5 microtubule-associated protein post source decay collision-induced dissociation dithiothreitol 4-morpholinepropanesulfonic acid 4-morpholineethanesulfonic acid glyceraldehyde-3-phosphate dehydrogenase. tubulin polymerization-promoting protein microtubule transmission electron microscopy extracellular signal-regulated protein kinase 2 cAMP-dependent protein kinase cyclin-dependent kinase 5 microtubule-associated protein post source decay collision-induced dissociation dithiothreitol 4-morpholinepropanesulfonic acid 4-morpholineethanesulfonic acid glyceraldehyde-3-phosphate dehydrogenase. Both α-synuclein and GAPDH directly bind to TPPP and co-localize in the Lewy body (5Kovács G.G. László L. Kovács J. Jensen P.H. Lindersson E. Botond G. Molnár T. Perczel A. Hudecz F. Mező G. Erdei A. Tirián L. Lehotzky A. Gelpi E. Budka H. Ovádi J. Neurobiol. Dis. 2004; 17: 155-162Crossref PubMed Scopus (113) Google Scholar, 6Oláh J. Tőkési N. Vincze O. Horváth I. Lehotzky A. Erdei A. Szájli E. Medzihradszky K.F. Orosz F. Kovács G.G. Ovádi J. FEBS Lett. 2006; 580: 5807-5814Crossref PubMed Scopus (30) Google Scholar, 7Lindersson E. Lundvig D. Petersen C. Madsen P. Nyengaard J.R. Hojrup P. Moos T. Otzen D. Gai W.P. Blumbergs P.C. Jensen P.H. J. Biol. Chem. 2005; 280: 5703-5715Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Our immunohistochemistry studies revealed enrichment of TPPP in α-synuclein-positive inclusions characteristic for synucleinopathies but not for tauopathies (5Kovács G.G. László L. Kovács J. Jensen P.H. Lindersson E. Botond G. Molnár T. Perczel A. Hudecz F. Mező G. Erdei A. Tirián L. Lehotzky A. Gelpi E. Budka H. Ovádi J. Neurobiol. Dis. 2004; 17: 155-162Crossref PubMed Scopus (113) Google Scholar). TPPP is similar to α-synuclein and tau (2Hlavanda E. Kovács J. Oláh J. Orosz F. Medzihradszky K.F. Ovádi J. Biochemistry. 2002; 41: 8657-8664Crossref PubMed Scopus (100) Google Scholar, 8Orosz F. Kovács G.G. Lehotzky A. Oláh J. Vincze O. Ovádi J. Biol. Cell. 2004; 96: 701-711Crossref PubMed Scopus (48) Google Scholar) as far as all of them belong to the family of the unstructured proteins. Protein phosphorylation has a significant impact in the etiology of neurodegenerative processes (9Murray A.W. Cell. 1998; 92: 157-159Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). For example, tau and α-synuclein, the major hallmarks of Parkinson and Alzheimer diseases, respectively, are targets of different protein kinases. One of the kinases phosphorylating tau with pathological relevance in Alzheimer disease is cyclin-dependent kinase 5 (Cdk5), which is abundant in brain tissue, associates with tau (10Veeranna G.J. Shetty K.T. Takahashi M. Grant P. Pant H.C. Brain Res. Mol. Brain Res. 2000; 76: 229-236Crossref PubMed Scopus (74) Google Scholar), and plays important role in the signaling of mitogen-activated protein kinases (MAP kinases) also called extracellular signal-regulated kinases (ERKs). MAP kinases and Cdk5 phosphorylate numerous KSPXK consensus motifs in diverse cytoskeletal proteins and cross-talk in the regulation of neuronal functions (11Sharma P. Veeranna Sharma M. Amin N.D. Sihag R.K. Grant P. Ahn N. Kulkarni A.B. Pant H.C. J. Biol. Chem. 2002; 277: 528-534Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The cAMP-dependent protein kinase (PKA) interacts with the so-called A kinase-anchoring proteins via its regulatory subunit, that targets the kinase activity to specific subcellular locations (12Langeberg L.K. Scott J.D. J. Cell Sci. 2005; 118: 3217-3220Crossref PubMed Scopus (49) Google Scholar) including MTs through a direct binding to MAP2 (13Scott J.D. Stofko R.E. McDonald J.R. Comer J.D. Vitalis E.A. Mangili J.A. J. Biol. Chem. 1990; 265: 21561-21566Abstract Full Text PDF PubMed Google Scholar, 14Hausken Z.E. Coghlan V.M. Hastings C.A. Reimann E.M. Scott J.D. J. Biol. Chem. 1994; 269: 24245-24251Abstract Full Text PDF PubMed Google Scholar). Currently there is limited information on the phosphorylation of TPPP, although it has been considered as a phosphoprotein (15Takahashi M. Tomizawa K. Ishiguro K. Sato K. Omori A. Sato S. Shiratsuchi A. Uchida T. Imahori K. FEBS Lett. 1991; 289: 37-43Crossref PubMed Scopus (64) Google Scholar). It was partially co-purified with tau protein kinase II (also denoted Cdk5) from bovine brain extract; and two of the Ser/Thr-Pro sites in synthetic peptides, segments of TPPP, were phosphorylated by Cdk5 (15Takahashi M. Tomizawa K. Ishiguro K. Sato K. Omori A. Sato S. Shiratsuchi A. Uchida T. Imahori K. FEBS Lett. 1991; 289: 37-43Crossref PubMed Scopus (64) Google Scholar). Martin et al. (16Martin C.P. Vazquez J. Avila J. Moreno F. Biochim. Biophys. Acta. 2002; 1586: 113-122Crossref PubMed Scopus (28) Google Scholar) suggested that rat brain TPPP could be phosphorylated near to stoichiometrically in vitro by Cdk5 and by PKA but practically not by glycogen synthase kinase 3. The aim of this work was to demonstrate that TPPP was able to promote the formation of intact-like MTs exhibiting a function characteristic of MAPs, and to establish the effect of phosphorylation on the unfolded "structure" of TPPP, on its interaction with tubulin as well as on its MT promoting and bundling activities. For these purposes we performed in vitro phosphorylation studies with human recombinant TPPP and with its truncated form, searched for the in vivo phosphorylation sites and interacting kinase partner(s), as well as studied the functional consequences of the specific phosphorylation events. Materials—DTT, iodoacetamide, NH4HCO3, and 2,5-dihydroxybenzoic acid were obtained from Sigma, the sequencing grade side-chain protected trypsin (modified by reductive methylation) was ordered from Promega. C18 ZipTip was from Millipore. MAP-free tubulin was purified from bovine brain as described in Ref. 17Na C.N. Timasheff S.N. Biochemistry. 1986; 25: 6214-6222Crossref PubMed Scopus (94) Google Scholar. Ni-NTA magnetic agarose beads were purchased from Qiagen. TiO2 was from SunChrom GmbH. An active complex of N-terminal His-tagged human Cdk5 and N-terminal glutathione S-transferase-tagged human p25 protein was obtained from Upstate Biochemicals. The two proteins were co-expressed in baculovirus-infected Sf21 insect cells. The catalytic subunit of PKA isolated from bovine heart was from Calbiochem. The catalytic subunit was active without the addition of cAMP. His-tagged activated human ERK2 expressed in Escherichia coli was from Calbiochem. [γ32P]ATP was purchased from the Institute of Isotopes. Fine chemicals and buffer components were from Sigma. DNA Manipulations—The coding region of human TPPP (5Kovács G.G. László L. Kovács J. Jensen P.H. Lindersson E. Botond G. Molnár T. Perczel A. Hudecz F. Mező G. Erdei A. Tirián L. Lehotzky A. Gelpi E. Budka H. Ovádi J. Neurobiol. Dis. 2004; 17: 155-162Crossref PubMed Scopus (113) Google Scholar) was inserted into the XhoI and BamHI restriction sites of pET15b vector (Novagen) producing pET15b-TPPP. A PCR fragment for expression of Δ3-43TPPP, the deletion mutant lacking residues 3-43, was generated using pET15b-TPPP as template with the following primers: 5′-GATACTCGAGATGGCTGCATCCCCTGAGCTCAGTGCCCTGGAGGAG-3′ and 5′-CCGTGGATCCCTACTTGCCCCCTTGCACCTTCTGGTCGTAGG-3′. The PCR fragment was inserted into the XhoI and BamHI restriction sites of pET-15b producing pET15b-Δ3-43TPPP. Correct ligations were verified by DNA sequencing. Protein Purification—Recombinant human TPPP and p20 (TPPP3) were expressed in E. coli BL21 (DE3) cells and isolated as described previously (4Vincze O. Tőkési N. J. Oláh Hlavanda E. Zotter Á. Horváth I. Lehotzky A. Tirián L. Medzihradszky K.F. Kovács J. Orosz F. Ovádi J. Biochemistry. 2006; 45: 13818-13826Crossref PubMed Scopus (68) Google Scholar, 5Kovács G.G. László L. Kovács J. Jensen P.H. Lindersson E. Botond G. Molnár T. Perczel A. Hudecz F. Mező G. Erdei A. Tirián L. Lehotzky A. Gelpi E. Budka H. Ovádi J. Neurobiol. Dis. 2004; 17: 155-162Crossref PubMed Scopus (113) Google Scholar). Δ3-43TPPP was expressed in E. coli and was purified on HIS-Selected™ Cartridge (Sigma H8286) as the full-length TPPP. Protein Determination—The protein concentration was measured by the Bradford method (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) using the Bio-Rad protein assay kit. Affinity Chromatography—Human recombinant TPPP was immobilized to CNBr-activated Sepharose 4B (Amersham Biosciences) and used for finding interacting proteins from bovine brain extract as described previously (6Oláh J. Tőkési N. Vincze O. Horváth I. Lehotzky A. Erdei A. Szájli E. Medzihradszky K.F. Orosz F. Kovács G.G. Ovádi J. FEBS Lett. 2006; 580: 5807-5814Crossref PubMed Scopus (30) Google Scholar). The bound proteins were eluted with 10 mm phosphate buffer, pH 7.0, containing 0.5 m NaCl, and the protein bands obtained by SDS-PAGE (19Laemmli U.K. Nature (Lond.). 1970; 227: 680-688Crossref PubMed Scopus (205531) Google Scholar) were analyzed by mass spectrometry. Protein Phosphorylation—30 μg of recombinant human TPPP or its truncated form (Δ3-43TPPP) or p20 protein (TPPP3) were phosphorylated with PKA, ERK2, and Cdk5 in vitro. A typical reaction mixture contained 50 mm Tris-HCl; pH 7.5 buffer supplemented with 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride, 1 mm EGTA, 10 mm NaF, 0.05 mm sodium vanadate, 25 mm MgCl2, 0.2 mm ATP, and 0.4 μg PKA or 0.2 μg of ERK2 in a final volume of 100 μl. In one set of experiments 30 μg of recombinant human TPPP was phosphorylated with 0.1 μg of ERK2 to reduce the degree of modification. Phosphorylation with Cdk5 was conducted in the presence of 12 mm MOPS, pH 7.0 buffer, 0.2 mm EDTA, 1 mm EGTA, 5 mm NaF, 0.2 mm sodium vanadate, 0.2 mm dithiothreitol, 27 mm MgCl2, 0.2 mm ATP, and 0.05 μg of kinase. About 107 cpm of [γ-32P]ATP was added to the mixture for radioactive labeling. The reactions were initiated by the addition of the kinase and were terminated with 50 mm EDTA. 5-μl samples were withdrawn at regular time intervals for quantitative analysis. Phosphate incorporation into the proteins was determined by the method of Witt and Roskoski (20Witt J.J. Roskoski R. Anal. Biochem. 1975; 66: 253-258Crossref PubMed Scopus (269) Google Scholar) using Whatmann P-81 filter paper. The radioactivity was counted by Tscherenkov radiation. Aliquots of the samples were separated by SDS-PAGE (19Laemmli U.K. Nature (Lond.). 1970; 227: 680-688Crossref PubMed Scopus (205531) Google Scholar). Dried gels were analyzed by autoradiography using RX Fuji medical x-ray films. According to the densitometric scanning of the films with a Bio-Rad Multi-Analyst apparatus more than 90% of the incorporated radioactivity was found in the monomeric and dimeric forms of the proteins. A prestained protein ladder (Fermentas) was used for the estimation of the molecular masses. Radioactive and cold phosphorylation reactions were run parallel under identical conditions. The degree of phosphorylation in the latter case was checked by the method of back phosphorylation (21Nestler E.J. Greengard P. Nature. 1983; 305: 583-588Crossref PubMed Scopus (278) Google Scholar). Only the non-radioactive samples were subjected to functional studies and MS. In-gel Digestion—Digestion with side chain-protected porcine trypsin proceeded at 37 °C for 4 h. In-solution Digestion—Protein was dissolved in 6 m guanidine-HCl, disulfide bridges were reduced with dithiothreitol and free sulfhydryls alkylated with iodoacetamide. Then sample was diluted with 25 mm NH4HCO3 and digested with side chain-protected porcine trypsin at 37 °C for 4 h. Methyl Esterification—The protocol of Ficarro (22Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Nat. Biotech. 2002; 20: 301-305Crossref PubMed Scopus (1478) Google Scholar) was applied. Phosphopeptide Enrichment by Immobilized Metal Ion Affinity Chromatography—In NTA magnetic agarose beads Ni2+ was displaced by Fe3+ as described by Thompson et al. (23Thompson A.J. Hart S.R. Franz C. Barnouin K. Ridley A. Cramer R. Anal. Chem. 2003; 75: 3232-3243Crossref PubMed Scopus (91) Google Scholar) with final washing steps with a mixture of equal volumes of water, methanol, and acetonitrile. Methyl-esterified sample dissolved in water/methanol/acetonitrile was loaded onto the beads and vortexed for 15 min, then beads were washed three times with the loading solvent. Phosphopeptides were eluted with 0.42% H3P04 in 50% acetonitrile. An aliquot of the eluate was either directly analyzed by MALDI-TOF MS or diluted with 0.1% formic acid and loaded on the trapping column for LC-MS/MS. Phosphopeptide Enrichment by TiO2—A modified protocol of Larsen et al. (24Larsen M.R. Thingholm T.E. Jensen O.N. Roepstorff P. Jorgensen T.J.D. Mol. Cell Proteomics. 2005; 4: 873-886Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar) was applied. The tryptic digest was dried down, re-dissolved in 1% trifluoroacetic acid/50% acetonitrile and mixed with TiO2 suspended in the same solvent. After a few minutes vortexing the supernatant was discarded, and TiO2 was washed three to five times with the same solvent. Phosphopeptides were eluted with 1% NH4OH. MALDI-TOF MS and PSD Analysis—These analyses were performed on a Bruker Reflex III MALDI-TOF mass spectrometer in 2,5-dihydroxy-benzoic acid matrix. Three point external calibration was used with standard peptides. PSD analysis was performed in 10-12 steps, lowering the reflectron voltage by 25% at each step, and then stitching the data together. While the MS conditions permitted monoisotopic mass measurements, in PSD mode average masses were determined. LC-MS/MS—Samples were analyzed on an Agilent 1100 nanoLC system on-line coupled to an XCT Plus ion trap mass spectrometer in information-dependent acquisition mode. For phosphopeptide analysis, occasionally a preferred mass list was included in data acquisition with m/z values calculated from MALDI-TOF data or derived from previous analyses. HPLC conditions were as follows: column: C18, 75 μm × 150 mm; flow rate 300 nl/min; gradient: 5-45% B in 16 min, up to 90% B for 3 min, then equilibrated at 5% B for 15 min; solvent A was 0.1% formic acid in water, solvent B: 0.1% formic acid in acetonitrile; trapping: 10 μl/min flow rate, for 5 min in solvent A. Data Base Search—MS and MS/MS data were searched against the Swissprot 51.3 (250,296 sequences) and the NCBI 20070120 (4462937 sequences) non-redundant protein data-bases using the Mascot search engine v2.1. Mass tolerance was set according to the type of instrument and acquisition mode. No species restriction was used. Data were also manually inspected. Bioinformatic Methods—For prediction of phosphorylation and docking sites MotifScan (25Obenauer J.C. Cantley L.C. Yaffe M.B. Nucleic Acids Res. 2003; 31: 3635-3641Crossref PubMed Scopus (1322) Google Scholar) was used. Multiple sequence alignment of vertebrate TPPPs was done by ClustalW (26Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (54908) Google Scholar). CD Measurements—CD spectra were acquired with a Jasco J-720 spectropolarimeter (Tokyo, Japan) in the 190-260 nm wavelength range employing 0.1-cm thermostated cuvettes at 25 °C, using 10 mm phosphate buffer (pH 7.0) as described previously (4Vincze O. Tőkési N. J. Oláh Hlavanda E. Zotter Á. Horváth I. Lehotzky A. Tirián L. Medzihradszky K.F. Kovács J. Orosz F. Ovádi J. Biochemistry. 2006; 45: 13818-13826Crossref PubMed Scopus (68) Google Scholar). During the titration of 1 μm tubulin the difference ellipticity at 207 nm was determined as a function of the concentration of TPPP. Difference ellipticities were calculated from the ellipticity measured at 207 nm in the mixtures of two proteins and in the samples of the individual proteins. Turbidity Measurements—A total of 7 μm MAP-free tubulin was polymerized by 3 μm TPPP before and after phosphorylation at 37 °C in 50 mm MES buffer, pH 6.6, containing 1 mm dithiothreitol, 1 mm EDTA, 1 mm MgCl2, and 50 mm KCl. In the absence of TPPP no tubulin assembly occurs. Absorbance was monitored at 350 nm by a Cary 50 spectrophotometer (Varian, Australia). Transmission Electron Microscopy (TEM)—The polymerized tubulin samples were centrifuged at 30,000 × g and 30 °C for 20 min and the pellet fraction was used for TEM. The pellets were fixed for 1 h in a mixture of 2% glutaraldehyde, 0.2% freshly prepared tannic acid, and 0.1 m sodium cacodylate (pH 7.4). After washing in cacodylate, they were postfixed in 0.5% OsO4, and embedded in Durcupan (Fluka, Switzerland). Thin sections were contrasted with uranyl acetate and lead citrate and examined in a Jeol CX 100 electron microscope (1Tirián L. Hlavanda E. Oláh J. Horváth I. Orosz F. Szabó B. Kovács J. Szabad J. Ovádi J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13976-13981Crossref PubMed Scopus (92) Google Scholar). In Vitro Phosphorylation of TPPP—Recombinant human TPPP was phosphorylated by protein kinases (ERK2, Cdk5, and PKA) in the presence of [γ-32P]ATP under conditions to reach maximal phosphorylation within 2 h (see "Experimental Procedures"). Fig. 1 shows typical SDS/PAGE protein staining and autoradiographic images. Two characteristic phosphoprotein bands of 26.5 and 63 kDa appeared in the pictures corresponding to monomeric and dimeric species. By the addition of an excess DTT to the samples all of the dimers were converted to monomers (data not shown). The radioactivity incorporated into TPPP was determined as described under "Experimental Procedures"; the stoichiometry of the phosphorylation was found to be 2.9 ± 0.3, 2.2 ± 0.3, and 0.9 ± 0.1 mol P/mol protein for ERK2, Cdk5, and PKA kinases, respectively (Fig. 1). The phosphorylated protein bands were excised from the gels and digested with trypsin. After phosphopeptide enrichment, samples were analyzed by MALDI-TOF MS, MALDI-TOF PSD, and by LC-MS/MS on an ion trap mass spectrometer. Phosphopeptides were first identified by the 80-Da mass shift in comparison to the predicted molecular masses of tryptic peptides. Their identity was further confirmed by the β-elimination of phosphoric acid (98 Da) from the precursor ion under collision-induced dissociation (CID) or post source decay (PSD) conditions, a characteristic fragmentation step of Ser- and Thr-phosphorylated peptides (27Covey T.R. Sushan B.I. Bonner R. Shroder W. Hucho F. Jörnvall H. Höög J.O. Gustavsson A.M. Methods in Protein Sequence Analysis. Birkhauser Verlag Basel, 1991: 411-447Google Scholar). The sites of phosphorylation were assigned considering the 80 Da mass shift of the appropriate peptide fragments (Table 1).TABLE 1Identification of phosphorylation sites in human recombinant TPPP phosphorylated in vitro by Cdk5, ERK2, and PKAMH+m/zChargeSequencePositionEnrichmentSiteKinase998.0499.52+AISpSPTVSR[157-165]TiO2Ser-160Cdk5, ERK21011.6506.32+AIpSSPTVSR′[157-165]Fe-NTASer-159PKA1559.0780.02+NVTVTD′VD′IVFSK′[90-102]Fe-NTAThr-92PKA3081.71027.93+RLpSLE′S.E.′GAGE′GAAASPE′LSALE′ E′AFRR′[30-57]Fe-NTASer-32PKA1874.5625.53+AANRTPPKpSPGDPSKDR[10-26]TiO2Ser-18Cdk5, ERK21462.3488.13+TPPKpSPGDPSKDR[14-26]TiO2Ser-18Cdk5, ERK21504.3502.13+TPPKpSPGD′PSKD′R′[14-26]Fe-NTASer-18Cdk5, ERK21218.6609.82+TPPKpSPGD′PSK′[14-24]Fe-NTASer-18Cdk5, ERK21915.91915.91+AANRpTPPKSPGD′PSKD′R′[10-26]Fe-NTAThr-14Cdk5 Open table in a new tab In agreement with the minimal consensus of ERK2 and Cdk5, Ser, and Thr residues followed by Pro were targeted by these kinases. From the total of 4 such sites in TPPP, Ser18, and Ser160 were phosphorylated by ERK2, while Thr14, Ser18, and Ser160 were modified by Cdk5. PKA phosphorylated Ser32 within the single PKA consensus sequence KRLS in addition to Thr92 and Ser159 residues, which are in a less favorable sequence environment and may represent minor or unspecific sites. Therefore, we conclude that under in vitro conditions Thr14, Ser18, Ser32, and Ser160 of human TPPP are the main phosphorylation sites of the MT-associated kinases. Our structural prediction (8Orosz F. Kovács G.G. Lehotzky A. Oláh J. Vincze O. Ovádi J. Biol. Cell. 2004; 96: 701-711Crossref PubMed Scopus (48) Google Scholar) suggested a quite extended region in the N terminus of TPPP (1-52 amino acids) with highly unfolded structure. As three of the main phosphorylation sites, Thr14, Ser18, and Ser32, are localized in this tail region we expressed a truncated form of the human TPPP that lacks the first 43 amino acid residues (Δ3-43TPPP) and investigated the consequences of this deletion. Fig. 1 demonstrates that the truncation of the protein significantly reduced both the rate and degree of its phosphorylation. A 0.4-0.5 mol of P/mol protein stoichiometry was reached after 2 h of incubation with ERK2, Cdk5, and PKA. As a negative control, the phosphorylation of recombinant human p20 (TPPP3), a homologue of TPPP that lacks the N-terminal tail, and the phosphorylation site corresponding to Ser160 in TPPP was also tested under similar conditions; and as expected virtually no 32P incorporation (0.02 mol of P/mol protein) was detected. These results suggest that ERK2 and Cdk5 phosphorylate the residues Thr14 and Ser18 in the N-terminal tail more vigorously than Ser160; and PKA efficiently modifies Ser32 that is also localized in the N terminus. Our data are consistent with the predictions by MotifScan (25Obenauer J.C. Cantley L.C. Yaffe M.B. Nucleic Acids Res. 2003; 31: 3635-3641Crossref PubMed Scopus (1322) Google Scholar) for the probability of the phosphorylation sites by PKA, ERK2, or Cdk5 (see Table 3).TABLE 3Summary of the phosphorylation sites in TPPPKinasesPhosphorylated sitesThrSerSerSerbab, bovine.hbh, human.mcm, mouse.bhmbhmbhm121415161819303231159160159ProbabilitydProbability of phosphorylation predicted by MotifScan. High stringency indicates that the motif identified in the query sequence is within the top 0.2% of all matching sequences contained in vertebrate Swiss-Prot protein database. Medium and low stringency scores correspond to the top 1% and 5% of sequence matches, respectively.CDK5MediumMediumLowERK2LowMediumLowPKAHighIn vitroCDK5++ePeptide phosphorylation (15). ++ePeptide phosphorylation (15). +ERK2++PKA+In vivo+ +fPhosphoproteomics (30-32). +fPhosphoproteomics (30-32).+ +fPhosphoproteomics (30-32). +fPhosphoproteomics (30-32).+ +fPhosphoproteomics (30-32). +fPhosphoproteomics (30-32).a b, bovine.b h, human.c m, mouse.d Probability of phosphorylation predicted by MotifScan. High stringency indicates that the motif identified in the query sequence is within the top 0.2% of all matching sequences contained in vertebrate Swiss-Prot protein database. Medium and low stringency scores correspond to the top 1% and 5% of sequence matches, respectively.e Peptide phosphorylation (15Takahashi M. Tomizawa K. Ishiguro K. Sato K. Omori A. Sato S. Shiratsuchi A. Uchida T. Imahori K. FEBS Lett. 1991; 289: 37-43Crossref PubMed Scopus (64) Google Scholar).f Phosphoproteomics (30DeGiorgis J.A. Jaffe H. Moreira J.E. Carlotti C.G. Leite J.P. Pant H.C. Dosemeci A. J. Proteome Res. 2005; 4: 306-315Crossref PubMed Scopus (48) Google Scholar, 31Collins M.O. Yu L. Coba M.P. Husi H. Campuzano I. Blackstock W.P. Choudhary J.S. Grant S.G. J. Biol. Chem. 2005; 280: 5972-5982Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 32Trinidad J.C. Specht C.G. Tha
TPPP/p25 is a brain-specific protein, which induces tubulin polymerization and microtubule (MT) bundling and is enriched in Lewy bodies characteristic of Parkinson's disease [Tirián et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 13976−13981]. We identified two human gene sequences, CG1−38 and p25β, which encoded homologous proteins, that we termed p20 and p18, respectively. These homologous proteins display 60% identity with tubulin polymerization promoting protein/p25 (TPPP/p25); however, the N-terminal segment of TPPP/p25 is missing. They could be clustered into three subfamilies present in mammals and other vertebrates. We cloned, isolated, and characterized the structural and functional properties of the recombinant human proteins at molecular, ultrastructural, and cellular levels using a number of tools. These data revealed that, while p20 behaved as a disorganized protein similarly to TPPP/p25, which was described as a flexible and inherently dynamic protein with a long unstructured N-terminal tail, p18 was featured in more ordered fashion. TPPP/p25 and p20 specifically attached to MTs causing MT bundling both in vitro and in vivo; p18 protein did not cross-link MTs, and it distributed homogeneously within the cytosol of the transfected HeLa cells. These data indicate that the two shorter homologues display distinct structural features that determine their associations to MTs. The properties of p20 resemble TPPP/p25. The bundling activity of these two proteins results in the stabilization of the microtubular network, which is likely related to their physiological functions.
TPPP/p25, the first representative of a new protein family, identified as a brain-specific unfolded protein induces aberrant microtubule assemblies in vitro, suppresses mitosis in Drosophila embryo and is accumulated in inclusion bodies of human pathological brain tissues. In this paper, we present prediction and additional experimental data that validate TPPP/p25 to be a new member of the "intrinsically unstructured" protein family. The comparison of these characteristics with that of alpha-synuclein and tau, involved also in neurodegenerative diseases, suggested that although the primary sequences of these proteins are entirely different, there are similarities in their well-defined unstructured segments interrupted by "stabilization centres", phosphorylation and tubulin binding motives. SK-N-MC neuroblastoma cells were transfected with pEGFP-TPPP/p25 construct and a stable clone denoted K4 was selected and used to establish the effect of this unstructured protein on the energy state/metabolism of the cells. Our data by analyzing the mitochondrial membrane polarization by fluorescence microscopy revealed that the high-energy phosphate production in K4 clone is not damaged by the TPPP/p25 expression. Biochemical analysis with cell homogenates provided quantitative data that the ATP level increased 1.5-fold and the activities of hexokinase, glucosephosphate isomerase, phosphofructokinase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase were 1.2 to 2.0-fold higher in K4 as compared to the control. Our modelling using these data and rate equations of the individual enzymes suggests that the TPPP/p25 expression stimulates glucose metabolism. At pathological conditions TPPP/p25 is localized in inclusion bodies in multiple system atrophy, it tightly co-localizes with alpha-synuclein, partially with tubulin and not with vimentin. The previous and the present studies obtained with immunohistochemistry with pathological human brain tissues rendered it possible to classify among pathological inclusions on the basis of immunolabelling of TPPP/p25, and suggest this protein to be a potential linkage between Parkinson's and Alzheimer's diseases.
The sensing, integrating and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeletal network. Cytoskeleton comprises fibrous protein networks of microtubules, actin and intermediate filaments. These filamentous polymer structures are highly dynamic and undergo constant and rapid reorganization during cellular processes. The microtubular system plays a crucial role in the brain, as it is involved in an enormous number of cellular events including cell differentiation and pathological inclusion formation. These multifarious functions of microtubules can be achieved by their decoration with proteins/enzymes that exert specific effects on the dynamics and organization of the cytoskeleton and mediate distinct functions due to their moonlighting features. This mini-review focuses on two aspects of the microtubule cytoskeleton. On the one hand, we describe the heteroassociation of tubulin/microtubules with metabolic enzymes, which in addition to their catalytic activities stabilize microtubule structures via their cross-linking functions. On the other hand, we focus on the recently identified moonlighting Tubulin Polymerization Promoting Protein, TPPP/p25. TPPP/p25 is a Microtubule Associated Protein and it displays distinct physiological or pathological (aberrant) functions; thus it is a prototype of Neomorphic Moonlighting Proteins. The expression of TPPP/p25 is finely controlled in the human brain; this protein is indispensable for the development of projections of oligodendrocytes that are responsible for the ensheathment of axons. The non-physiological, higher or lower TPPP/p25 level leads to distinct CNS diseases. Mechanisms contributing to the control of microtubule stability and dynamics by metabolic enzymes and TPPP/p25 will be discussed.
