G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
microtubule-associated protein 4
G00000551 (Mus musculus)

Databases (7)

ENSG00000047849 (Ensembl human gene)
4134 (Entrez Gene)
968 (G2Cdb plasticity & disease)
MAP4 (GeneCards)
157132 (OMIM)
Marker Symbol
HGNC:6862 (HGNC)
Protein Sequence
P27816 (UniProt)

Literature (43)

Pubmed - other

  • Identification of neuroglycan C and interacting partners as potential susceptibility genes for schizophrenia in a Southern Chinese population.

    So HC, Fong PY, Chen RY, Hui TC, Ng MY, Cherny SS, Mak WW, Cheung EF, Chan RC, Chen EY, Li T and Sham PC

    Department of Psychiatry, University of Hong Kong, Hong Kong SAR, China.

    Chromosome 3p was reported by previous studies as one of the regions showing strong evidence of linkage with schizophrenia. We performed a fine-mapping association study of a 6-Mb high-LD and gene-rich region on 3p in a Southern Chinese sample of 489 schizophrenia patients and 519 controls to search for susceptibility genes. In the initial screen, 4 SNPs out of the 144 tag SNPs genotyped were nominally significant (P < 0.05). One of the most significant SNPs (rs3732530, P = 0.0048) was a non-synonymous SNP in the neuroglycan C (NGC, also known as CSPG5) gene, which belongs to the neuregulin family. The gene prioritization program Endeavor ranked NGC 8th out of the 129 genes in the 6-Mb region and the highest among the genes within the same LD block. Further genotyping of NGC revealed 3 more SNPs to be nominally associated with schizophrenia. Three other genes (NRG1, ErbB3, ErbB4) involved in the neuregulin pathways were subsequently genotyped. Interaction analysis by multifactor dimensionality reduction (MDR) revealed a significant two-SNP interaction between NGC and NRG1 (P = 0.015) and three-SNP interactions between NRG1 and ErbB4 (P = 0.009). The gene NGC is exclusively expressed in the brain. It is implicated in neurodevelopment in rats and was previously shown to promote neurite outgrowth. Methamphetamine, a drug that may induce psychotic symptoms, was reported to alter the expression of NGC. Taken together, these results suggest that NGC may be a novel candidate gene, and neuregulin signaling pathways may play an important role in schizophrenia.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2010;153B;1;103-13

  • Sequential use of transcriptional profiling, expression quantitative trait mapping, and gene association implicates MMP20 in human kidney aging.

    Wheeler HE, Metter EJ, Tanaka T, Absher D, Higgins J, Zahn JM, Wilhelmy J, Davis RW, Singleton A, Myers RM, Ferrucci L and Kim SK

    Department of Genetics, Stanford University Medical Center, Stanford, California, USA.

    Kidneys age at different rates, such that some people show little or no effects of aging whereas others show rapid functional decline. We sequentially used transcriptional profiling and expression quantitative trait loci (eQTL) mapping to narrow down which genes to test for association with kidney aging. We first performed whole-genome transcriptional profiling to find 630 genes that change expression with age in the kidney. Using two methods to detect eQTLs, we found 101 of these age-regulated genes contain expression-associated SNPs. We tested the eQTLs for association with kidney aging, measured by glomerular filtration rate (GFR) using combined data from the Baltimore Longitudinal Study of Aging (BLSA) and the InCHIANTI study. We found a SNP association (rs1711437 in MMP20) with kidney aging (uncorrected p = 3.6 x 10(-5), empirical p = 0.01) that explains 1%-2% of the variance in GFR among individuals. The results of this sequential analysis may provide the first evidence for a gene association with kidney aging in humans.

    Funded by: Intramural NIH HHS; NIA NIH HHS: R01 AG025941, R01 AG025941-01A2; NIMHD NIH HHS: 263 MD 821336, 263 MD 9164, R01 MD009164

    PLoS genetics 2009;5;10;e1000685

  • Beta-tubulin isotype classes II and V expression patterns in nonsmall cell lung carcinomas.

    Cucchiarelli V, Hiser L, Smith H, Frankfurter A, Spano A, Correia JJ and Lobert S

    The University of Mississippi Medical Center, Department of Biochemistry, Jackson, Mississippi, USA.

    Previous studies suggest that beta-tubulin isotype protein levels could be useful as indicators of nonsmall cell lung cancer (NSCLC) aggressiveness. However, measurement of protein amounts in tissue samples by staining techniques is semiquantitative at best. Since technologies for measuring mRNA levels have become more efficient and quantitative, we wanted to determine whether beta-tubulin message levels may be useful as biomarkers. Quantitative real-time RT-PCR was used to measure the seven classes of beta-tubulin isotypes, stathmin and MAP4 mRNA levels in 64 NSCLC and 12 normal lung tissue samples. We found significantly higher fractions of beta-tubulin classes II and V mRNA compared to the other isotypes in all lung tumor samples (P < 0.05). In addition, the ratio of beta-tubulin classes II/V mRNA was significantly higher in NSCLCs compared to normal lung tissues (P < 0.001). The data suggest that the ratio of beta-tubulin classes II and V mRNA could be useful as a biomarker for NSCLC tumor differentiation and/or NSCLC aggressiveness. Furthermore, the ratio of MAP4 to stathmin mRNA was found to be higher in diseased lung tissues compared to normal lung tissues, suggesting this ratio might also be used as a clinically relevant biomarker for NSCLCs.

    Cell motility and the cytoskeleton 2008;65;8;675-85

  • Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening.

    Wu C, Ma MH, Brown KR, Geisler M, Li L, Tzeng E, Jia CY, Jurisica I and Li SS

    Department of Biochemistry and the Siebens-Drake Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

    Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.

    Proteomics 2007;7;11;1775-85

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

    Beausoleil SA, Villén J, Gerber SA, Rush J and Gygi SP

    Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, Massachusetts 02115, USA.

    Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

    Funded by: NHGRI NIH HHS: HG03456; NIGMS NIH HHS: GM67945

    Nature biotechnology 2006;24;10;1285-92

  • A novel fusion of the MALT1 gene and the microtubule-associated protein 4 (MAP4) gene occurs in diffuse large B-cell lymphoma.

    Murga Penas EM, Kawadler H, Siebert R, Frank M, Ye H, Hinz K, Becher C, Hummel M, Barth TF, Bokemeyer C, Stein H, Trümper L, Möller P, Marynen P, Du MQ, Yang X, Hansmann ML and Dierlamm J

    Department of Oncology and Hematology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.

    Rearrangements of the MALT1 gene by the t(11;18)(q21;q21) and t(14;18)(q32;q21) are the most frequent structural chromosomal abnormalities in MALT lymphomas. These translocations lead to fusions of BIRC3-MALT1 and IGH-MALT1 respectively, and activate the NF-kappaB pathway. Among 122 diffuse large B-cell lymphomas and 28 Burkitt's lymphomas screened by interphase FISH, we found two cases with a break within MALT1, but without a t(11;18) or a t(14;18). Molecular genetic analyses in one of these cases revealed a novel "in frame" fusion of exon 9 of MALT1 and exon 9 of the microtubule-associated protein 4 (MAP4) gene. The translocation was accompanied by a deletion of MALT1 sequences distal to the breakpoint including the caspase-like domain, which is essential for activation of NF-kappaB. As a result of the deletion, the reciprocal 5'MAP4-3'MALT1 transcript was not present, demonstrating that the 5'MALT1-3'MAP4 fusion represents the pathogenetically relevant transcript. Immunohistochemistry with amino-terminal and carboxy-terminal MALT1 antibodies, indicated a strong expression of the chimeric MALT1-MAP4 protein. Moreover, NF-kappaB activation was not increased in this case as shown by the levels of IkappaBalpha phosphorylation and NEMO ubiquitination. Our data demonstrate that the pathogenetic consequences of the novel MALT1-MAP4 fusion are different from those of the known MALT1-associated chromosomal rearrangements and do not involve NF-kappaB activation.

    Genes, chromosomes & cancer 2006;45;9;863-73

  • Phosphoproteome analysis of the human mitotic spindle.

    Nousiainen M, Silljé HH, Sauer G, Nigg EA and Körner R

    Department of Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.

    During cell division, the mitotic spindle segregates the sister chromatids into two nascent cells, such that each daughter cell inherits one complete set of chromosomes. Errors in spindle formation can result in both chromosome missegregation and cytokinesis defects and hence lead to genomic instability. To ensure the correct function of the spindle, the activity and localization of spindle associated proteins has to be tightly regulated in time and space. Reversible phosphorylation has been shown to be one of the key regulatory mechanisms for the organization of the mitotic spindle. The relatively low number of identified in vivo phosphorylation sites of spindle components, however, has hampered functional analysis of regulatory spindle networks. A more complete inventory of the phosphorylation sites of spindle-associated proteins would therefore constitute an important advance. Here, we describe the mass spectrometry-based identification of in vivo phosphorylation sites from purified human mitotic spindles. In total, 736 phosphorylation sites were identified, of which 312 could be attributed to known spindle proteins. Among these are phosphorylation sites that were previously shown to be important for the regulation of spindle-associated proteins. Importantly, this data set also comprises 279 novel phosphorylation sites of known spindle proteins for future functional studies. This inventory of spindle phosphorylation sites should thus make an important contribution to a better understanding of the molecular mechanisms that regulate the formation, function, and integrity of the mitotic spindle.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;14;5391-6

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • Mammalian septins regulate microtubule stability through interaction with the microtubule-binding protein MAP4.

    Kremer BE, Haystead T and Macara IG

    Center for Cell Signaling, Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

    Mammalian septins constitute a family of at least 12 GTP-binding proteins that can form hetero-oligomers and that are sometimes found in association with actin or microtubule filaments. However, their functions are not understood. Using RNA interference, we found that suppression of septin expression in HeLa cells caused a pronounced increase in microtubule stability. Mass spectroscopic analysis of proteins coprecipitating with Sept6 identified the microtubule-associated protein MAP4 as a septin binding partner. A small, proline-rich region in the C-terminal half of MAP4 bound directly to a Sept 2:6:7 heterotrimer, and to the Sept2 monomer. The trimer blocked the ability of this MAP4 fragment to bind and bundle microtubules in vitro. In intact cells, MAP4 was required for the stabilization of microtubules induced by septin depletion. Moreover, septin depletion increased the number of cells with abnormal nuclei, and this effect was blocked by gene silencing of MAP4. These data identify a novel molecular function for septins in mammalian cells: the modulation of microtubule dynamics through interaction with MAP4.

    Funded by: NIGMS NIH HHS: GM-66306, R01 GM066306, T32 GM007267

    Molecular biology of the cell 2005;16;10;4648-59

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Inhibition of beta-adrenergic receptor trafficking in adult cardiocytes by MAP4 decoration of microtubules.

    Cheng G, Qiao F, Gallien TN, Kuppuswamy D and Cooper G

    Gazes Cardiac Research Institute, Cardiology Division, Medical University of South Carolina, and Department of Veterans Affairs Medical Center, Charleston, South Carolina 29403, USA.

    Decreased beta-adrenergic receptor (beta-AR) number occurs both in animal models of cardiac hypertrophy and failure and in patients. beta-AR recycling is an important mechanism for the beta-AR resensitization that maintains a normal complement of cell surface beta-ARs. We have shown that 1) in severe pressure overload cardiac hypertrophy, there is extensive microtubule-associated protein 4 (MAP4) decoration of a dense microtubule network; and 2) MAP4 microtubule decoration inhibits muscarinic acetylcholine receptor recycling in neuroblastoma cells. We asked here whether MAP4 microtubule decoration inhibits beta-AR recycling in adult cardiocytes. [(3)H]CGP-12177 was used as a beta-AR ligand, and feline cardiocytes were isolated and infected with adenovirus containing MAP4 (AdMAP4) or beta-galactosidase (Adbeta-gal) cDNA. MAP4 decorated the microtubules extensively only in AdMAP4 cardiocytes. beta-AR agonist exposure reduced cell surface beta-AR number comparably in AdMAP4 and Adbeta-gal cardiocytes; however, after agonist withdrawal, the cell surface beta-AR number recovered to 78.4 +/- 2.9% of the pretreatment value in Adbeta-gal cardiocytes but only to 56.8 +/- 1.4% in AdMAP4 cardiocytes (P < 0.01). This result was confirmed in cardiocytes isolated from transgenic mice having cardiac-restricted MAP4 overexpression. In functional terms of cAMP generation, beta-AR agonist responsiveness of AdMAP4 cells was 47% less than that of Adbeta-gal cells. We conclude that MAP4 microtubule decoration interferes with beta-AR recycling and that this may be one mechanism for beta-AR downregulation in heart failure.

    Funded by: NHLBI NIH HHS: HL-48788

    American journal of physiology. Heart and circulatory physiology 2005;288;3;H1193-202

  • Automated yeast two-hybrid screening for nuclear receptor-interacting proteins.

    Albers M, Kranz H, Kober I, Kaiser C, Klink M, Suckow J, Kern R and Koegl M

    PheneX Pharmaceuticals AG, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany.

    High throughput analysis of protein-protein interactions is an important sector of hypothesis-generating research. Using an improved and automated version of the yeast two-hybrid system, we completed a large interaction screening project with a focus on nuclear receptors and their cofactors. A total of 425 independent yeast two-hybrid cDNA library screens resulted in 6425 potential interacting protein fragments involved in 1613 different interaction pairs. We show that simple statistical parameters can be used to narrow down the data set to a high confidence set of 377 interaction pairs where validated interactions are enriched to 61% of all pairs. Within the high confidence set, there are 64 novel proteins potentially binding to nuclear receptors or their cofactors. We discuss several examples of high interest, and we expect that communication of this huge data set will help to complement our knowledge of the protein interaction repertoire of this family of transcription factors and instigate the characterization of the various novel candidate interactors.

    Molecular & cellular proteomics : MCP 2005;4;2;205-13

  • Truncation of the projection domain of MAP4 (microtubule-associated protein 4) leads to attenuation of microtubule dynamic instability.

    Permana S, Hisanaga S, Nagatomo Y, Iida J, Hotani H and Itoh TJ

    Division of Biological Sciences, Graduate School of Science, Nagoya University, Japan.

    MAP4, a ubiquitous heat-stable MAP, is composed of an asymmetric structure common to the heat-stable MAPs, consisting of an N-terminal projection (PJ) domain and a C-terminal microtubule (MT)-binding (MTB) domain. Although the MTB domain has been intensively studied, the role of the PJ domain, which protrudes from MT-wall and does not bind to MTs, remains unclear. We investigated the roles of the PJ domain on the dynamic instability of MTs by dark-field microscopy using various PJ domain deletion constructs of human MAP4 (PJ1, PJ2, Na-MTB and KDM-MTB). There was no obvious difference in the dynamic instability between the wtMAP4 and any fragments at 0.1 microM, the minimum concentration required to stabilize MTs. The individual MTs stochastically altered between polymerization and depolymerization phases with similar profiles of length change as had been observed in the presence of MAP2 or tau. We also examined the effects at the increased concentrations of 0.7 microM, and found that in some cases the dynamic instability was almost entirely attenuated. The length of both the polymerization and depolymerization phases decreased and "pause-phases" were occasionally observed, especially in the case of PJ1, PJ2 or Na-MTB. No obvious change was observed in the increased concentration of wtMAP4 and KDM-MTB. Additionally, the profiles of MT length change were quite different in 0.7 microM PJ2. Relatively rapid and long depolymerization phases were sometimes observed among quite slow length changes. Perhaps, this unusual profile could be due to the uneven distribution of PJ2 along the MT lattice. These results indicate that the PJ domain of MAP4 participates in the regulation of the dynamic instability.

    Cell structure and function 2005;29;5-6;147-57

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Large-scale characterization of HeLa cell nuclear phosphoproteins.

    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

    Funded by: NHGRI NIH HHS: HG00041, K22 HG000041, T32 HG000041; NIGMS NIH HHS: GM67945, GMS6203, R01 GM056203, R01 GM067945

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;33;12130-5

  • Robust phosphoproteomic profiling of tyrosine phosphorylation sites from human T cells using immobilized metal affinity chromatography and tandem mass spectrometry.

    Brill LM, Salomon AR, Ficarro SB, Mukherji M, Stettler-Gill M and Peters EC

    Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA. lbrill@gnf.org

    Protein tyrosine phosphorylation cascades are difficult to analyze and are critical for cell signaling in higher eukaryotes. Methodology for profiling tyrosine phosphorylation, considered herein as the assignment of multiple protein tyrosine phosphorylation sites in single analyses, was reported recently (Salomon, A. R.; Ficarro, S. B.; Brill, L. M.; Brinker, A.; Phung, Q. T.; Ericson, C.; Sauer, K.; Brock, A.; Horn, D. M.; Schultz, P. G.; Peters, E. C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 443-448). The technology platform included the use of immunoprecipitation, immobilized metal affinity chromatography (IMAC), liquid chromatography, and tandem mass spectrometry. In the present report, we show that when using complex mixtures of peptides from human cells, methylation improved the selectivity of IMAC for phosphopeptides and eliminated the acidic bias that occurred with unmethylated peptides. The IMAC procedure was significantly improved by desalting methylated peptides, followed by gradient elution of the peptides to a larger IMAC column. These improvements resulted in assignment of approximately 3-fold more tyrosine phosphorylation sites, from human cell lysates, than the previous methodology. Nearly 70 tyrosine-phosphorylated peptides from proteins in human T cells were assigned in single analyses. These proteins had unknown functions or were associated with a plethora of fundamental cellular processes. This robust technology platform should be broadly applicable to profiling the dynamics of tyrosine phosphorylation.

    Analytical chemistry 2004;76;10;2763-72

  • MARK4 is a novel microtubule-associated proteins/microtubule affinity-regulating kinase that binds to the cellular microtubule network and to centrosomes.

    Trinczek B, Brajenovic M, Ebneth A and Drewes G

    Department of Medicinal Chemistry, University of Kansas, Malott Hall, Lawrence, Kansas 66045, USA.

    The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule-binding domain of tau that is critical for microtubule binding. Here, we report the cloning and expression of a novel human paralog, MARK4, which shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%), and MARK4 readily phosphorylates tau and the related microtubule-associated protein 2 (MAP2) and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, we show that a tandem affinity-purified MARK4 protein complex contains alpha-, beta-, and gamma-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. We suggest that although the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimer's disease.

    The Journal of biological chemistry 2004;279;7;5915-23

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • An unappreciated role for RNA surveillance.

    Hillman RT, Green RE and Brenner SE

    Department of Bioengineering, University of California, Berkeley, CA 94720-3102, USA.

    Background: Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA surveillance mechanism that detects and degrades mRNAs with premature termination codons (PTC+ mRNAs). In mammals, a termination codon is recognized as premature if it lies more than about 50 nucleotides upstream of the final intron position. More than a third of reliably inferred alternative splicing events in humans have been shown to result in PTC+ mRNA isoforms. As the mechanistic details of NMD have only recently been elucidated, we hypothesized that many PTC+ isoforms may have been cloned, characterized and deposited in the public databases, even though they would be targeted for degradation in vivo.

    Results: We analyzed the human alternative protein isoforms described in the SWISS-PROT database and found that 144 (5.8% of 2,483) isoform sequences amenable to analysis, from 107 (7.9% of 1,363) SWISS-PROT entries, derive from PTC+ mRNA.

    Conclusions: For several of the PTC+ isoforms we identified, existing experimental evidence can be reinterpreted and is consistent with the action of NMD to degrade the transcripts. Several genes with mRNA isoforms that we identified as PTC+--calpain-10, the CDC-like kinases (CLKs) and LARD--show how previous experimental results may be understood in light of NMD.

    Funded by: NHGRI NIH HHS: K22 HG000056, K22 HG00056, T32 HG000047, T32 HG00047

    Genome biology 2004;5;2;R8

  • Phenotypic consequences of beta1-tubulin expression and MAP4 decoration of microtubules in adult cardiocytes.

    Takahashi M, Shiraishi H, Ishibashi Y, Blade KL, McDermott PJ, Menick DR, Kuppuswamy D and Cooper G

    Gazes Cardiac Research Institute, PO Box 250773, Medical University of South Carolina, 114 Doughty Street, Charleston, SC 29403, USA.

    In pressure-overload cardiac hypertrophy, microtubule network densification is one cause of contractile dysfunction. Cardiac transcriptional upregulation of beta1-tubulin rather than the constitutive beta4-tubulin and of microtubule-associated protein (MAP)4 accompanies hypertrophy, with extensive microtubule decoration by MAP4. Because MAP4 stabilizes microtubules, and because the isoform-variable carboxy terminus of beta-tubulin binds to MAP4, we wished to determine whether one or both of these proteins has etiologic significance for cardiac microtubule network densification. Recombinant adenoviruses encoding beta1-tubulin, beta4-tubulin, and MAP4 were used to infect isolated cardiocytes. Overexpressed MAP4 caused a shift of tubulin dimers to the polymerized fraction and formation of a dense, stable microtubule network. Overexpressed beta1- or beta4-tubulin had neither any independent effect on these variables nor any effect additive to that of simultaneously overexpressed MAP4. Results from transgenic mice with cardiac overexpression of beta1-tubulin or MAP4 were confirmatory, but unlike the effects of brief adenovirus-mediated MAP4 overexpression in isolated cardiocytes, MAP4 transgenic hearts showed a marked increase in total alpha- and beta-tubulin. Thus MAP4 overexpression caused increased tubulin expression, formation of stable microtubules, and altered microtubule network properties, such that MAP4 upregulation may be one cause for the dense, stable microtubule network characteristic of pressure-overloaded, hypertrophied cardiocytes.

    Funded by: NHLBI NIH HHS: HL 48788

    American journal of physiology. Heart and circulatory physiology 2003;285;5;H2072-83

  • Adenovirus 2 E1B-55K protein relieves p53-mediated transcriptional repression of the survivin and MAP4 promoters.

    Punga T and Akusjärvi G

    Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, 751 23 Uppsala, Sweden. tanel.punga@imbim.uu.se

    It is well established that adenovirus E1B-55K protein functions as an inhibitor of the tumor suppressor protein p53 by binding and inactivating p53 as a transcriptional activator protein. Here we show that the adenovirus 2 E1B-55K protein also blocks p53 as a transcriptional repressor protein of the survivin and the MAP4 promoters. The repression is dependent on the ability of E1B-55K to bind to p53 and is enhanced by coexpression of the adenovirus E4orf6 protein. Overexpression of the transcriptional corepressor protein Sin3A partially relieves the inhibitory effect of E1B-55K, suggesting that E1B-55K blocks p53 functions by interfering with the Sin3 complex.

    FEBS letters 2003;552;2-3;214-8

  • Interphase and monoastral-mitotic phenotypes of overexpressed MAP4 are modulated by free tubulin concentrations.

    Holmfeldt P, Brattsand G and Gullberg M

    Department of Molecular Biology, University of Umea, S-901 87 Umea, Sweden.

    The microtubule-associated protein 4 (MAP4) has recently been shown to counteract destabilization of interphase microtubules caused by catastrophe promotion but not by tubulin sequestering. To address how MAP4 discriminates between destabilization of microtubules by these two mechanisms, we have evaluated the combined phenotypes of MAP4 coexpressed with Op18/stathmin family member derivatives with either catastrophe-promoting or sequestering activities. This approach relies on the finding that overexpression of MAP4 alone stabilizes microtubules during all phases of the cell cycle in human leukemia cells, and causes a potent mitotic block and a dramatic, previously unobserved, phenotype characterized by large monoastral spindles. Coexpression of either catastrophe-promoting or tubulin-sequestration-specific Op18 derivatives was found to modulate the activity of ectopic MAP4 during mitosis, but with differential functional outcome. Interestingly, the tubulin-sequestering derivative suppressed the monoastral mitotic phenotype of MAP4 (i.e. coexpression facilitated the formation of functional spindles). To evaluate whether this phenotypic suppression could be explained by tubulin-sequestration-dependent modulation of MAP4 activity, a plasma-membrane-targeted, tubulin-sequestering chimera was constructed to decrease the cytosolic free tubulin concentration substantially. This chimera likewise suppressed the monoastral phenotype caused by overexpression of MAP4, suggesting a direct downregulation of MAP4 activity by reduced free tubulin concentrations.

    Journal of cell science 2003;116;Pt 18;3701-11

  • The number of repeat sequences in microtubule-associated protein 4 affects the microtubule surface properties.

    Tokuraku K, Matsushima K, Matui T, Nakagawa H, Katsuki M, Majima R and Kotani S

    Department of Chemical Science and Engineering, Miyakonojo National College of Technology, 473-1 Yoshio-cho, Miyakonojo, Miyazaki 885-8567, Japan. tokuraku@miyakonojo-nct.ac.jp

    The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a Repeat region with tandemly organized repeat sequences. In this study, we focused on the variations of the Repeat region, and searched for MAP4 isoforms with diverse Repeat region organizations. We successfully isolated four types of MAP4 cDNAs, which differed from each other in both the number and the arrangement of the repeat sequences, from a single source (bovine adrenal gland). To examine the functional differences among the isoforms, we prepared the microtubule-binding domain polypeptides of three of the four isoforms, and examined their activities. The isoform fragments showed similar degrees of microtubule assembly promoting activity and microtubule binding affinity. This result suggested that the Repeat region variation is not important for the control of microtubule dynamics, which is believed to be the main function of MAPs. On the other hand, the microtubule bundle-forming activity differed among the isoform fragments. The bundle formation was augmented by increasing the number of repeat sequences in the fragments. Based on these results, we propose the hypothesis that the role of the MAP4 isoforms is to regulate the surface charge of microtubules.

    The Journal of biological chemistry 2003;278;32;29609-18

  • The projection domain of MAP4 suppresses the microtubule-bundling activity of the microtubule-binding domain.

    Iida J, Itoh TJ, Hotani H, Nishiyama K, Murofushi H, Bulinski JC and Hisanaga S

    Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachiohji, Tokyo 192-0397, Japan.

    Microtubule-associated protein 4 (MAP4), a major MAP expressed in proliferating non-neuronal cells, consists of an N-terminal projection (PJ) domain and a C-terminal microtubule-binding (MTB) domain. The PJ domain of MAP4 is divided into three regions; the N-terminal acidic region (the Na-region), the multiple KDM-repeated sequence region (the KDM-region), and the b-region followed by the MTB domain. To investigate roles of the PJ domain, we prepared three truncated forms of human MAP4 with different PJ domain lengths; PJ1, PJ2 and MTB with deletion of about one-third, two-third and all of the PJ domain, respectively, and examined their effects on bundle formation of microtubules (MTs). MTs polymerized by full length MAP4 were singly distributed as observed by both negative staining electron microscopy and dark field microscopy. MTs with PJ1 were also separated in solution but became pairs when pelleted by centrifugation. PJ2 formed planar two-dimensional bundles consisting of several MTs (the 2D-bundle). MTB induced large bundles of many MTs, tightly packed without space in between (termed the 3D-bundle). To study how the PJ domain decreases the bundle-forming activity of the MTB domain of MAP4, we made three additional deletion-mutants of MAP4, called Na-MTB, KDM-MTB and Na-PJ2. Na-MTB and KDM-MTB, in which the KDM/b-region and both of Na- and b-regions were deleted respectively, were prepared by fusing the Na-region or KDM-region to MTB. Both of Na-MTB and KDM-MTB suppressed the 3D-bundle formation as effectively as PJ2. MTs polymerized with Na-PJ2, the KDM-deletion mutant made by adding the Na-region to PJ2, were singular and did not become bundles. These results indicated that the PJ domain kept individual MTs separated by suppressing the bundle-forming ability of the MTB domain. The suppressive activity of the PJ domain was correlated with the length, but not the amino acid sequence, of the PJ.

    Journal of molecular biology 2002;320;1;97-106

  • MAP4 counteracts microtubule catastrophe promotion but not tubulin-sequestering activity in intact cells.

    Holmfeldt P, Brattsand G and Gullberg M

    Department of Molecular Biology, Umeå University, Sweden.

    Microtubules are polar polymers that continually switch between phases of elongation and shortening, a property referred to as dynamic instability. The ubiquitous microtubule associated protein 4 (MAP4) shows rescue-promoting activity during in vitro assembly of microtubules (i.e., promotes transitions from shortening to elongation), but its regulatory role in intact cells is poorly defined. Here, we demonstrate that ectopic MAP4 promotes outgrowth of extended MTs during beta1-integrin-induced cell spreading. An inducible cotransfection protocol was employed to further analyze the regulatory role of MAP4 in human leukemia cells with microtubules partially destabilized by either ectopic tubulin-sequestering proteins or proteins that promote catastrophes (i.e., transitions from elongation to shortening). Coexpression of proteins that sequester free tubulin heterodimers with different efficiencies was found to abolish microtubule stabilization by MAP4. In contrast, however, the microtubule-stabilizing activity of MAP4 was found to suppress the activities of two distinct and specific catastrophe promoters, namely, XKCM1 and a nonsequestering truncation derivative of Op18/stathmin. These observations reveal specificity in the microtubule-stabilizing activity of MAP4 that differentiates between two mechanistically distinct types of MT destabilization.

    Current biology : CB 2002;12;12;1034-9

  • Phosphorylation of MAP4 affects microtubule properties and cell cycle progression.

    Chang W, Gruber D, Chari S, Kitazawa H, Hamazumi Y, Hisanaga S and Bulinski JC

    Department of Biological Sciences, College of Arts & Sciences, Columbia University, New York, NY 10027-2450, USA.

    In human cells, MAP4, a microtubule-associated protein ubiquitously expressed in proliferating cells, has been shown to undergo in vivo phosphorylation. Two phosphorylation sites, serines 696 and 787, lie within the proline-rich region of its microtubule-binding domain. To test the hypothesis that phosphorylation at these sites influences microtubule properties or cell cycle progression, we prepared stable cell lines that inducibly express versions of MAP4 in which phosphorylation of these two serines was prevented by their replacement with alanine, lysine, or glutamate residues (AA-, KK-, or EE-MAP4). All nonphosphorylatable mutant forms of MAP4 expressed in mouse Ltk- cells were localized to MT arrays that were unremarkable in appearance. Expression of nonphosphorylatable mutants of MAP4 did not affect cell doubling time; however, expression of some mutants altered progression into or through cell division. Interactions of mutant MAP4 with MTs were examined in vitro. KK mutant MAP4 bound MTs more avidly than its wild-type counterpart, WT-MAP4. In vivo MT polymer also differed among the mutants: MTs in cells expressing the KK- and AA-MAP4 forms were more resistant to nocodazole depolymerization than those in cells expressing EE- or WT-MAP4 forms. Our results demonstrate that phosphorylation alters MAP4 properties and suggest a raison d'être for phosphorylation of the MAP4 microtubule-binding domain during cell cycle progression.

    Funded by: NCI NIH HHS: T32 CA 09503; NHLBI NIH HHS: HL 62617; NIGMS NIH HHS: T32 GM 08224

    Journal of cell science 2001;114;Pt 15;2879-87

  • Ser787 in the proline-rich region of human MAP4 is a critical phosphorylation site that reduces its activity to promote tubulin polymerization.

    Kitazawa H, Iida J, Uchida A, Haino-Fukushima K, Itoh TJ, Hotani H, Ookata K, Murofushi H, Bulinski JC, Kishimoto T and Hisanaga S

    Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Hachiohji, Japan.

    p34cdc2 kinase-phosphorylation sites in the microtubule (MT)-binding region of MAP4 were determined by peptide sequence of phosphorylated MTB3, a fragment containing the carboxy-terminal half of human MAP4. In addition to two phosphopeptides containing Ser696 and Ser787 which were previously indicated to be in vivo phosphorylation sites, two novel phosphopeptides, containing Thr892 or Thr901 and Thr917 as possible phosphorylation sites, were isolated, though only in in vitro phosphorylation. The role of phosphorylation at Ser696 and Ser787, which were differently phosphorylated during the cell cycle (Ookata et al., (1997). Biochemistry, 36: 15873-15883), was investigated in MT-polymerization, using MAP4 Ser to Glu mutants, which mimic phosphorylation at each site. Mutation of Ser787 to Glu strikingly reduced the MAP4's MT-polymerization activity, while Glu-mutation at Ser696 did not. These results suggest that Ser787 could be the critical phosphorylation site causing MTs to be dynamic at mitosis.

    Cell structure and function 2000;25;1;33-9

  • Phosphorylation of MAP2c and MAP4 by MARK kinases leads to the destabilization of microtubules in cells.

    Ebneth A, Drewes G, Mandelkow EM and Mandelkow E

    Max-Planck Unit for Structural Molecular Biology, Hamburg, Germany.

    Microtubules serve as transport tracks in molecular mechanisms governing cellular shape and polarity. Rapid transitions between stable and dynamic microtubules are regulated by several factors, including microtubule-associated proteins (MAPs). We have shown that MAP/microtubule affinity regulating kinases (MARK) can phosphorylate the microtubule-associated-proteins MAP4, MAP2c, and tau on their microtubule-binding domain in vitro. This leads to their detachment from microtubules (MT) and an increased dynamic instability of MT. Here we show that MARK protein kinases phosphorylate MAP2 and MAP4 on their microtubule-binding domain in transfected CHO cells. In CHO cells expressing MARK1 or MARK2 under control of an inducible promoter, MARK2 phosphorylates an endogenous MAP4-related protein. Prolonged expression of MARK2 results in microtubule-disruption, detachment of cells from the substratum, and cell death. Concomitant with microtubule disruption, we also observed a breakdown of the vimentin network, whereas actin fibers remained unaffected. Thus, MARK seems to play an important role in controlling cytoskeletal dynamics.

    Cell motility and the cytoskeleton 1999;44;3;209-24

  • Microtubule-associated protein 4 (MAP4) regulates assembly, protomer-polymer partitioning and synthesis of tubulin in cultured cells.

    Nguyen HL, Gruber D and Bulinski JC

    Departments of Pathology and Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, BB1213-630 W. 168th St, New York, NY 10032-3702, USA. jcb4@columbia.edu

    We depleted MAP4, a ubiquitously expressed microtubule (MT)-associated protein previously shown to be capable of stabilizing MTs, from HeLa cells by stably expressing antisense RNA. These HeLa-AS cells, in which the MAP4 level was decreased to 33% of the wild-type level, displayed decreased content of total tubulin (65% of the wild-type level). The partitioning of cellular tubulin into protomer and polymer was altered in HeLa-AS cells: polymeric tubulin was decreased to 46% of the level in control cells, while protomeric tubulin was increased to 226% of the level in control cells. Tubulin protein synthesis was decreased, consistent with the tubulin autoregulation model, which proposes that tubulin protomer inhibits its own synthesis. Following release from drug-induced depolymerization, MTs in HeLa-AS cells reformed more slowly, and showed an increased focus on the centrosome, as compared to control cells. HeLa-AS cells also appeared to be less bipolar in shape and flatter than control cells. Our data suggest that MAP4 regulates assembly level of MTs and, perhaps through this mechanism, is involved in controlling spreading and shape of cells.

    Funded by: NCI NIH HHS: CA-70951, T32-CA09503

    Journal of cell science 1999;112 ( Pt 12);1813-24

  • MAP4 is the in vivo substrate for CDC2 kinase in HeLa cells: identification of an M-phase specific and a cell cycle-independent phosphorylation site in MAP4.

    Ookata K, Hisanaga S, Sugita M, Okuyama A, Murofushi H, Kitazawa H, Chari S, Bulinski JC and Kishimoto T

    Laboratory of Cell and Developmental Biology, Faculty of Biosciences, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226, Japan.

    We reported previously that cdc2 kinase decreased the microtubule-stabilizing ability of a major HeLa cell microtubule-associated protein, MAP4, by phosphorylation in vitro [Ookata, K., et al. (1995) J. Cell Biol. 128, 849-862]. An important question raised by this study is whether MAP4 is indeed phosphorylated by cdc2 kinase at mitosis in vivo. We present here evidence that cdc2 kinase is the major M-phase MAP4 kinase, and, further, we identify two phosphorylation sites within the proline-rich domain of MAP4. Metabolic 32P labeling showed the increased phosphorylation of MAP4 at mitosis. A specific inhibitor of cdc2 kinase, butyrolactone I, inhibited phosphorylation of MAP4 both in mitotic HeLa cells and in the mitotic HeLa cell extract. The phosphopeptide map analysis revealed the high similarity of in vivo labeled mitotic MAP4 to that phosphorylated by cdc2 kinase in vitro. Ser-696 and Ser-787, both of which lie within SPXK consensus sequences for cdc2 kinase, were identified as phosphorylation sites in the proline-rich region of MAP4 in vivo and in vitro. Immunoblotting with antibodies that recognize the phosphorylation state of Ser-696 or Ser-787 showed that Ser-787 in the SPSK sequence was specifically phosphorylated at mitosis while Ser-696 in the SPEK sequence was phosphorylated both at mitosis and in interphase. These results suggest that cdc2 kinase directly regulates microtubule dynamics at mitosis through phosphorylation of MAP4 at a number of sites, including Ser-787.

    Biochemistry 1997;36;50;15873-83

  • Overexpression of MAP4 inhibits organelle motility and trafficking in vivo.

    Bulinski JC, McGraw TE, Gruber D, Nguyen HL and Sheetz MP

    Department of Anatomy, Columbia University, College of Physicians & Surgeons, New York, NY 10032, USA. JCB4@columbia.edu

    We previously prepared cell lines that inducibly overexpress MAP4, a microtubule (MT)-associated protein widely expressed in non-neuronal cells. Overexpression of either the full-length MAP4 molecule or its MT-binding domain, MTB, stabilized MTs and retarded cell growth, suggesting that overexpressed MAP4 impacts on MT-dependent functions in vivo. To test this hypothesis, we examined MT-based vesicle movements in living cells, using high resolution DIC microscopy. Overexpression of either MAP4 or MTB yielded a dose-dependent reduction in the frequency of MT-dependent organelle movements, relative to control cells. At steady state, both MAP4- and MTB-overexpressing cells showed unusual distributions of transferrin, LDL, dextran, and Golgi elements, as compared to control cells. MAP4 preferentially inhibited receptor-dependent uptake and degradation of LDL, and repositioning of Golgi elements after disruption by the drug, brefeldin A. L-MOCK cells treated with Taxol to stabilize the MTs to an extent equivalent to MAP4 overexpression did not show similar inhibition of vesicle motility or organellar trafficking, suggesting that deficits in organelle movements in vivo represent a direct effect of the presence of MAP4 or MTB, rather than an indirect effect of the stabilization of MTs by overexpressed MAP constructs. Our results show that MAP4 has the capacity to affect transport along MTs in vivo; these findings suggest a potential mechanism by which MAP4 could contribute to polarization or morphogenesis of cells.

    Funded by: NIA NIH HHS: T32 AG00189

    Journal of cell science 1997;110 ( Pt 24);3055-64

  • Large-scale concatenation cDNA sequencing.

    Yu W, Andersson B, Worley KC, Muzny DM, Ding Y, Liu W, Ricafrente JY, Wentland MA, Lennon G and Gibbs RA

    A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

    Funded by: NHGRI NIH HHS: 1F32 HG00169-01, F32 HG000169, F33 HG000210, P30 HG00210-05, R01 HG00823

    Genome research 1997;7;4;353-8

  • Phosphorylation of microtubule-associated proteins MAP2 and MAP4 by the protein kinase p110mark. Phosphorylation sites and regulation of microtubule dynamics.

    Illenberger S, Drewes G, Trinczek B, Biernat J, Meyer HE, Olmsted JB, Mandelkow EM and Mandelkow E

    Max-Planck-Unit for Structural Molecular Biology, Hamburg, Germany.

    The phosphorylation of microtubule-associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule stability. We have shown recently that a novel protein kinase, termed p110 microtubule-affinity regulating kinase ("MARK"), phosphorylates microtubule-associated protein tau at the KXGS motifs in the region of internal repeats and causes the detachment of tau from microtubules (Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H.E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Here we show that p110mark phosphorylates analogous KXGS sites in the microtubule binding domains of the neuronal MAP2 and the ubiquitous MAP4. Phosphorylation in vitro leads to the dissociation of MAP2 and MAP4 from microtubules and to a pronounced increase in dynamic instability. Thus, the phosphorylation of the repeated motifs in the microtubule binding domains of MAPs by p110mark might provide a mechanism for the regulation of microtubule dynamics in cells.

    The Journal of biological chemistry 1996;271;18;10834-43

  • A "double adaptor" method for improved shotgun library construction.

    Andersson B, Wentland MA, Ricafrente JY, Liu W and Gibbs RA

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.

    The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

    Funded by: NHGRI NIH HHS: R01 HG00823

    Analytical biochemistry 1996;236;1;107-13

  • Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics.

    Ookata K, Hisanaga S, Bulinski JC, Murofushi H, Aizawa H, Itoh TJ, Hotani H, Okumura E, Tachibana K and Kishimoto T

    Laboratory of Cell and Developmental Biology, Faculty of Biosciences, Tokyo Institute of Technology, Yokohama, Japan.

    We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.

    The Journal of cell biology 1995;128;5;849-62

  • Differential expression of alternatively spliced forms of MAP4: a repertoire of structurally different microtubule-binding domains.

    Chapin SJ, Lue CM, Yu MT and Bulinski JC

    Department of Anatomy & Cell Biology, College of Physicians & Surgeons, Columbia University, New York, New York 10032.

    We previously reported that the microtubule (MT)-binding domain of microtubule-associated protein 4 (MAP4) contains three 18-amino acid imperfect repeats that are homologous to the repeats found in the MT-binding domains of the neuronal MAPs, MAP2 and tau [Chapin, S. J., & Bulinski, J. C. (1991) J. Cell Sci. 98, 27-36]. Here we report the isolation of clones encoding additional isoforms of MAP4, which differ in the number of repeated elements contained within their MT-binding domains. In addition to clones encoding three repeats, we isolated clones encoding a four-repeat isoform, whose MT-binding domain bears a striking similarity to the four-repeat isoform of tau. Other MAP4 clones that we isolated encode five repeats. The additional repeat in the five-repeat isoform of MAP4 is quite unusual in its amino acid sequence; this unusual repeat was also found by Aizawa et al. [Aizawa, H., et al. (1990) J. Biol. Chem. 265, 13849-13855] among the repeats encoded by bovine MAP4 clones possessing four repeats. In humans, MAP4 was recently shown to be encoded by a single-copy gene [West, R. R., et al. (1991) J. Biol. Chem. 266, 21886-21896]; we demonstrated that the human MAP4 gene is located on human chromosome 3p21. Expression of multiple MAP4 isoforms from this gene, which appears to result from alternative RNA splicing, was investigated by RNase protection analysis of mammalian cell lines and rat tissues. The five-repeat isoform was the only form detectable in most cell lines, and it was the most abundant isoform expressed in rat lung, liver, kidney, spleen, and testis. However, in rat brain, heart, and skeletal muscle, although the five-repeat isoform was expressed at all developmental stages examined, the tau-like four-repeat isoform was also expressed, and its expression increased during development. The three-repeat isoform was expressed in heart and, to a lesser extent, in brain, skeletal muscle, and lung. Our results demonstrate that several different MAP4 isoforms are expressed in the rat in different tissues and at various developmental stages. Furthermore, our data suggest that differential expression of MAP4 isoforms possessing distinct MT-binding domains may be involved in the changes in MT dynamics or function that are known to accompany differentiation.

    Biochemistry 1995;34;7;2289-301

  • Cellular microtubules heterogeneous in their content of microtubule-associated protein 4 (MAP4).

    Chapin SJ and Bulinski JC

    Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, NY 10032.

    Previous immunolocalization studies using many primate cultured cell lines demonstrated that a microtubule-associated protein of M(r) approximately 210,000 which is now called MAP4, is present along the length of microtubules in interphase and mitotic cells [Bulinski and Borisy (1980) J. Cell Biol. 87:802-808; DeBrabander et al. (1981) J. Cell Biol. 91:438-455]. Since MAP4 has been implicated as a microtubule stabilizer, we asked whether all classes of microtubules possess an equal complement of MAP4. We have reexamined the cellular distribution of MAP4, using both conventional double-label immunofluorescence and an antibody blocking technique [Schulze and Kirschner (1987) J. Cell Biol. 104:277-288] to highlight microtubules lacking, or depleted in, MAP4. These techniques have revealed that thin processes extending from monkey kidney cells (TC-7), and those made by human neuroblastoma cells (IMR-32) in response to retinoic acid, are often deficient in MAP4 immunoreactivity. Since both types of cellular processes contain stable microtubules, which are enriched in detyrosinated (Glu) tubulin, we tested the ability of MAP4 to bind to microtubules made from pure Glu and pure tyrosinated (Tyr) tubulin in vitro. MAP4 bound to both types of microtubules, and the similar saturation level of MAP4 binding to Glu and Tyr microtubules suggested that differential binding to these forms of tubulin does not contribute directly to a mechanism for segregation of MAP4 on microtubules in vivo. In TC-7 cells, we also observed MAP4-depletion on single microtubules, distal regions of broad cytoplasmic extensions, and midbodies of dividing cells. MAP4 depletion may reflect recent, rapid growth of microtubules to which MAP4 has not yet bound, or the presence of other MAPs that may compete with MAP4 for binding sites on the MT. We suggest that different levels of MAP4 on microtubules may directly modulate microtubule dynamics within single cells, as well as other microtubule functions such as those involving microtubule motor activity.

    Funded by: NCI NIH HHS: CA 09056-12, CA39755

    Cell motility and the cytoskeleton 1994;27;2;133-49

  • Microtubule stabilization by assembly-promoting microtubule-associated proteins: a repeat performance.

    Chapin SJ and Bulinski JC

    Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.

    Cell motility and the cytoskeleton 1992;23;4;236-43

  • A model for microtubule-associated protein 4 structure. Domains defined by comparisons of human, mouse, and bovine sequences.

    West RR, Tenbarge KM and Olmsted JB

    Department of Biology, University of Rochester, New York 14627.

    cDNAs encoding human and mouse microtubule-associated protein 4 (MAP 4) were isolated. MAP 4 is encoded by a single gene. Multiple MAP 4 mRNAs are transcribed that are differentially expressed among mouse tissues. Open reading frames for the human and mouse MAP 4 clones indicate three distinct regions consisting of related sequences with different motifs. Approximately 30% of the protein is tandem related repeats of approximately 14 amino acids. Another region contains clusters of serine and proline. Four 18-mer repeats characteristic of the microtubule-binding domains of MAP 2 and tau are located at the carboxyl-terminal portion of MAP 4. Amino acid sequence analysis revealed that human and mouse MAP 4 are homologs of the bovine 190-kDa MAP/MAP U (Aizawa, H., Emori, Y., Murofushi, H., Kawasakai, H., Sakai, H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855). Mouse and human MAP 4 and the bovine 190-kDa MAP are approximately 75% similar, indicating that these proteins are all members of the same class. Domains with extremely high conservation (greater than or equal to 88%) are: 1) the extreme amino terminus; 2) a proline-rich region between the KDM and S,P domains; 3) the microtubule-binding domain; and 4) the extreme carboxyl terminus.

    Funded by: NIGMS NIH HHS: GM 22214

    The Journal of biological chemistry 1991;266;32;21886-96

  • Non-neuronal 210 x 10(3) Mr microtubule-associated protein (MAP4) contains a domain homologous to the microtubule-binding domains of neuronal MAP2 and tau.

    Chapin SJ and Bulinski JC

    Department of Biology, University of California, Los Angeles 90024.

    A polyclonal antiserum raised against a HeLa cell microtubule-associated protein of Mr 210,000 (210 kD MAP or MAP4), an abundant non-neuronal MAP, was used to isolate cDNA clones encoding MAP4 from a human fetal brain lambda gt11 cDNA expression library. The largest of these clones, pMAP4.245, contains an insert of 4.1 kb and encodes a 245 kD beta-galactosidase fusion protein. Evidence that pMAP4.245 encodes MAP4 sequences includes immunoabsorption of MAP4 antibodies with the pMAP4.245 fusion protein, as well as identity of protein sequences obtained from HeLa 210 kD MAP4 with amino acid sequences encoded by pMAP4.245. The MAP4.245 cDNA hybridizes to several large (approximately 6-9 kb) transcripts on Northern blots of HeLa cell RNA. DNA sequencing of overlapping MAP4 cDNA clones revealed a long open reading frame containing a C-terminal region with three imperfect 18-amino acid repeats; this region is homologous to a motif present in the microtubule (MT)-binding domain of two prominent neuronal MAPs, MAP2 and tau. The pMAP4.245 sequence also encoded a series of unrelated repeats, located in the MAP's projection domain, N-terminal to the MT-binding domain. MAP4.245 fusion proteins bound to MTs in vitro, while fusion proteins that contained only the projection domain repeats failed to bind specifically to MTs. Thus, the major human non-neuronal MAP resembles two neuronal MAPs in its MT-binding domain, while most of the molecule has sequences, and presumably functions, distinct from those of the neuronal MAPs.

    Funded by: NCI NIH HHS: CA09056

    Journal of cell science 1991;98 ( Pt 1);27-36

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

Cookies Policy | Terms and Conditions. This site is hosted by Edinburgh University and the Genes to Cognition Programme.