G2Cdb::Gene report

Gene id
G00002021
Gene symbol
DCTN2 (HGNC)
Species
Homo sapiens
Description
dynactin 2 (p50)
Orthologue
G00000772 (Mus musculus)

Databases (7)

Gene
ENSG00000175203 (Ensembl human gene)
10540 (Entrez Gene)
1175 (G2Cdb plasticity & disease)
DCTN2 (GeneCards)
Literature
607376 (OMIM)
Marker Symbol
HGNC:2712 (HGNC)
Protein Sequence
Q13561 (UniProt)

Synonyms (2)

  • DCTN-50
  • RBP50

Literature (37)

Pubmed - other

  • Proteomic analysis reveals Hrs ubiquitin-interacting motif-mediated ubiquitin signaling in multiple cellular processes.

    Pridgeon JW, Webber EA, Sha D, Li L and Chin LS

    Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA.

    Despite the critical importance of protein ubiquitination in the regulation of diverse cellular processes, the molecular mechanisms by which cells recognize and transmit ubiquitin signals remain poorly understood. The endosomal sorting machinery component hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) contains a ubiquitin-interacting motif (UIM), which is believed to bind ubiquitinated membrane cargo proteins and mediate their sorting to the lysosomal degradation pathway. To gain insight into the role of Hrs UIM-mediated ubiquitin signaling in cells, we performed a proteomic screen for Hrs UIM-interacting ubiquitinated proteins in human brain by using an in vitro expression cloning screening approach. We have identified 48 ubiquitinated proteins that are specifically recognized by the UIM domain of Hrs. Among them, 12 are membrane proteins that are likely to be Hrs cargo proteins, and four are membrane protein-associated adaptor proteins whose ubiquitination may act as a signal to target their associated membrane cargo for Hrs-mediated endosomal sorting. Other classes of the identified proteins include components of the vesicular trafficking machinery, cell signaling molecules, proteins associated with the cytoskeleton and cytoskeleton-dependent transport, and enzymes involved in ubiquitination and metabolism, suggesting the involvement of Hrs UIM-mediated ubiquitin signaling in the regulation of multiple cellular processes. We have characterized the ubiquitination of two identified proteins, Munc18-1 and Hsc70, and their interaction with Hrs UIM, and provided functional evidence supporting a role for Hsc70 in the regulation of Hrs-mediated endosome-to-lysosome trafficking.

    Funded by: NIGMS NIH HHS: GM082828, R01 GM082828, R01 GM082828-01A1, R01 GM082828-02, R01 GM082828-02S1; NINDS NIH HHS: NS047575, NS050650, R01 NS047575, R01 NS047575-01, R01 NS047575-02, R01 NS047575-03, R01 NS047575-04, R01 NS050650, R01 NS050650-01A1, R01 NS050650-02, R01 NS050650-03, R01 NS050650-04, T32 NS007480, T32 NS007480-05, T32 NS007480-06, T32 NS007480-07, T32 NS007480-08, T32 NS007480-09, T32NS007480

    The FEBS journal 2009;276;1;118-31

  • N-terminal region of ZW10 serves not only as a determinant for localization but also as a link with dynein function.

    Inoue M, Arasaki K, Ueda A, Aoki T and Tagaya M

    School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan.

    ZW10 interacts with dynamitin, a subunit of the dynein accessory complex dynactin, and functions in termination of the spindle checkpoint during mitosis and in membrane transport between the endoplasmic reticulum (ER) and Golgi apparatus during interphase. Its associations with kinetochores and ER membranes are mediated by Zwint-1 and RINT-1, respectively. A previous yeast two-hybrid study showed that the C-terminal region of ZW10 interacts with dynamitin, and part of this region has been used as an inhibitor of ZW10 function. In the present study, we reinvestigated the interaction between ZW10 and dynamitin, and showed that the N-terminal region of ZW10 is the major binding site for dynamitin and, like full-length ZW10, could potentially move along microtubules to the centrosomal area in a dynein-dynactin-dependent manner. Competitive binding experiments demonstrated that dynamitin and RINT-1 occupy the same N-terminal region of ZW10 in a mutually exclusive fashion. Consistent with this, over-expression of RINT-1 interfered with the dynein-dynactin-mediated movement of ZW10 to the centrosomal area. Given that the N-terminal region of ZW10 also interacts with Zwint-1, this region may be important for switching partners; one partner is a determinant for localization (kinetochore and ER) and the other links ZW10 to dynein function.

    Genes to cells : devoted to molecular & cellular mechanisms 2008;13;8;905-14

  • Dynamitin mutagenesis reveals protein-protein interactions important for dynactin structure.

    Maier KC, Godfrey JE, Echeverri CJ, Cheong FK and Schroer TA

    Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.

    Dynactin is a highly conserved, multiprotein complex that works in conjunction with microtubule-based motors to power a variety of intracellular motile events. Dynamitin (p50) is a core element of dynactin structure. In the present study, we use targeted mutagenesis to evaluate how dynamitin's different structural domains contribute to its ability to self-associate, interact with dynactin and assemble into a complex with its close binding partner, p24. We show that these interactions involve three distinct structural elements: (i) a previously unidentified dimerization motif in the N-terminal 100 amino acids, (ii) an alpha-helical motif spanning aa 106-162 and (iii) the C-terminal half of the molecule (aa 213-406), which is predicted to fold into an antiparallel alpha-helix bundle. The N-terminal half of dynamitin by itself is sufficient to disrupt dynactin, although very high concentrations are required. The ability of mutations in dynamitin's interaction domains to disrupt dynactin in vitro was found to correlate with their inhibitory effects when expressed in cells. We determined that the dynactin subunit, p24, governs dynamitin oligomerization by binding dynamitin along its length. This suppresses aberrant multimerization and drives formation of a protein complex that is identical to the native dynactin shoulder.

    Funded by: NIGMS NIH HHS: R01 GM044589, R01 GM044589-15, R01 GM044589-16, R01 GM044589-17, R01GM44589, R37 GM047434, R37GM047434

    Traffic (Copenhagen, Denmark) 2008;9;4;481-91

  • Disrupted in Schizophrenia 1 Interactome: evidence for the close connectivity of risk genes and a potential synaptic basis for schizophrenia.

    Camargo LM, Collura V, Rain JC, Mizuguchi K, Hermjakob H, Kerrien S, Bonnert TP, Whiting PJ and Brandon NJ

    Merck Research Labs, Merck & Co., Boston, MA 02115, USA. miguel_camargo@merck.com

    Disrupted in Schizophrenia 1 (DISC1) is a schizophrenia risk gene associated with cognitive deficits in both schizophrenics and the normal ageing population. In this study, we have generated a network of protein-protein interactions (PPIs) around DISC1. This has been achieved by utilising iterative yeast-two hybrid (Y2H) screens, combined with detailed pathway and functional analysis. This so-called 'DISC1 interactome' contains many novel PPIs and provides a molecular framework to explore the function of DISC1. The network implicates DISC1 in processes of cytoskeletal stability and organisation, intracellular transport and cell-cycle/division. In particular, DISC1 looks to have a PPI profile consistent with that of an essential synaptic protein, which fits well with the underlying molecular pathology observed at the synaptic level and the cognitive deficits seen behaviourally in schizophrenics. Utilising a similar approach with dysbindin (DTNBP1), a second schizophrenia risk gene, we show that dysbindin and DISC1 share common PPIs suggesting they may affect common biological processes and that the function of schizophrenia risk genes may converge.

    Molecular psychiatry 2007;12;1;74-86

  • GSK-3beta-regulated interaction of BICD with dynein is involved in microtubule anchorage at centrosome.

    Fumoto K, Hoogenraad CC and Kikuchi A

    Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.

    Microtubule arrays direct intracellular organization and define cellular polarity. Here, we show a novel function of glycogen synthase kinase-3beta (GSK-3beta) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein-dynactin and to function in the intracellular vesicle trafficking. Our data revealed that GSK-3beta is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3beta or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3beta knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3beta and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3beta may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization.

    The EMBO journal 2006;25;24;5670-82

  • The PITSLRE/CDK11p58 protein kinase promotes centrosome maturation and bipolar spindle formation.

    Petretti C, Savoian M, Montembault E, Glover DM, Prigent C and Giet R

    CNRS UMR 6061 Université de Rennes I, Equipe Labellisée Ligue Nationale Contre le Cancer, IFR140 GFAS, Faculté de Médecine, France.

    The CDK11 (cyclin-dependent kinase 11) gene has an internal ribosome entry site (IRES), allowing the expression of two protein kinases. The longer 110-kDa isoform is expressed at constant levels during the cell cycle and the shorter 58-kDa isoform is expressed only during G2 and M phases. By means of RNA interference (RNAi), we show that the CDK11 gene is required for mitotic spindle formation. CDK11 RNAi leads to mitotic checkpoint activation. Mitotic cells are arrested with short or monopolar spindles. gamma-Tubulin as well as Plk1 and Aurora A protein kinase levels are greatly reduced at centrosomes, resulting in microtubule nucleation defects. We show that the mitotic CDK11(p58) isoform, but not the CDK11(p110) isoform, associates with mitotic centrosomes and rescues the phenotypes resulting from CDK11 RNAi. This work demonstrates for the first time the role of CDK11(p58) in centrosome maturation and bipolar spindle morphogenesis.

    EMBO reports 2006;7;4;418-24

  • Phenotypic changes associated with DYNACTIN-2 (DCTN2) over expression characterise SJSA-1 osteosarcoma cells.

    Bransfield KL, Askham JM, Leek JP, Robinson PA and Mighell AJ

    Molecular Medicine Unit, The University of Leeds, St. James's University Hospital, Leeds, United Kingdom.

    DYNACTIN-2 (DCTN2) localises to chromosome 12q13-q15, a region prone to stable amplification in several cancers. Transient DCTN2 overexpression has a significant impact on cellular phenotype primarily due to disruption of the DYNEIN-dynactin motor. Changes reported include alterations of microtubule-directed movement of molecular (e.g. TP53) and organelle (e.g. Golgi) cargoes towards the nucleus, centrosome biology, cellular movement and mitosis with a potential predisposition to mitotic block and polyploidy. These changes would be expected to be of relevance to carcinogenesis. To investigate this, we report the first study of DCTN2 genomic amplification and sustained DCTN2 overexpression in cancer cells. QFMPCR was employed to characterise the extent of chromosome 12q13-q15 amplicons in SJSA-1, SJRH30, U373MG and CCF-STTG1 cancer cells. DCTN2 amplification was present in SJSA-1, U373MG and SJRH30 cells, yet was incomplete at the 5'-end in SJRH30 cells. Only SJSA-1 cells were characterised by DCTN2 overexpression on Western blot analyses. Microscopy studies distinguished SJSA-1 cells by greater DCTN2 immunofluorescence and diminished centrosome and 58K protein Golgi-marker focus compared to SJRH30 cells. Indirect evidence derived from the published work of others indicated that TP53 transport into the nucleus was unimpaired. Furthermore, we observed that SJSA-1 cells were easy to propagate. In conclusion, persistent DCTN2 overexpression can be tolerated in SJSA-1 cancer cells despite phenotypic abnormalities predicted from transient overexpression studies. This preliminary study does not support a major role for DCTN2 overexpression in carcinogenesis, although further studies would be necessary to confirm this.

    Molecular carcinogenesis 2006;45;3;157-63

  • 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

  • RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins.

    Khanna H, Hurd TW, Lillo C, Shu X, Parapuram SK, He S, Akimoto M, Wright AF, Margolis B, Williams DS and Swaroop A

    Department of Ophthalmology & Visual Sciences, University of Michigan, Ann Arbor, Michigan 48105, USA.

    Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of photoreceptor connecting cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, structural maintenance of chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal photoreceptors and Madin-Darby canine kidney cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150Glued, and p50-dynamitin. Inhibition of dynein function by overexpressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia.

    Funded by: NEI NIH HHS: EY07003, EY07961, EY12598, EY13408, F31 EY007003, P30 EY007003, P30 EY012598, R01 EY007042, R01 EY007961, R01 EY013408; NIDDK NIH HHS: DK069605, R01 DK069605

    The Journal of biological chemistry 2005;280;39;33580-7

  • 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

  • Requirement for microtubules and dynein motors in the earliest stages of peroxisome biogenesis.

    Brocard CB, Boucher KK, Jedeszko C, Kim PK and Walton PA

    Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, Canada N6A 5C1.

    Our aim was to determine the role of microtubules in the biogenesis of peroxisomes. Fusion experiments between human PEX16- and PEX1-mutant cells in the presence of nocodazol implied that microtubules were not required for import of proteins into the peroxisomal matrix after cell fusion complementation. We further studied the importance of microtubules in the early stages of peroxisome biogenesis following the microinjection complementation of PEX16-mutant cells. In the absence of nocodazol, nuclear microinjection of plasmids expressing EGFP-SKL and Pex16p in PEX16-mutant cells resulted in the accumulation of EGFP-SKL into newly formed peroxisomes. However, pretreatment of the cells with nocodazol, prior to microinjection, resulted in the inhibition of complementation of the PEX16 mutant and the cytosolic location of the EGFP-SKL. In addition, coexpression of a dominant-negative CC1 subunit of the dynein/dynactin motor complex resulted in the inability to complement PEX16-mutant cells. Both of these treatments resulted in the cytosolic localization of expressed Pex16p. Our results demonstrate that the formation of peroxisomes via the preperoxisomal compartment is dependent upon microtubules and minus-end-directed motor proteins and that the inhibition described above occurs at a step that precedes the association of Pex16p with the vesicles that would otherwise become the peroxisomes.

    Traffic (Copenhagen, Denmark) 2005;6;5;386-95

  • Coupling of ER exit to microtubules through direct interaction of COPII with dynactin.

    Watson P, Forster R, Palmer KJ, Pepperkok R and Stephens DJ

    Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.

    Transport of proteins from the endoplasmic reticulum (ER) to the Golgi is mediated by the sequential action of two coat complexes: COPII concentrates cargo for secretion at ER export sites, then COPI is subsequently recruited to nascent carriers and retrieves recycling proteins back to the ER. These carriers then move towards the Golgi along microtubules, driven by the dynein/dynactin complexes. Here we show that the Sec23p component of the COPII complex directly interacts with the dynactin complex through the carboxy-terminal cargo-binding domain of p150(Glued). Functional assays, including measurements of the rate of recycling of COPII on the ER membrane and quantitative analyses of secretion, indicate that this interaction underlies functional coupling of ER export to microtubules. Together, our data suggest a mechanism by which membranes of the early secretory pathway can be linked to motors and microtubules for subsequent organization and movement to the Golgi apparatus.

    Funded by: Wellcome Trust: 071233

    Nature cell biology 2005;7;1;48-55

  • Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites.

    Ficarro SB, Salomon AR, Brill LM, Mason DE, Stettler-Gill M, Brock A and Peters EC

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

    A versatile integrated system has been developed for the automated enrichment and analysis of phosphopeptides by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). This system utilizes two independently controlled high-performance liquid chromatography (HPLC) pumps, an autosampler and microvalves to prepare and elute samples into an ion trap mass spectrometer. The use of robust reversed-phase HPLC columns with integrated ESI emitter tips enables the reproducible detection and identification of low-femtomole quantities of phosphopeptides. The entire system is coordinated through a simple user interface by customized software. The ruggedness of the system is demonstrated by highly reproducible analyses of single and multi-protein digests, while its utility is demonstrated by the thorough evaluation of the relative immunoprecipitation efficiencies of several commercially available anti-phosphotyrosine antibodies.

    Rapid communications in mass spectrometry : RCM 2005;19;1;57-71

  • Phosphoproteomic analysis of the developing mouse brain.

    Ballif BA, Villén J, Beausoleil SA, Schwartz D and Gygi SP

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

    Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.

    Funded by: NHGRI NIH HHS: HG00041

    Molecular & cellular proteomics : MCP 2004;3;11;1093-101

  • 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

  • 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

  • Interaction of Cep135 with a p50 dynactin subunit in mammalian centrosomes.

    Uetake Y, Terada Y, Matuliene J and Kuriyama R

    Department of Genetics, Cell Biology, Development, University of Minnesota, Minneapolis 55455, USA.

    Cep135 is a 135-kDa, coiled-coil centrosome protein important for microtubule organization in mammalian cells [Ohta et al., 2002: J. Cell Biol. 156:87-99]. To identify Cep135-interacting molecules, we screened yeast two-hybrid libraries. One clone encoded dynamitin, a p50 dynactin subunit, which localized at the centrosome and has been shown to be involved in anchoring microtubules to centrosomes. The central domain of p50 binds to the C-terminal sequence of Cep135; this was further confirmed by immunoprecipitation and immunostaining of CHO cells co-expressing the binding domains for Cep135 and p50. Exogenous p50 lacking the Cep 135-binding domain failed to locate at the centrosome, suggesting that Cep135 is required for initial targeting of the centrosome. Altered levels of Cep135 and p50 by RNAi and protein overexpression caused the release of endogenous partner molecules from centrosomes. This also resulted in dislocation of other centrosomal molecules, such as gamma-tubulin and pericentrin, ultimately leading to disorganization of microtubule patterns. These results suggest that Cep135 and p50 play an important role in assembly and maintenance of functional microtubule-organizing centers.

    Funded by: NIGMS NIH HHS: GM55735

    Cell motility and the cytoskeleton 2004;58;1;53-66

  • 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

  • Polo-like kinase 1 regulates Nlp, a centrosome protein involved in microtubule nucleation.

    Casenghi M, Meraldi P, Weinhart U, Duncan PI, Körner R and Nigg EA

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

    In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.

    Developmental cell 2003;5;1;113-25

  • Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

    Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. kris.gevaert@rug.ac.be

    Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

    Nature biotechnology 2003;21;5;566-9

  • The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes.

    Short B, Preisinger C, Schaletzky J, Kopajtich R and Barr FA

    Department of Cell Biology, Max-Planck-Institute of Biochemistry, Martinsried, Germany.

    Dynactin is a multisubunit protein complex required for the activity of dynein in diverse intracellular motility processes, including membrane transport. Dynactin can bind to vesicles and liposomes containing acidic phospholipids, but general properties such as this are unlikely to explain the regulated recruitment of dynactin to specific sites on organelle membranes. Additional factors must therefore exist to control this process. Candidates for these factors are the Rab GTPases, which function in the tethering of vesicles to their target organelle prior to membrane fusion. In particular, Rab27a tethers melanosomes to the actin cytoskeleton. Other Rabs have been implicated in microtubule-dependent organelle motility; Rab7 controls lysosomal transport, and Rab6 is involved in microtubule-dependent transport pathways through the Golgi and from endosomes to the Golgi. We demonstrate that dynactin binds to Rab6 and shows a Rab6-dependent recruitment to Golgi membranes. Other Golgi Rabs do not bind to dynactin and are unable to support its recruitment to membranes. Rab6 therefore functions as a specificity or tethering factor controlling the recruitment of dynactin to membranes.

    Current biology : CB 2002;12;20;1792-5

  • Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex.

    Takahashi M, Yamagiwa A, Nishimura T, Mukai H and Ono Y

    Biosignal Research Center, Kobe University, Japan.

    Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC.

    Molecular biology of the cell 2002;13;9;3235-45

  • Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function.

    Tai CY, Dujardin DL, Faulkner NE and Vallee RB

    University of Massachusetts Medical School, Department of Cell Biology, Worcester, MA 01605, USA.

    Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170-dynein interactions and in coordinating dynein cargo-binding and motor activities.

    Funded by: NICHD NIH HHS: HD61982; NIGMS NIH HHS: GM47434, R01 GM047434, R37 GM047434

    The Journal of cell biology 2002;156;6;959-68

  • Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes.

    Hoogenraad CC, Akhmanova A, Howell SA, Dortland BR, De Zeeuw CI, Willemsen R, Visser P, Grosveld F and Galjart N

    MGC Department of Cell Biology, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands.

    Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein- dynactin interaction on the surface of membranous organelles, by associating with these complexes.

    The EMBO journal 2001;20;15;4041-54

  • Colocalization of dynactin subunits P150Glued and P50 with melanosomes in normal human melanocytes.

    Vancoillie G, Lambert J, Haeghen YV, Westbroek W, Mulder A, Koerten HK, Mommaas AM, Van Oostveldt P and Naeyaert JM

    Department of Dermatology, University Hospital, Ghent, Belgium.

    Melanocytic dendrites consist of a central core of microtubules (MT) and a subcortical actin network. In previous reports we showed the presence of MT-associated motor proteins kinesin and cytoplasmic dynein on the melanosomal surface, forming a link with MT (Vancoillie et al. J Invest Dermatol 2000;114:421-429; Vancoillie et al. Br J Dermatol 2000;143:258-306). We could also demonstrate the association of kinectin, the kinesin receptor, with melanosomes. The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. Therefore, in this study, we investigated the in vitro expression of dynactin subunits P150Glued and P50 in normal human epidermal melanocytes, keratinocytes, and dermal fibroblasts by reverse transcription-polymerase chain reaction and northern blot analysis. In an attempt to gain an insight into the subcellular localization of dynactin, immunofluorescence and immunoelectron microscopy (IEM) studies were performed. The two isoforms of P150Glued and P50 are expressed in all studied skin cells. Immunofluorescence staining shows punctate distributions for P150Glued and P50 in melanocytes. P150Glued shows a clear centrosomal staining and accentuation in the dendrite tips. P50 is also accentuated in the perinuclear area and dendrite tips. Immunofluorescence double-labeling with a melanosome marker showed apparent colocalization of both P150Glued and P50 with melanosomes. By IEM, P50 is detected on the surface of the majority of melanosomes in melanocytes. The colocalization of different subunits of the dynactin complex with melanosomes is consistent with the earlier finding of cytoplasmic dynein association with melanosomes and supports the hypothesis that this complex could form a link between cytoplasmic dynein and the melanosomal membrane.

    Pigment cell research 2000;13;6;449-57

  • The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion.

    Mayor T, Stierhof YD, Tanaka K, Fry AM and Nigg EA

    Department of Molecular Biology, Sciences II, University of Geneva, CH-1211 Geneva, Switzerland.

    Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.

    The Journal of cell biology 2000;151;4;837-46

  • Protein kinase C-regulated dynamitin-macrophage-enriched myristoylated alanine-rice C kinase substrate interaction is involved in macrophage cell spreading.

    Yue L, Lu S, Garces J, Jin T and Li J

    Department of Oral Biology, College of Dentistry, the Cancer Center and Department of Microbiology & Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.

    Macrophage spreading requires the microtubule cytoskeleton and protein kinase C (PKC). The mechanism of involvement of the microtubules and PKC in this event is not fully understood. Dynamitin is a subunit of dynactin, which is important for linking the microtubule-dependent motor protein dynein to vesicle membranes. We report that dynamitin is a Ca(2+)/calmodulin-binding protein and that dynamitin binds directly to macrophage-enriched myristoylated alanine-rice C kinase substrate (MacMARCKS), a membrane-associated PKC substrate involved in macrophage spreading and integrin activation. Dynamitin was found to copurify with MacMARCKS both during MacMARCKS purification with conventional chromatography and during the immunoabsorption of MacMARCKS using anti-MacMARCKS antibody. Vice versa, MacMARCKS was also found to cosediment with the 20 S dynactin complex. We determined that the effector domain of MacMARCKS is required to interact with the N-terminal domain of dynamitin. MacMARCKS and dynamitin also partially colocalized at peripheral regions of macrophages and in the cell-cell border of 293 epithelial cells. Treatment with phorbol esters abolished this colocalization. Disrupting the interaction with a short peptide derived from the MacMARCKS-binding domain of dynamitin caused macrophages to spread and flatten. These data suggest that the dynamitin-MacMARCKS interaction is involved in cell spreading. Furthermore, the regulation of this interaction by PKC and Ca(2+)/calmodulin provides a possible regulatory mechanism for cell adhesion and spreading.

    Funded by: NIGMS NIH HHS: GM54715

    The Journal of biological chemistry 2000;275;31;23948-56

  • Formation of spindle poles by dynein/dynactin-dependent transport of NuMA.

    Merdes A, Heald R, Samejima K, Earnshaw WC and Cleveland DW

    ICMB, University of Edinburgh, Scotland. a.merdes@ed.ac.uk

    NuMA is a large nuclear protein whose relocation to the spindle poles is required for bipolar mitotic spindle assembly. We show here that this process depends on directed NuMA transport toward microtubule minus ends powered by cytoplasmic dynein and its activator dynactin. Upon nuclear envelope breakdown, large cytoplasmic aggregates of green fluorescent protein (GFP)-tagged NuMA stream poleward along spindle fibers in association with the actin-related protein 1 (Arp1) protein of the dynactin complex and cytoplasmic dynein. Immunoprecipitations and gel filtration demonstrate the assembly of a reversible, mitosis-specific complex of NuMA with dynein and dynactin. NuMA transport is required for spindle pole assembly and maintenance, since disruption of the dynactin complex (by increasing the amount of the dynamitin subunit) or dynein function (with an antibody) strongly inhibits NuMA translocation and accumulation and disrupts spindle pole assembly.

    Funded by: NIGMS NIH HHS: GM 29513, R01 GM029513, R37 GM029513; Wellcome Trust: 073915

    The Journal of cell biology 2000;149;4;851-62

  • A dynactin subunit with a highly conserved cysteine-rich motif interacts directly with Arp1.

    Karki S, Tokito MK and Holzbaur EL

    Department of Animal Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    Dynactin is a multisubunit complex and a required cofactor for most, or all, of the cellular processes powered by the microtubule-based motor cytoplasmic dynein. Using a dynein affinity column, the previously uncharacterized p62 subunit of dynactin was isolated and microsequenced. Two peptide sequences were used to clone human cDNAs encoding p62. Sequence analysis of the predicted human polypeptide of 53 kDa revealed a highly conserved pattern of eleven cysteine residues, eight of which fit the consensus sequence for a Zn(2+)-binding RING domain. We have characterized p62 as an integral component of 20 S dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments demonstrate that p62 binds directly to the Arp1 subunit of dynactin. Immunocytochemistry with antibodies to p62 demonstrates that this polypeptide has a punctate cytoplasmic distribution as well as centrosomal distribution typical of dynactin. In transfected cells, overexpression of p62 did not disrupt microtubule organization or the integrity of the Golgi but did cause both cytosolic and nuclear distribution of the protein, suggesting that this polypeptide may be targeted to the nucleus at very high expression levels.

    Funded by: NIGMS NIH HHS: GM48661

    The Journal of biological chemistry 2000;275;7;4834-9

  • Analysis of dynactin subcomplexes reveals a novel actin-related protein associated with the arp1 minifilament pointed end.

    Eckley DM, Gill SR, Melkonian KA, Bingham JB, Goodson HV, Heuser JE and Schroer TA

    Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.

    The multisubunit protein, dynactin, is a critical component of the cytoplasmic dynein motor machinery. Dynactin contains two distinct structural domains: a projecting sidearm that interacts with dynein and an actin-like minifilament backbone that is thought to bind cargo. Here, we use biochemical, ultrastructural, and molecular cloning techniques to obtain a comprehensive picture of dynactin composition and structure. Treatment of purified dynactin with recombinant dynamitin yields two assemblies: the actin-related protein, Arp1, minifilament and the p150(Glued) sidearm. Both contain dynamitin. Treatment of dynactin with the chaotropic salt, potassium iodide, completely depolymerizes the Arp1 minifilament to reveal multiple protein complexes that contain the remaining dynactin subunits. The shoulder/sidearm complex contains p150(Glued), dynamitin, and p24 subunits and is ultrastructurally similar to dynactin's flexible projecting sidearm. The dynactin shoulder complex, which contains dynamitin and p24, is an elongated, flexible assembly that may link the shoulder/sidearm complex to the Arp1 minifilament. Pointed-end complex contains p62, p27, and p25 subunits, plus a novel actin-related protein, Arp11. p62, p27, and p25 contain predicted cargo-binding motifs, while the Arp11 sequence suggests a pointed-end capping activity. These isolated dynactin subdomains will be useful tools for further analysis of dynactin assembly and function.

    Funded by: NIGMS NIH HHS: R0 GM44598

    The Journal of cell biology 1999;147;2;307-20

  • The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain.

    Berrueta L, Tirnauer JS, Schuyler SC, Pellman D and Bierer BE

    Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachussetts 02115, USA.

    Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein [1], a domain of APC that is commonly deleted in colorectal neoplasia [2]. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 [3] and Saccharomyces cerevisiae Bim1p [4]. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex [5]. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle [6], in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis [7] [8] [9] [10]. Here, we demonstrate that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.

    Current biology : CB 1999;9;8;425-8

  • ZW10 helps recruit dynactin and dynein to the kinetochore.

    Starr DA, Williams BC, Hays TS and Goldberg ML

    Section of Genetics and Development, Cornell University, Ithaca, New York 14853-2703, USA.

    Mutations in the Drosophila melanogaster zw10 gene, which encodes a conserved, essential kinetochore component, abolish the ability of dynein to localize to kinetochores. Several similarities between the behavior of ZW10 protein and dynein further support a role for ZW10 in the recruitment of dynein to the kinetochore: (a) in response to bipolar tension across the chromosomes, both proteins mostly leave the kinetochore at metaphase, when their association with the spindle becomes apparent; (b) ZW10 and dynein both bind to functional neocentromeres of structurally acentric minichromosomes; and (c) the localization of both ZW10 and dynein to the kinetochore is abolished in cells mutant for the gene rough deal. ZW10's role in the recruitment of dynein to the kinetochore is likely to be reasonably direct, because dynamitin, the p50 subunit of the dynactin complex, interacts with ZW10 in a yeast two-hybrid screen. Since in zw10 mutants no defects in chromosome behavior are observed before anaphase onset, our results suggest that dynein at the kinetochore is essential for neither microtubule capture nor congression to the metaphase plate. Instead, dynein's role at the kinetochore is more likely to be involved in the coordination of chromosome separation and/or poleward movement at anaphase onset.

    Funded by: NIGMS NIH HHS: GM 07617, GM 48430, R01 GM048430, T32 GM007617

    The Journal of cell biology 1998;142;3;763-74

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis.

    Echeverri CJ, Paschal BM, Vaughan KT and Vallee RB

    Cell Biology Group, Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545, USA.

    Dynactin is a multi-subunit complex which has been implicated in cytoplasmic dynein function, though its mechanism of action is unknown. In this study, we have characterized the 50-kD subunit of dynactin, and analyzed the effects of its overexpression on mitosis in living cells. Rat and human cDNA clones revealed p50 to be novel and highly conserved, containing three predicted coiled-coil domains. Immunofluorescence staining of dynactin and cytoplasmic dynein components in cultured vertebrate cells showed that both complexes are recruited to kinetochores during prometaphase, and concentrate near spindle poles thereafter. Overexpression of p50 in COS-7 cells disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings represent clear evidence for dynactin and cytoplasmic dynein codistribution within cells, and for the presence of dynactin at kinetochores. The data also provide direct in vivo evidence for a role for vertebrate dynactin in modulating cytoplasmic dynein binding to an organelle, and implicate both dynactin and dynein in chromosome alignment and spindle organization.

    Funded by: NIGMS NIH HHS: GM 15941, GM 43474

    The Journal of cell biology 1996;132;4;617-33

  • Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.

    Golsteyn RM, Mundt KE, Fry AM and Nigg EA

    Swiss Institute for Experimental Cancer Research (ISREC), Epalinges.

    Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

    The Journal of cell biology 1995;129;6;1617-28

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

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.