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. 1995 Dec;6(12):1673–1684. doi: 10.1091/mbc.6.12.1673

CP60: a microtubule-associated protein that is localized to the centrosome in a cell cycle-specific manner.

D R Kellogg 1, K Oegema 1, J Raff 1, K Schneider 1, B M Alberts 1
PMCID: PMC301324  PMID: 8590797

Abstract

DMAP190 is a microtubule-associated protein from Drosophila that is localized to the centrosome. In a previous study, we used affinity chromatography to identify proteins that interact with DMAP190, and identified a 60-kDa protein that we named DMAP60 (Kellogg and Alberts, 1992). Like DMAP190, DMAP60 interacts with microtubules and is localized to the centrosome, and the two proteins associate as part of a multiprotein complex. We now report the cloning and sequencing of the cDNA encoding DMAP60. The amino acid sequence of DMAP60 is not homologous to any protein in the database, although it contains six consensus sites for phosphorylation by cyclin-dependent kinases. As judged by in situ hybridization, the gene for DMAP60 maps to chromosomal region 46A. In agreement with others working on Drosophila centrosomal proteins, we have changed the names for DMAP190 and DMAP60 to CP190 and CP60, respectively, to give these proteins a consistent nomenclature. Antibodies that recognize CP60 reveal that it is localized to the centrosome in a cell cycle-dependent manner. The amount of CP60 at the centrosome is maximal during anaphase and telophase, and then drops dramatically during late telophase or early interphase. This dramatic disappearance of CP60 may be due to specific proteolysis, because CP60 contains a sequence of amino acids similar to the "destruction box" that targets cyclins for proteolysis at the end of mitosis. Starting with nuclear cycle 12, CP60 and CP190 are both found in the nucleus during interphase. CP60 isolated from Drosophila embryos is highly phosphorylated, and dephosphorylated CP60 is a good substrate for cyclin B/p34cdc2 kinase complexes. A second kinase activity capable of phosphorylating CP60 is present in the CP60/CP190 multiprotein complex. We find that bacterially expressed CP60 binds to purified microtubules, and this binding is blocked by CP60 phosphorylation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson C. W., Baum P. R., Gesteland R. F. Processing of adenovirus 2-induced proteins. J Virol. 1973 Aug;12(2):241–252. doi: 10.1128/jvi.12.2.241-252.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnes G., Louie K. A., Botstein D. Yeast proteins associated with microtubules in vitro and in vivo. Mol Biol Cell. 1992 Jan;3(1):29–47. doi: 10.1091/mbc.3.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Brown K. D., Coulson R. M., Yen T. J., Cleveland D. W. Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis. J Cell Biol. 1994 Jun;125(6):1303–1312. doi: 10.1083/jcb.125.6.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown N. H., Kafatos F. C. Functional cDNA libraries from Drosophila embryos. J Mol Biol. 1988 Sep 20;203(2):425–437. doi: 10.1016/0022-2836(88)90010-1. [DOI] [PubMed] [Google Scholar]
  6. Butner K. A., Kirschner M. W. Tau protein binds to microtubules through a flexible array of distributed weak sites. J Cell Biol. 1991 Nov;115(3):717–730. doi: 10.1083/jcb.115.3.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Choi K. Y., Satterberg B., Lyons D. M., Elion E. A. Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. Cell. 1994 Aug 12;78(3):499–512. doi: 10.1016/0092-8674(94)90427-8. [DOI] [PubMed] [Google Scholar]
  8. Evans T., Rosenthal E. T., Youngblom J., Distel D., Hunt T. Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell. 1983 Jun;33(2):389–396. doi: 10.1016/0092-8674(83)90420-8. [DOI] [PubMed] [Google Scholar]
  9. Foe V. E., Alberts B. M. Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. J Cell Sci. 1983 May;61:31–70. doi: 10.1242/jcs.61.1.31. [DOI] [PubMed] [Google Scholar]
  10. Frasch M., Glover D. M., Saumweber H. Nuclear antigens follow different pathways into daughter nuclei during mitosis in early Drosophila embryos. J Cell Sci. 1986 Jun;82:155–172. doi: 10.1242/jcs.82.1.155. [DOI] [PubMed] [Google Scholar]
  11. Gibbons I. R., Asai D. J., Ching N. S., Dolecki G. J., Mocz G., Phillipson C. A., Ren H., Tang W. J., Gibbons B. H. A PCR procedure to determine the sequence of large polypeptides by rapid walking through a cDNA library. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8563–8567. doi: 10.1073/pnas.88.19.8563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Glotzer M., Murray A. W., Kirschner M. W. Cyclin is degraded by the ubiquitin pathway. Nature. 1991 Jan 10;349(6305):132–138. doi: 10.1038/349132a0. [DOI] [PubMed] [Google Scholar]
  13. Gotoh Y., Nishida E., Yamashita T., Hoshi M., Kawakami M., Sakai H. Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells. Eur J Biochem. 1990 Nov 13;193(3):661–669. doi: 10.1111/j.1432-1033.1990.tb19384.x. [DOI] [PubMed] [Google Scholar]
  14. Heider H., Hug C., Lucocq J. M. A 40-kDa myelin basic protein kinase, distinct from erk1 and erk2, is activated in mitotic HeLa cells. Eur J Biochem. 1994 Jan 15;219(1-2):513–520. doi: 10.1111/j.1432-1033.1994.tb19966.x. [DOI] [PubMed] [Google Scholar]
  15. Holloway S. L., Glotzer M., King R. W., Murray A. W. Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor. Cell. 1993 Jul 2;73(7):1393–1402. doi: 10.1016/0092-8674(93)90364-v. [DOI] [PubMed] [Google Scholar]
  16. Kalt A., Schliwa M. Molecular components of the centrosome. Trends Cell Biol. 1993 Apr;3(4):118–128. doi: 10.1016/0962-8924(93)90174-y. [DOI] [PubMed] [Google Scholar]
  17. Kellogg D. R., Alberts B. M. Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies. Mol Biol Cell. 1992 Jan;3(1):1–11. doi: 10.1091/mbc.3.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kellogg D. R., Kikuchi A., Fujii-Nakata T., Turck C. W., Murray A. W. Members of the NAP/SET family of proteins interact specifically with B-type cyclins. J Cell Biol. 1995 Aug;130(3):661–673. doi: 10.1083/jcb.130.3.661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kellogg D. R., Mitchison T. J., Alberts B. M. Behaviour of microtubules and actin filaments in living Drosophila embryos. Development. 1988 Aug;103(4):675–686. doi: 10.1242/dev.103.4.675. [DOI] [PubMed] [Google Scholar]
  20. Kellogg D. R., Moritz M., Alberts B. M. The centrosome and cellular organization. Annu Rev Biochem. 1994;63:639–674. doi: 10.1146/annurev.bi.63.070194.003231. [DOI] [PubMed] [Google Scholar]
  21. Kuriyama R., Borisy G. G. Microtubule-nucleating activity of centrosomes in Chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle. J Cell Biol. 1981 Dec;91(3 Pt 1):822–826. doi: 10.1083/jcb.91.3.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kuriyama R., Nislow C. Molecular components of the mitotic spindle. Bioessays. 1992 Feb;14(2):81–88. doi: 10.1002/bies.950140203. [DOI] [PubMed] [Google Scholar]
  23. Liao H., Winkfein R. J., Mack G., Rattner J. B., Yen T. J. CENP-F is a protein of the nuclear matrix that assembles onto kinetochores at late G2 and is rapidly degraded after mitosis. J Cell Biol. 1995 Aug;130(3):507–518. doi: 10.1083/jcb.130.3.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Marcus S., Polverino A., Barr M., Wigler M. Complexes between STE5 and components of the pheromone-responsive mitogen-activated protein kinase module. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7762–7766. doi: 10.1073/pnas.91.16.7762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marsh L., Neiman A. M., Herskowitz I. Signal transduction during pheromone response in yeast. Annu Rev Cell Biol. 1991;7:699–728. doi: 10.1146/annurev.cb.07.110191.003411. [DOI] [PubMed] [Google Scholar]
  26. Miller K. G., Kiehart D. P. Fly division. J Cell Biol. 1995 Oct;131(1):1–5. doi: 10.1083/jcb.131.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Minshull J., Sun H., Tonks N. K., Murray A. W. A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts. Cell. 1994 Nov 4;79(3):475–486. doi: 10.1016/0092-8674(94)90256-9. [DOI] [PubMed] [Google Scholar]
  28. Mitchison T., Kirschner M. Microtubule assembly nucleated by isolated centrosomes. Nature. 1984 Nov 15;312(5991):232–237. doi: 10.1038/312232a0. [DOI] [PubMed] [Google Scholar]
  29. Morris C. F., Hama-Inaba H., Mace D., Sinha N. K., Alberts B. Purification of the gene 43, 44, 45, and 62 proteins of the bacteriophage T4 DNA replication apparatus. J Biol Chem. 1979 Jul 25;254(14):6787–6796. [PubMed] [Google Scholar]
  30. Nigg E. A. Cellular substrates of p34(cdc2) and its companion cyclin-dependent kinases. Trends Cell Biol. 1993 Sep;3(9):296–301. doi: 10.1016/0962-8924(93)90011-o. [DOI] [PubMed] [Google Scholar]
  31. Nislow C., Lombillo V. A., Kuriyama R., McIntosh J. R. A plus-end-directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles. Nature. 1992 Oct 8;359(6395):543–547. doi: 10.1038/359543a0. [DOI] [PubMed] [Google Scholar]
  32. Reed S. I. Pheromone signaling pathways in yeast. Curr Opin Genet Dev. 1991 Oct;1(3):391–396. doi: 10.1016/s0959-437x(05)80305-3. [DOI] [PubMed] [Google Scholar]
  33. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  34. Steinhauer W. R., Walsh R. C., Kalfayan L. J. Sequence and structure of the Drosophila melanogaster ovarian tumor gene and generation of an antibody specific for the ovarian tumor protein. Mol Cell Biol. 1989 Dec;9(12):5726–5732. doi: 10.1128/mcb.9.12.5726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Theurkauf W. E. Behavior of structurally divergent alpha-tubulin isotypes during Drosophila embryogenesis: evidence for post-translational regulation of isotype abundance. Dev Biol. 1992 Nov;154(1):205–217. doi: 10.1016/0012-1606(92)90060-t. [DOI] [PubMed] [Google Scholar]
  36. Vandre D. D., Davis F. M., Rao P. N., Borisy G. G. Phosphoproteins are components of mitotic microtubule organizing centers. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4439–4443. doi: 10.1073/pnas.81.14.4439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vorobjev I. A., Nadezhdina E. S. The centrosome and its role in the organization of microtubules. Int Rev Cytol. 1987;106:227–293. doi: 10.1016/s0074-7696(08)61714-3. [DOI] [PubMed] [Google Scholar]
  38. Voronova A. F., Buss J. E., Patschinsky T., Hunter T., Sefton B. M. Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells. Mol Cell Biol. 1984 Dec;4(12):2705–2713. doi: 10.1128/mcb.4.12.2705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Whitfield W. G., Millar S. E., Saumweber H., Frasch M., Glover D. M. Cloning of a gene encoding an antigen associated with the centrosome in Drosophila. J Cell Sci. 1988 Apr;89(Pt 4):467–480. doi: 10.1242/jcs.89.4.467. [DOI] [PubMed] [Google Scholar]
  40. Yang C. H., Lambie E. J., Snyder M. NuMA: an unusually long coiled-coil related protein in the mammalian nucleus. J Cell Biol. 1992 Mar;116(6):1303–1317. doi: 10.1083/jcb.116.6.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]

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