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Mutation of the predicted p34cdc2 phosphorylation sites in NuMA impair the assembly of the mitotic spindle and block mitosis
D.A. Compton, C. Luo
Journal of Cell Science 1995 108: 621-633;

Summary

NuMA is a 236 kDa intranuclear protein that is distributed into each daughter cell during mitosis through association with the pericentrosomal region of the mitotic spindle. NuMA's interaction with the microtubules of the mitotic spindle is mediated through its 45 kDa carboxyl-terminal globular tail, and there is indirect evidence suggesting that NuMA's interaction with the mitotic spindle is controlled in a mitosis-specific manner. Consistent with this evidence is the fact that all four of the predicted p34cdc2 consensus phosphorylation sites in the NuMA protein are located in the carboxyl-terminal globular domain, and we demonstrate here that NuMA is phosphorylated in a mitosis-specific fashion in vivo. To test if the predicted p34cdc2 phosphorylation sites are necessary for NuMA's mitosis-specific interaction with the mitotic spindle, we have introduced mutations into the human NuMA cDNA that convert these predicted p34cdc2 phosphorylation sites from threonine or serine residues into alanine residues, and subsequently determined the cell cycle-dependent localization of these altered NuMA proteins following their expression in tissue culture cells. While none of these specific mutations in the NuMA sequence alters the faithful targeting of the protein into the interphase nucleus, mutation of threonine residue 2040 alone or in combination with mutations in other potential p34cdc2 phosphorylation sites abolishes NuMA's ability to associate normally with the microtubules of the mitotic spindle. Instead of binding to the mitotic spindle these mutant forms of NuMA concentrate at the plasma membrane of the mitotic cell. Cells expressing these mutant forms of NuMA have disorganized mitotic spindles, fail to complete cytokinesis normally, and assemble micronuclei in the subsequent interphase. These data suggest that NuMA's interaction with the microtubules of the mitotic spindle is controlled by cell cycle-dependent phosphorylation in addition to differential subcellular compartmentalization, and the characteristics of the dominant negative phenotype induced by these mutant forms of NuMA support a role for NuMA in the organization of the mitotic spindle apparatus.

REFERENCES

    1. Bernat R. L.,
    2. Borisy G. G.,
    3. Rothfield N. F. and
    4. Earnshaw W. C.
    (1990). Injection of anticentromere antibodies in interphase disrupts events required for chromosome movements at mitosis. J. Cell Biol 111, 1519–1533
    OpenUrlAbstract/FREE Full Text
    1. Capecchi M. R.
    (1980). High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22, 479–488
    OpenUrlCrossRefPubMedWeb of Science
    1. Compton D. A.,
    2. Yen T. J. and
    3. Cleveland D. W.
    (1991). Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds. J. Cell Biol 112, 1083–1097
    OpenUrlAbstract/FREE Full Text
    1. Compton D. A.,
    2. Szilak I. and
    3. Cleveland D. W.
    (1992). Primary structure of NuMA, an intranuclear protein that defines a novel pathway for segregation of proteins at mitosis. J. Cell Biol 116, 1395–1408
    OpenUrlAbstract/FREE Full Text
    1. Compton D. A. and
    2. Cleveland D. W.
    (1993). NuMA is required for the proper completion of mitosis. J. Cell Biol 120, 947–957
    OpenUrlAbstract/FREE Full Text
    1. Compton D. A. and
    2. Cleveland D. W.
    (1994). NuMA, a nuclear protein involved in mitosis and nuclear reformation. Curr. Opin. Cell Biol 6, 343–346
    OpenUrlCrossRefPubMedWeb of Science
    1. Gerace L. and
    2. Burke B.
    (1988). Functional organization of the nuclear envelope. Annu. Rev. Cell Biol 4, 335–374
    OpenUrlCrossRefWeb of Science
    1. Gill S. R.,
    2. Schroer T. A.,
    3. Szilak I.,
    4. Steuer E. R.,
    5. Sheetz M. P. and
    6. Cleveland D. W.
    (1991). Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility by cytoplasmic dynein. J. Cell Biol 115, 1639–1650
    OpenUrlAbstract/FREE Full Text
    1. Kallajoki M.,
    2. Weber K. and
    3. Osborn M.
    (1991). A 210 kD nuclear matrix protein is a functional part of the mitotic spindle; a microinjection study using SPN monoclonal antibodies. EMBO J 10, 3351–3362
    OpenUrlPubMedWeb of Science
    1. Kallajoki M.,
    2. Weber K. and
    3. Osborn M.
    (1992). Ability to organize microtubules in taxol-treated mitotic PtK2 cells goes with the SPN antigen and not with the centrosome. J. Cell Sci 102, 91–102
    OpenUrlAbstract/FREE Full Text
    1. Kallajoki M.,
    2. Harborth J.,
    3. Weber K. and
    4. Osborn M.
    (1993). Microinjection of a monoclonal antibody against SPN antigen, now identified by peptide sequences as the NuMA protein, induces micronuclei in PtK2 cells. J. Cell Sci 104, 139–150
    OpenUrlAbstract/FREE Full Text
    1. Laemmli U. K.
    (1970). Cleavage of structural proteins during assembly at the head of the bacteriophage T4. Nature 227, 680–682
    OpenUrlCrossRefPubMedWeb of Science
    1. Lydersen B. K. and
    2. Pettijohn D. E.
    (1980). Human specific nuclear protein that associates with the polar region of the mitotic apparatus: distribution in a human/hamster hybrid cell. Cell 22, 489–499
    OpenUrlCrossRefPubMedWeb of Science
    1. Maekawa T.,
    2. Leslie R. and
    3. Kuriyama R.
    (1991). Identification of a minus-end specific microtubule-associated protein located at the mitotic spindle poles in cultured mammalian cells. Eur. J. Cell Biol 54, 255–267
    OpenUrlPubMedWeb of Science
    1. Maekawa T. and
    2. Kuriyama R.
    (1993). Primary structure and microtubule-interacting domain of the SP-H antigen: a mitotic map located at the spindle pole and characterized as a homologous protein to NuMA. J. Cell Sci 105, 589–600
    OpenUrlAbstract/FREE Full Text
    1. McKeon F.
    (1991). Nuclear lamin proteins: domains required for nuclear targeting, assembly, and cell-cycle-regulated dynamics. Curr. Opin. Cell Biol 3, 82–86
    OpenUrlCrossRefPubMed
    1. Mitchison T. J.
    (1989). Mitosis: basic concepts. Curr. Opin. Cell Biol 1, 67–74
    OpenUrlCrossRefPubMed
    1. Monero S. and
    2. Nurse P.
    (1990). Substrates for p34cdc2: in vitro veritas?. Cell 61, 549–551
    OpenUrlCrossRefPubMedWeb of Science
    1. Newport J. W. and
    2. Forbes D. J.
    (1987). The nucleus: structure, function and dynamics. Annu. Rev. Biochem 56, 535–565
    OpenUrlCrossRefPubMedWeb of Science
    1. Nigg E. A.
    (1992). Assembly-disassembly of the nuclear lamina. Curr. Opin. Cell Biol 4, 105–109
    OpenUrlCrossRefPubMed
    1. Nishimoto T.,
    2. Eilen E. and
    3. Basilico C.
    (1978). Premature chromosome condensation in a tsDNAmutant of BHK cells. Cell 15, 475–483
    OpenUrlCrossRefPubMedWeb of Science
    1. Price C. M. and
    2. Pettijohn D. E.
    (1986). Redistribution of the nuclear mitotic apparatus protein (NuMA) during mitosis and nuclear assembly. Exp. Cell Res 166, 295–311
    OpenUrlCrossRefPubMedWeb of Science
    1. Rieder C. L.
    (1991). Mitosis: towards an understanding of chromosome behavior. Curr. Opin. Cell Biol 3, 59–66
    OpenUrlCrossRefPubMed
    1. Rieder C. L. and
    2. Palazzo R. E.
    (1992). Colcemid and the cell cycle. J. Cell Sci 102, 387–392
    OpenUrlFREE Full Text
    1. Sawin K. E. and
    2. Mitchison T. J.
    (1991). Mitotic spindle assembly by two different pathways in vitro. J. Cell Biol 112, 925–940
    OpenUrlAbstract/FREE Full Text
    1. Sawin K. E.,
    2. LeGuillic K.,
    3. Philippe M. and
    4. Mitchison T. J.
    (1992). Mitotic spindle organization by a plus-end-directed microtubule motor. Nature 359, 540–543
    OpenUrlCrossRefPubMedWeb of Science
    1. Tang T. K.,
    2. Tang C. C.,
    3. Chen Y.-L. and
    4. Wu C.-W.
    (1993). Nuclearproteins of the bovine epithelium. II. The NuMA gene gives rise to multiple mRNAs and gene products reactive with monoclonal antibody W1. J. Cell Sci 104, 249–260
    OpenUrlAbstract/FREE Full Text
    1. Tousson A.,
    2. Zeng C.,
    3. Brinkley B. R. and
    4. Valdivia M. M.
    (1991). Centrophilin, a novel mitotic spindle protein involved in microtubule nucleation. J. Cell Biol 112, 427–440
    OpenUrlAbstract/FREE Full Text
    1. Yang C. H.,
    2. Lambie E. J. and
    3. Snyder M.
    (1992). NuMA: an unusually long coiled-coil related protein in the mammalian cell nucleus. J. Cell Biol 116, 1303–1317
    OpenUrlAbstract/FREE Full Text
    1. Yang C. H. and
    2. Snyder M.
    (1992). The nuclear-mitotic apparatus protein (NuMA) is important in the establishment and maintenance of the bipolar mitotic spindle apparatus. Mol. Biol. Cell 3, 1259–1267
    OpenUrlAbstract/FREE Full Text
    1. Zeng C.,
    2. He D.,
    3. Berget S. M. and
    4. Brinkley B. R.
    (1994). Nuclear-mitotic apparatus protein: A structural protein interface between the nucleoskeleton and RNA splicing. Proc. Nat. Acad. Sci. USA 91, 1505–1509
    OpenUrlAbstract/FREE Full Text
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Journal Articles
Mutation of the predicted p34cdc2 phosphorylation sites in NuMA impair the assembly of the mitotic spindle and block mitosis
D.A. Compton, C. Luo
Journal of Cell Science 1995 108: 621-633;
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