Phosphorylation of NUMA occurs during nuclear breakdown and not mitotic spindle assembly.

NuMA, the nuclear mitotic apparatus protein, is a component of the nuclear matrix at interphase that redistributes to the spindle poles at mitosis. While the function of NuMA is not known, it has been implicated in spindle organization during mitosis and nuclear reformation. Phosphorylation is thought to play a regulatory role in NuMA function. In this study, NuMA phosphorylation was examined through the cell cycle using highly synchronized cells. In intact cells labeled with 32P-orthophosphate, NuMA appeared as a 250 kDa phosphoprotein in interphase that shifted to a higher apparent molecular mass in mitosis. The shift was due to phosphorylation as shown by reduction of the shifted band to interphase mobility by phosphatase treatment. This phosphorylation event occurred roughly at the G2/M transition at the time of NuMA's release from the nucleus and its redistribution to the mitotic spindle. However, mitotic phosphorylation did not require spindle formation since the phosphorylated species was detected in nocodazole-treated cells lacking microtubule spindles. Dephosphorylation of NuMA occurred in two distinct steps, after lamin B assembled into the nuclear lamina, in early G1 and at the end of G1. Based on the timing of the phosphorylation and dephosphorylation observed in this study, we propose that they may play a role in nuclear events such as nuclear organization, transcription, or initiation of DNA replication at G1/S.

[1]  N. Gonatas,et al.  THE ULTRASTRUCTURE OF A MAMMALIAN CELL DURING THE MITOTIC CYCLE , 1964, The Journal of cell biology.

[2]  W. Ostertag,et al.  The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. , 1990, Molecular and cellular biology.

[3]  Estimation of phosphorylation stoichiometry by separation of phosphorylated isoforms. , 1991 .

[4]  W. T. Chen,et al.  Nuclear proteins of the bovine esophageal epithelium. I. Monoclonal antibody W2 specifically reacts with condensed nuclei of differentiated superficial cells. , 1993, Journal of cell science.

[5]  D. Compton,et al.  NuMA, a nuclear protein involved in mitosis and nuclear reformation. , 1994, Current opinion in cell biology.

[6]  E. Harlow,et al.  The retinoblastoma protein is phosphorylated during specific phases of the cell cycle , 1989, Cell.

[7]  F. McKeon,et al.  Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis , 1990, Cell.

[8]  M. Kirschner,et al.  Identification of cell cycle-regulated phosphorylation sites on nuclear lamin C , 1990, Cell.

[9]  R. Kuriyama,et al.  Identification of a minus end-specific microtubule-associated protein located at the mitotic poles in cultured mammalian cells. , 1991, European journal of cell biology.

[10]  C. Wu,et al.  Nuclear proteins of the bovine esophageal epithelium. II. The NuMA gene gives rise to multiple mRNAs and gene products reactive with monoclonal antibody W1. , 1993, Journal of cell science.

[11]  R Berezney,et al.  Identification of a nuclear protein matrix. , 1974, Biochemical and biophysical research communications.

[12]  D. Pettijohn,et al.  Specific attachment of nuclear-mitotic apparatus protein to metaphase chromosomes and mitotic spindle poles: possible function in nuclear reassembly. , 1983, Journal of molecular biology.

[13]  C. Wu,et al.  Nuclear mitotic apparatus protein (NuMA): spindle association, nuclear targeting and differential subcellular localization of various NuMA isoforms. , 1994, Journal of cell science.

[14]  D. Compton,et al.  Primary structure of NuMA, an intranuclear protein that defines a novel pathway for segregation of proteins at mitosis , 1992, The Journal of cell biology.

[15]  R. Evans.  Cyclic AMP‐dependent protein kinase‐induced vimentin filament disassembly involves modification of the N‐terminal domain of intermediate filament subunits , 1988, FEBS letters.

[16]  A. Cherniack,et al.  Disassembly of Son-of-sevenless Proteins from Grb2 during p21 Desensitization by Insulin (*) , 1995, The Journal of Biological Chemistry.

[17]  K. Weber,et al.  A 210 kDa nuclear matrix protein is a functional part of the mitotic spindle; a microinjection study using SPN monoclonal antibodies. , 1991, The EMBO journal.

[18]  B. Brinkley,et al.  Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation , 1991, The Journal of cell biology.

[19]  R. Kuriyama,et al.  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. , 1993, Journal of cell science.

[20]  M. Inagaki,et al.  Intermediate filament reconstitution in vitro. The role of phosphorylation on the assembly-disassembly of desmin. , 1988, The Journal of biological chemistry.

[21]  D. Pettijohn,et al.  Human-specific nuclear protein that associates with the polar region of the mitotic apparatus: Distribution in a human/hamster hybrid cell , 1980, Cell.

[22]  M. Snyder,et al.  The nuclear-mitotic apparatus protein is important in the establishment and maintenance of the bipolar mitotic spindle apparatus. , 1992, Molecular biology of the cell.

[23]  R. Berezney,et al.  Nuclear matrix: isolation and characterization of a framework structure from rat liver nuclei , 1977, The Journal of cell biology.

[24]  M. Kirschner,et al.  Mitosis in transition , 1994, Cell.

[25]  D. Compton,et al.  Mutation of the predicted p34cdc2 phosphorylation sites in NuMA impair the assembly of the mitotic spindle and block mitosis. , 1995, Journal of cell science.

[26]  J. Spudich,et al.  Intermolecular versus intramolecular interactions of Dictyostelium myosin: possible regulation by heavy chain phosphorylation , 1989, The Journal of cell biology.

[27]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[28]  B. Brinkley,et al.  Localization of NuMA protein isoforms in the nuclear matrix of mammalian cells. , 1994, Cell motility and the cytoskeleton.

[29]  K. Weber,et al.  Ability to organize microtubules in taxol-treated mitotic PtK2 cells goes with the SPN antigen and not with the centrosome. , 1992, Journal of cell science.

[30]  J. Harborth,et al.  Microinjection of a monoclonal antibody against SPN antigen, now identified by peptide sequences as the NuMA protein, induces micronuclei in PtK2 cells. , 1993, Journal of cell science.

[31]  F. Bischoff,et al.  Isolation of human NuMA protein , 1994, FEBS letters.

[32]  M. Kirschner,et al.  Induction of early mitotic events in a cell-free system , 1985, Cell.

[33]  D. Pettijohn,et al.  Redistribution of the nuclear mitotic apparatus protein (NuMA) during mitosis and nuclear assembly. Properties of purified NuMA protein. , 1986, Experimental cell research.

[34]  J. Mullins,et al.  Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor nocodazole. Nocodazole accumulated mitotic cells. , 1980, Experimental cell research.

[35]  D. Pettijohn,et al.  NuMA protein is a human autoantigen. , 1984, Arthritis and rheumatism.

[36]  D. Compton,et al.  Binding of matrix attachment regions to lamin polymers involves single-stranded regions and the minor groove , 1994, Molecular and cellular biology.

[37]  M. Kirschner,et al.  Pericentrin, a highly conserved centrosome protein involved in microtubule organization , 1994, Cell.

[38]  W. Dewey,et al.  A method for freezing synchronous mitotic and G1 cells. , 1987, Experimental cell research.

[39]  D A Parry,et al.  NuMA/centrophilin: sequence analysis of the coiled-coil rod domain. , 1994, Biophysical journal.

[40]  D. Compton,et al.  Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds , 1991, The Journal of cell biology.

[41]  J. Lawrence,et al.  Assignment of the nuclear mitotic apparatus protein NuMA gene to human chromosome 11q13. , 1993, Genomics.

[42]  M. Snyder,et al.  NuMA: an unusually long coiled-coil related protein in the mammalian nucleus , 1992, The Journal of cell biology.

[43]  L. Gerace,et al.  A cell free system to study reassembly of the nuclear envelope at the end of mitosis , 1986, Cell.

[44]  J. Labbé,et al.  In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase , 1990, Cell.

[45]  R. Hancock,et al.  Nuclear matrix attachment regions and topoisomerase II binding and reaction sites in the vicinity of a chicken DNA replication origin. , 1991, Biochemical and biophysical research communications.

[46]  W. Dewey,et al.  X-ray induction of 8-azaguanine-resistant mutants in synchronous Chinese hamster ovary cells. , 1980, Radiation research.

[47]  G. Blobel,et al.  The nuclear envelope lamina is reversibly depolymerized during mitosis , 1980, Cell.