Certain proteins or activities are present in mitotic cells but not in interphase cells. These proteins may be synthesized or activated, or both, just prior to mitosis and are responsible for the breakdown of the nuclear envelope and the condensation of chromosomes. To learn more about the nature of these proteins, we raised monoclonal antibodies to mitotic cells. Spleen cells from mice immunized with a 0.15 M NaCl extract of synchronized mitotic HeLa cells were fused with SP2/0-Agl4 mouse myeloma cells, and hybrids were selected in medium containing hypoxanthine, methotrexate, thymidine, and glycine. Two different hybridoma clones secreting antibodies reactive with mitotic and meiotic cells from every species tested were isolated. Chromosomes as well as cytoplasm in mitotic cells reacted with the antibodies, as detected by indirect immunofluorescence. The proteins from mitotic cells were separated by electrophoresis in NaDodSO4/polyacrylamide slab gels, transferred to nitrocellulose sheets, and stained immunochemically. The two antibodies, designated MPM-1 and MPM-2, recognize a family of polypeptides with apparent molecular masses of 0.40 to >200 kilodaltons (kDa). Both antibodies reacted strongly with three polypeptide bands of 182 kDa, 118 kDa, and 70 kDa. Only mitotic cells exhibited the protein bands that were recognized by the antibodies. All these bands were found to be phosphoproteins as shown by 32P labeling and autoradiography and their removal by alkaline phosphatase treatment.The nucleus of a mammalian cell undergoes profound reorganization when the cell enters mitosis. The nuclear envelope breaks down, and the chromatin condenses into discrete chromosomes. Although the initiation of mitosis is dependent on both RNA and protein synthesis until 2 hr and 1 hr, respectively, before mitosis, the addition of inhibitors of RNA and protein synthesis to cells after these times does not affect either initiation or completion of mitosis (1, 2).The fusion of a mitotic cell with an interphase cell can induce nuclear envelope breakdown and premature chromosome condensation of the interphase nucleus (3). During this process, [3H]tryptophan-labeled proteins from mitotic cells labeled at G2 phase become associated with the prematurely condensed chromosomes, whereas the grain density on the metaphase chromosomes is not changed (4). Chromosome condensation and germinal vesicle (nucleus) breakdown can be induced in frog oocytes by the injection of extracts from mitotic but not interphase cells (5). The activity is labile to heat, sensitive to protease, insensitive to nuclease, and stabilized by phosphatase inhibitors and is found both in the cytoplasm and on the chromosomes of mitotic cells (6). These results suggest that certain nonhistone proteins, possibly phosphoproteins, of mitotic cells are not present in interphase cells. Al-Bader et al. (7) have reported the presence of such mitotic-specific proteins by using two-dimensional gel electrophoresis.The objective of this study was to raise monoclonal antib...

No abstract

No abstract

The MPM2 monoclonal antibody binds to a phospho amino acid-containing epitope present on more than 40 proteins of M-phase eukaryotic cells. We have developed a technique for cloning cDNAs encoding MPM2-reactive phosphoproteins from bacteriophage A expression libraries. Proteins from phage plaques were adsorbed to nitroceflulose filters, phosphorylated by M-phase kinases, and screened for MPM2 binding. Partial-length cDNAs encoding two MPM2-reactive proteins termed MPM2-reactive phosphoproteins 1 and 2 (MPP1 and MPP2) were isolated. The deduced MPP1 and MPP2 amino acid sequences are not dosely related to any previously described proteins. To determine which amino acid stretches contained the MPM2 epitope, sequences from a 15 amino acid peptide expression library were selected for binding to MPM2 after phosphorylation by M-phase kinases. A string of five amino acids was similar among all selected peptides, and the sequence reflecting the most frequent amino acid at each position was Leu-Thr-Pro-Leu-Lys (LTPLK). MPP1 and MPP2 proteins, respectively, contained five and nine sites closely related to LTPLK, including two that were common to both proteins, (F/T)TPLQ and SSP(I/S)D. Peptides containing LTPLK and FTPLQ were strongly phosphorylated by M-phase, but not interphase, cytosolic kinases, and the phosphorylated peptides were bound by MPM2. Thus, we have identified M-phase-specific phosphorylation sites bound by MPM2 and two putative M-phase phosphoproteins containing these sites.Progression ofcells from interphase to mitosis involves major alterations in cellular structures and activities. The G2/M transition is induced by M-phase-promoting factor (MPF), which is composed of a protein kinase, p34cdc2, and its associated regulatory subunit, cyclin (reviewed in ref. 1). Upon entry into M phase, many proteins are phosphorylated directly by MPF or indirectly by kinases that are activated by MPF. Forty or more of the M-phase phosphoproteins are reactive with MPM2, a monoclonal antibody that binds an epitope containing a phospho amino acid (2). The proteins are synthesized during interphase and become MPM2-reactive when cells enter M phase, apparently due to the action of an M-phase-specific kinase (3, 4). It is likely that phosphorylation at MPM2 antigenic sites regulates mitotic processes, because some of the MPM2 antigens are present in components of the mitotic machinery such as the centrosome, kinetochores, mitotic spindle, and chromosome axis (5, 6), and microinjected MPM2 inhibits entry into and exit from mitosis (3,4). As yet very few of the MPM2-reactive proteins have been identified (7,8), and definition of more would contribute to understanding of the mitotic process.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Protein phosphorylation has been suggested as an important control mechanism for the events leading toward the initiation and completion of mitosis. Using a monoclonal antibody recognizing a class of phosphoproteins abundant in mitotic cells, we demonstrated the localization of a subset of these phosphoproteins to several discrete mitotic structures. Patchy immunofluorescence was present in the interphase nuclei, but a significant increase in nuclear immunofluorescence was apparent at prophase. Subsequent mitotic stages demonstrated that immunoreactive material was particularly apparent at microtubule organizing centers, namely, centrosomes, kinetochores, and midbodies. Intense centrosomal localization occurred at the prophase-prometaphase transition and persisted until the reformation of the nuclear membrane in early G1. The cytoplasm of mitotic cells also contained immunoreactive material in sharp contrast to interphase cells that exhibited no cytoplasmic fluorescent staining. Much of the diffuse immunofluorescent cytoplasmic material was removed by a brief lysis of the cells with 0.15% Triton X-100 prior to fixation. The localization of the remaining immunoreactive material after detergent lysis to mitotic microtubule organizing centers suggests that they contain phosphoprotein structural components important, perhaps, in the mitotic phase-interphase transition.The regulatory events controlling the initiation and completion of mitosis are poorly understood. It is apparent, however, that such varied cellular responses as reorganization of the microtubule network, chromosome condensation, centriole duplication, breakdown of the nuclear membrane, formation of the mitotic spindle, and cytokinesis must be coordinated closely for the cell to progress through mitosis.One possible mechanism for controlling these mitotic events would be through protein phosphorylation and dephosphorylation. In several systems, increased levels of protein phosphorylation are cell-cycle dependent phenomena. During meiotic maturation of Xenopus laevis oocytes, the incorporation of 32p into protein is maximal prior to mitosis (1). Similarly, in HeLa cells, nuclear matrix proteins are phosphorylated to a maximal extent during the premitotic (G2) phase (2). More specifically, the three major protein components of the nuclear lamina, lamins A, B, and C, incorporate 32p maximally at the time of nuclear-envelope dissolution (3). The presence of other mitosis-specific proteins has been detected also by two-dimensional gel electrophoretic analysis of synchronized HeLa cell populations (4).Davis et al. (5) have reported on the properties of a monoclonal antibody that recognized a set of phosphorylated proteins abundant in mitotic cells. Using one of these monoclonal antibodies, MPM-2 (5), we report here the localization of immunoreactive material to the centrosome, the kinetochore, and the midbody of mitotic cells. These results support the concept that specific protein phosphorylation may coordinate the transition of cells from interphas...