Phosphoproteins are components of mitotic microtubule organizing centers.

Abstract

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...

Nucleolin functions in nucleolus formation and chromosome congression

“…Consistent with this hypothesis, abrogation of kinetochore-microtubule attachments by depleting certain kinetochore proteins, including CENP- Protein phosphorylation has been proposed as an important control mechanism for the events leading to the initiation and completion of mitosis (Vandre et al, 1984). Numerous phosphoproteins relocate to the spindle poles during mitosis (Vandre et al, 1984;Vandré and Burry, 1992). Meanwhile, nucleolin is highly phosphorylated by CDC2 during mitosis (Belenguer et al, 1990;Peter et al, 1990).…”

“…Protein phosphorylation has been proposed as an important control mechanism for the events leading to the initiation and completion of mitosis (Vandre et al, 1984). Numerous phosphoproteins relocate to the spindle poles during mitosis (Vandre et al, 1984;Vandré and Burry, 1992).…”

“…Numerous phosphoproteins relocate to the spindle poles during mitosis (Vandre et al, 1984;Vandré and Burry, 1992). Meanwhile, nucleolin is highly phosphorylated by CDC2 during mitosis (Belenguer et al, 1990;Peter et al, 1990).…”

“…Since nucleolin is important for the stability of kinetochore-microtubule attachments, we suspect that the mitotic spindle disorganization observed in nucleolin-depleted cells is the consequence of a defect in the kinetochoremicrotubule attachments. Consistent with this hypothesis, abrogation of kinetochore-microtubule attachments by depleting certain kinetochore proteins, including CENP- Protein phosphorylation has been proposed as an important control mechanism for the events leading to the initiation and completion of mitosis (Vandre et al, 1984). Numerous phosphoproteins relocate to the spindle poles during mitosis (Vandre et al, 1984;Vandré and Burry, 1992).…”

Threonine phosphorylation is associated with mitosis in HeLa cells

“…During mitosis, a number of proteins, histone H1 [22], lamins [23], microtubule-associated proteins or MAPs, proteins associated with centrosomes, kinetochores, and midbodies, and other nonhistone proteins [24][25][26], are phosphorylated. In fact, the MPM-2 antibody recognizes a number of phosphopeptides, ranging in molecular mass from 55 to >210 kDa, whose individual roles or functions during mitosis have not yet been identified.…”