Using an expression cloning technique, we isolated cDNAs for eight M phase phosphoproteins (MPPs 4-11). We then used affinity-purified antibodies to fusion proteins to characterize the intracellular localization and some biochemical properties of these proteins and two others that we identified previously (MPPs 1-2). Each antibody immunoprecipitated one or two protein species of a characteristic size ranging from 17,000 to 220,000 Da. Each MPP, when immunoprecipitated from lysates of M phase cells, was reactive with MPM2, a monoclonal antibody that recognizes a group of related M phase phosphorylation sites, including F-phosphoT-P-L-Q. This reactivity indicated that all the MPPS encoded genuine M phase phosphoproteins. When antibodies to the MPPS were used for immunofluorescence microscopy, each anti-MPP gave a characteristic pattern of localization. In interphase, several of the MPPs were nuclear proteins, whereas others were cytoplasmic or distributed throughout the cell. Three MPPS were strikingly localized to interphase structures: MPP7 to centers of DNA replication, MPP9 to the Golgi complex, and MPP10 to nucleoli. In mitosis, most of the MPPs were distributed throughout the cells. Further studies of the 10 MPPs, most of which are previously undescribed, are expected to provide new understandings of the process of cell division.
In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves toward the microtubule plus-end at a 0.07 m/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis.Eukaryotic cells exhibit dramatic changes of microtubule organization and dynamics as they enter mitosis (2, 3). These changes are timely and spatially coordinated with nucleus and membranes alterations by the tight control of M phase-promoting factor, whose catalytic component, the p34 cdc2 or Cdk1 kinase becomes activated at the G 2 /M transition (4, 5). Extensive progress has been made to describe the complex circuitry of phosphatases and kinases, which regulates Cdk1 activation (6, 7) and the downstream molecular pathways (8, 9).Microtubule dynamics are an intrinsic property of the polymer of tubulin and are highly regulated by the balance of the activity of different factors throughout the cell cycle (10 -12). Several microtubule-associated proteins have been described to promote tubulin assembly and polymer stabilization or destabilization (13,14). Besides their roles in intracellular trafficking of organelles and vesicles during interphase, dyneins and kinesin-related proteins (KRPs), 1 microtubule-based molecular motors, play important roles in cell division. At each stage of mitosis or meiosis, dyneins and various KRPs interact with microtubules in order to ensure centrosome separation, spindle formation and maintenance, chromosome congression, and cytokinesis completion (15-19).However, whereas it is well established that the p34 cdc2 kinase is centrally involved in the regulation of microtubule dynamics during mitosis (20), only a few Cdc2 substrates with plausible involvement in the control of microtubule dynamics have been identified so far. The p34 cdc2 kinase phosphorylates the ubiquitous microtubule-associated protein 4 during M phase (21), and this phosphorylation abolishes microtubuleassociated protein 4 microtubule stabilizing activity (22). There is evidence that the phosphorylation of the microtubule destabilizing protein Stathmin/Op18 by p34 cdc2 is important for mitotic progression (23). Similar phosphorylation of the mitotic KRP Eg5 is required for Eg5-dependent centrosome migration and bipolar spindle formation in vivo (17). These data suggest that mitotic kinases regulate microtubule dynamics and organization by phosphorylating various microtubule-interacting proteins, and this has been an incentive for the systematic search of mitotic phosphoproteins.We have recently identified a subset of M phase phosphoproteins by expression library screening using the MPM2 monoclonal antibody, which recognizes a phosphoepitope present on a set of 40 -...
Neuronal differentiation and function require extensive stabilization of the microtubule cytoskeleton. Neurons contain a large proportion of microtubules that resist the cold and depolymerizing drugs and exhibit slow subunit turnover. The origin of this stabilization is unclear. Here we have examined the role of STOP, a calmodulin-regulated protein previously isolated from cold-stable brain microtubules. We find that neuronal cells express increasing levels of STOP and of STOP variants during differentiation. These STOP proteins are associated with a large proportion of microtubules in neuronal cells, and are concentrated on cold-stable, drug-resistant, and long-lived polymers. STOP inhibition abolishes microtubule cold and drug stability in established neurites and impairs neurite formation. Thus, STOP proteins are responsible for microtubule stabilization in neurons, and are apparently required for normal neurite formation.
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