Abstract. Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/ threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycledependent activity and subcellular localization of Plkl, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plkl kinase activity. We show that the activity of human Plkl is cell cycle regulated, Plkl activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plkl binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plkl associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plkl with the spindle is highly dynamic and that Plkl may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plkl may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation. URING mitosis, replicated chromosomes (sister chromatids) segregate such that each daughter cell receives one complete copy of the genome. Chromosome segregation is a highly complex and dynamic process that relies on the assembly and function of a microtubulebased mitotic spindle apparatus (for reviews see Mclntosh

Purpose: In the current study, we examined the in vivo effects of AZD1152, a novel and specific inhibitor of Aurora kinase activity (with selectivity for Aurora B). Experimental Design: The pharmacodynamic effects and efficacy of AZD1152 were determined in a panel of human tumor xenograft models. AZD1152 was dosed via several parenteral (s.c. osmotic mini-pump, i.p., and i.v.) routes. Results: AZD1152 potently inhibited the growth of human colon, lung, and hematologic tumor xenografts (mean tumor growth inhibition range, 55% to z100%; P < 0.05) in immunodeficient mice. Detailed pharmacodynamic analysis in colorectal SW620 tumor-bearing athymic rats treated i.v. with AZD1152 revealed a temporal sequence of phenotypic events in tumors: transient suppression of histone H3 phosphorylation followed by accumulation of 4N DNA in cells (2.4-fold higher compared with controls) and then an increased proportion of polyploid cells (>4N DNA, 2.3-fold higher compared with controls). Histologic analysis showed aberrant cell division that was concurrent with an increase in apoptosis in AZD1152-treated tumors. Bone marrow analyses revealed transient myelosuppression with the drug that was fully reversible following cessation of AZD1152 treatment. Conclusions: These data suggest that selective targeting of Aurora B kinase may be a promising therapeutic approach for the treatment of a range of malignancies. In addition to the suppression of histone H3 phosphorylation, determination of tumor cell polyploidy and apoptosis may be useful biomarkers for this class of therapeutic agent. AZD1152 is currently in phase I trials.

The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two separate CSN-dependent mechanisms. During unperturbed S phase, Spd1 degradation is independent of checkpoint proteins. In irradiated G2 cells, Spd1 degradation requires the DNA damage checkpoint. The signalosome copurifies with Pcu4 (cullin 4). Pcu4, Csn1, and Csn2 promote the degradation of Spd1, identifying a new function for the signalosome as a regulator of Pcu4-containing E3 ubiquitin ligase. The COP9 signalosome (CSN) complex was originally identified as a negative regulator of photomorphogenesis in plants (for review, see Schwechheimer and Deng 2001). Subsequently, it was purified from human cell extracts during attempts to isolate the 19S regulatory lid complex of the proteosome (Seeger et al. 1998). The human signalosome consists of eight core subunits, each sharing significant homology with a corresponding subunit in the regulatory 19S lid complex of the proteosome . The purified CSN complex can cleave the ubiquitin-like Nedd8 protein from cullins Wee et al. 2002). Csn5 contains a putative metalloprotease motif that is presumed to mediate deneddylation activity (Cope et al. 2002). Cullins are subunits of E3 ubiquitin ligases (Feldman et al. 1997;Skowyra et al. 1997), and deneddylation of cullins decreases SCF E3 ubiquitin ligase activity (Osaka et al. 2000). SCF E3 complexes typically consist of a cullin, the Rbx1 RING domain protein that binds an E2 enzyme Skowyra et al. 1999), and an adapter protein, Skp1, that binds an F-box protein that determines the substrate specificity (Skowyra et al. 1997).In Arabidopsis, the signalosome is involved in the degradation of the two bZIP transcription factors (Hy5, HyH) that lie at the top of a transcriptional cascade required to induce ∼ 30% of Arabidopsis genes during photomorphogenesis (Holm et al. 2002). An E2-like protein, Cop10, and an E3 RING protein, Cop1, are also required to degrade Hy5 and HyH, which occurs when seedlings are germinated in the dark (Osterlund et al. 2000;Holm et al. 2002;Suzuki et al. 2002). The biochemical role of the signalosome is unknown, although a correlation with Cop1 nuclear localization (von Arnim et al. 1997) and the associations between the signalosome and E3 ubiquitin ligases Schwechheimer et al. 2002) suggest a regulatory role in ubiquitination that may be linked to subcellular localization (Chamovitz et al. 1996;Hellmann and Estelle 2002).A highly conserved signalosome complex was identified in the fission yeast Schizosaccharomyces pombe (Mundt et al. 1999) and subsequently shown to be required to remove the Nedd8 ubiquitin-like protein fr...

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The COP9/signalosome complex is conserved from plant to mammalian cells. In Arabidopsis, it regulates the nuclear abundance of COP1, a transcriptional repressor of photomorphogenic development [1] [2]. All COP (constitutive photomorphogenesis) mutants inappropriately express genes that are normally repressed in the dark. Eight subunits (Sgn1-Sgn8) of the homologous mammalian complex have been purified [3] [4]. Several of these have been previously identified through genetic or protein interaction screens. No coherent model for COP9/signalosome function has yet emerged, but a relationship with cell-cycle progression by transcriptional regulation, protein localisation or protein stability is possible. Interestingly, the COP9/signalosome subunits possess domain homology to subunits of the proteasome regulatory lid complex [5] [6]. Database searches indicate that only Sgn5/JAB1 is present in Saccharomyces cerevisiae, precluding genetic analysis of the complex in cell-cycle regulation. Here we identify a subunit of the signalosome in the fission yeast Schizosaccharomyces pombe through an analysis of the DNA-integrity checkpoint. We provide evidence for the conservation of the COP9/signalosome complex in fission yeast and demonstrate that it functions during S-phase progression.