Dynamic Phosphorylation of NudC by Aurora B in Cytokinesis.

Nuclear distribution protein C (NudC) is a mitotic regulator that plays a role in cytokinesis. However, how NudC is regulated during cytokinesis remains unclear. Here, we show that NudC is phosphorylated by Aurora B, a kinase critical for cell abscission. NudC is co-localized with Aurora B at the midbody and co-immunoprecipitated with Aurora B in mitosis. Inhibition of Aurora B by ZM447439 reduced NudC phosphorylation, suggesting that NudC is an Aurora B substrate in vivo. We identified T40 on NudC as an Aurora B phosphorylation site. NudC depletion resulted in cytokinesis failure with a dramatic elongation of the intercellular bridge between daughter cells, sustained Aurora B activity at the midbody, and reduced cell abscission. These cytokinetic defects can be rescued by the ectopic expression of wild-type NudC. Reconstitution with T40A phospho-defective NudC was found to rescue the cytokinesis defect. In contrast, reconstitution with the T40D phospho-mimetic NudC was inefficient in supporting the completion of cytokinesis. These results suggest that that dynamic phosphorylation of NudC by Aurora B regulates cytokinesis

Prophylactic application of CpG oligonucleotides augments the early host response and confers protection in acute melioidosis.

Prophylactic administration of CpG oligodeoxynucleotides (CpG ODNs) is known to confer protection against lethal sepsis caused by Burkholderia pseudomallei in the mouse model. The mechanisms whereby CpG regulates the innate immune response to provide protection against B. pseudomallei, however, are poorly characterized. In the present study, we demonstrate that intranasal treatment of mice with Class C CpG, results in recruitment of inflammatory monocytes and neutrophils to the lung at 48 h post-treatment. Mice infected with B. pseudomallei 48 h post-CpG treatment had reduced organ bacterial load and significantly altered cytokine and chemokine profiles concomitant with protection as compared to control animals. CpG administration reduced the robust production of chemokines and pro-inflammatory cytokines in blood, lung and spleen, observed following infection of non-treated animals. Death of control animals coincided with the time of peak cytokine production (day 1-3), while a moderate; sustained cytokine production in CpG-treated animals was associated with survival. In general, CpG treatment resulted in diminished expression of cytokines and chemokines post-infection, though IL-12p40 was released in larger quantities in CpG treated animals. In contrast to CpG-treated animals, the lungs of infected control animals were infiltrated with leukocytes, especially neutrophils, and large numbers of necrotic lesions were observed in lung sections. Therapeutic treatment of B. pseudomallei-infected animals with CpG at 24 h post-infection did not impact survival compared to control animals. In summary, protection of CpG-treated animals was associated with recruitment of inflammatory monocytes and neutrophils into the lungs prior to infection. These responses correspond with early control of bacterial growth, a dampened inflammatory cytokine/chemokine response, reduced lung pathology, and greatly increased survival. In contrast, a delay in recruitment of inflammatory cell populations, despite a robust production of pro-inflammatory cytokines, was associated with poorly controlled bacterial growth, severe lung pathology, and death of control animals

NudC is phosphorylated by Aurora B <i>in vitro</i> and <i>in vivo</i>.

<p>(A) HeLa cells were transfected with FLAG-Aurora B wild type (WT) or a kinase dead (K106R) mutant Aurora B for 24 h. Aurora B was immunoprecipitated using anti-FLAG antibody and used in IP kinase assays. Substrates used were GST-NudC (lanes 4–6), histone H3 (lanes 1–3) as a positive control, and GST (lane 7) as a negative control. Aurora B WT was also incubated with 2 μM of ZM447439 as a specificity control (lanes 3 and 6). Samples were transferred to a filter, stained by Ponceau S (lower panel) and analyzed by autoradiography (upper panel). *, degradation product. Data are reproducible in 3 independent experiments. (B) HeLa cells were synchronized by an overnight incubation with 100 ng/ml nocodazole (M, mitotic) as indicated. Cells (1 X 10⁶) were labeled with ³²P orthophosphate for 4 h in the presence or absence of 2 μM ZM447439 (ZM). Cell lysates (300 μg at 1 mg/ml) were immunoprecipitated for NudC, transferred to a filter, analyzed by autoradiography, and immunoblotted for NudC. ³²P-NudC was quantified as ³²P-NudC/total immunoprecipitated NudC and normalized against NudC signals in asynchronously cycling (Asy) cells.</p

PCR primers for GST-human NudC truncation constructs.

<p>PCR primers for GST-human NudC truncation constructs.</p

NudC is phosphorylated by Aurora B on T40.

<p>(A) A series of GST-NudC truncations were constructed based on functional domains in human NudC. N1 –N4, NudC truncations that retain the N terminal 49 amino acids (a.a.) but contain various deletions from the C terminus. C1 –C4, NudC truncations that retain most or the entire C terminal nuclear movement domain but contain various deletions from the N terminus. Numbers within brackets refer to amino acid residues in the human NudC protein. CC, coiled-coiled; AR, acidic rich; p23-like CHORD-Sgt domain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153455#pone.0153455.ref051" target="_blank">51</a>]; NMD, conserved nuclear movement domain. (B) GST-NudC full-length (FL), N- and C- terminal truncation series depicted in (A) were used in Aurora B IP kinase assays. Reactions were transferred to filters, analyzed by autoradiography, blotted for Aurora B, and stained by Ponceau S. Substrates used were GST-NudC (lanes 2–9), histone H3 (lane 1) as a positive control and GST (lane 10) as a negative control. Arrowheads, ³²P-labeled GST-NudC proteins in the autoradiogram corresponding to the GST-NudC proteins in the Ponceau stain. *, degradation product. The levels of ³²P-GST-NudC signals (autoradiogram)/total GST-NudC (Ponceau) normalized against that of GST-NudC full-length (set as 1) were quantified (mean ± s.e.m.) from 3 independent experiments, except for GST-NudC-N2 which was obtained from one experiment (data not shown). **, p < 0.001; ***, p < 0.04. (C) GST-NudC-N1 was used in Aurora B IP kinase assays. (D) NudC protein sequences from various species share a high degree of sequence homology surrounding amino acid T40. (E) GST-NudC-N1 wild type (WT) and GST-NudC-N1 containing T40A mutation were used in Aurora B IP kinase assays. GST, negative control. Data in C and E are representative of 3 independent experiments.</p