CDK11p58 Is Required for Centriole Duplication and Plk4 Recruitment to Mitotic Centrosomes

BACKGROUND: CDK11(p58) is a mitotic protein kinase, which has been shown to be required for different mitotic events such as centrosome maturation, chromatid cohesion and cytokinesis. METHODOLOGY/PRINCIPAL FINDINGS: In addition to these previously described roles, our study shows that CDK11(p58) inhibition induces a failure in the centriole duplication process in different human cell lines. We propose that this effect is mediated by the defective centrosomal recruitment of proteins at the onset of mitosis. Indeed, Plk4 protein kinase and the centrosomal protein Cep192, which are key components of the centriole duplication machinery, showed reduced levels at centrosomes of mitotic CDK11-depleted cells. CDK11(p58), which accumulates only in the vicinity of mitotic centrosomes, directly interacts with the centriole-associated protein kinase Plk4 that regulates centriole number in cells. In addition, we show that centriole from CDK11 defective cells are not able to be over duplicated following Plk4 overexpression. CONCLUSION/SIGNIFICANCE: We thus propose that CDK11 is required for centriole duplication by two non-mutually-exclusive mechanisms. On one hand, the observed duplication defect could be caused indirectly by a failure of the centrosome to fully maturate during mitosis. On the other hand, CDK11(p58) could also directly regulate key centriole components such as Plk4 during mitosis to trigger essential mitotic centriole modifications, required for centriole duplication during subsequent interphase

Nucleophosmin/B23 activates Aurora A at the centrosome through phosphorylation of serine 89.: Activation of Aurora-A by Nucleophosmin

International audienceAurora A (AurA) is a major mitotic protein kinase involved in centrosome maturation and spindle assembly. Nucleophosmin/B23 (NPM) is a pleiotropic nucleolar protein involved in a variety of cellular processes including centrosome maturation. In the present study, we report that NPM is a strong activator of AurA kinase activity. NPM and AurA coimmunoprecipitate and colocalize to centrosomes in G2 phase, where AurA becomes active. In contrast with previously characterized AurA activators, NPM does not trigger autophosphorylation of AurA on threonine 288. NPM induces phosphorylation of AurA on serine 89, and this phosphorylation is necessary for activation of AurA. These data were confirmed in vivo, as depletion of NPM by ribonucleic acid interference eliminated phosphorylation of CDC25B on S353 at the centrosome, indicating a local loss of AurA activity. Our data demonstrate that NPM is a strong activator of AurA kinase activity at the centrosome and support a novel mechanism of activation for AurA

Preparation and characterization of a human aurora-A kinase monoclonal antibody.

International audienceWe have developed monoclonal antibodies against the human aurora-A serine/threonine kinase. After immunization of a mouse, a fusion was performed to obtain hybridomas that were selected because they produced immunoglobulin positively reacting against the protein used for immunization. We isolated one particular monoclonal that we named 35C1 using a series of selective assays. The first criteria of the screen for monoclonals was an ELISA (Enzyme Linked Immunosorbant Assay) assay performed in 96-well plates against the purified recombinant histidine-tagged aurora-A. The second was a positive Western blot against the same recombinant protein. The third criteria was a positive western blot against an HeLa cell extract, the selected monoclonal should detect only one protein migrating at 46 kDa (kiloDalton) on SDS (Sodium Dodecyl Sulfate)-polyacrylamide gel electrophoresis. Finally, the monoclonal had to bind to duplicated centrosomes and spindle poles in human MCF7 cultured cells by indirect immunofluorescence. At this stage several monoclonals were still positive. We then increased the selectivity by searching for antibodies that were able to cross-react with the mouse aurora-A kinase both by western blot and indirect immunofluorescence. We selected and cloned the 35C1 hybridoma to produce the antibody. Further characterization of the 35C1 antibody revealed that it was able to immunoprecipitate the kinase, that it did not inhibit the aurora-A kinase activity and consequently could be used to measure the aurora-A kinase activity in vivo after immunoprecipitation

The absence of p53 aggravates polyploidy and centrosome number abnormality induced by Aurora-C overexpression.

International audienceAurora-C is the third member of the aurora serine/threonine kinase family and was found only in mammals. Because Aurora-C is overexpressed in many different types of cancer cells we decided to analyze the consequences of Aurora-C overexpression in human cells. We first investigated the subcellular localization of overexpressed GFP-Aurora-C in mitosis and interphase in HeLa cells. As expected, during mitosis, we found that Aurora-C mimics Aurora-B. Surprisingly, in few interphase cells, we found that Aurora-C localized to the centrosome, like Aurora-A. We then examined the phenotype generated by Aurora-C overexpression. Basically it looked similar to the phenotypes observed after overexpression of the other Aurora kinases. We observed an augmentation of polyploid cells containing more than two centrosomes. More interestingly this phenotype was aggravated in the absence of a functional p53. Although the physiological function of Aurora-C in somatic cells remains to be clarified, our results, just like for the two other Aurora kinases, raised the question of a role of Aurora-C in the development and progression of cancer especially in the presence of mutated p53

Aurora A kinase interacts with and phosphorylates VHL protein

International audienceThe serine/threonin kinase Aurora A is an oncoprotein, whereas von Hippel-Lindau protein (pVHL) is a tumor suppressor protein. Both proteins have the same localization during mitosis: in the mitotic spindle and the centrosome. These two proteins also have common functions, such as the regulation of the cell cycle, the stability of the mitotic spindle and both intervene in the functioning of centrosomes. In this study we have analyzed the interaction between Aurora A and pVHL with immunoprecipitation and in vitro phosphorylation experiments. We have confirmed that the immunoprecipitation of pVHL from Hek 293 cell extracts were coupled with Aurora A. In addition, the interaction between the two proteins has been tested by analyzing the phosphorylation of pVHL in vitro by Aurora A. The results revealed that pVHL was phosphorylated by Aurora A. In conclusion, the study demonstrated that Aurora A interacts with and phosphorylates pVHL. Given the role of these two proteins in cell division as well as their status in cancer, this interaction requires further investigation

Aurora-C interacts with and phosphorylates the transforming acidic coiled-coil 1 protein.

International audienceAurora-C, a member of the Aurora kinase family, is implicated in the regulation of mitosis. In contrast to Aurora-A and Aurora-B its cellular localization and functions are poorly characterized. TACC1 protein belongs to the transforming acidic coiled-coil family shown to interact with the Aurora kinases. In the present study we analyzed the interaction between Aurora-C and TACC1 by means of immunofluorescence (IF), co-immunoprecipitation (IP) and in vitro phosphorylation experiments. We demonstrated that Aurora-C and TACC1 proteins co-localize to the midbody of HeLa cells during cytokinesis. Immunoprecipitated TACC1 from HeLa cell extracts was associated with Aurora-C. In addition, the interaction of the two proteins was tested by analyzing the phosphorylation of TACC1 in vitro. The results demonstrated that TACC1 is phosphorylated by Aurora-C on a serine at position 228. In conclusion, the study demonstrated that TACC1 localizes at the midbody during cytokinesis and interacts with and is a substrate of Aurora-C, which warrant further investigation in order to elucidate the functional significance of this interaction

Aurora A overexpression and pVHL reduced expression are correlated with a bad kidney cancer prognosis.

International audienceWe investigate the expression and localization of the tumor suppressor protein pVHL as well as the oncoprotein Aurora A kinase in kidney cancer. Both Aurora A kinase and pVHL protein status were evaluated using immunohistochemistry. The Aurora A expression is correlated with the Fuhrman grade and the TNM stage, while the pVHL expression is correlated with the capsule rupture and the TNM stage. Aurora A kinase expression increases in malignant tissue comparing to the non-malignant one. And there is a decrease in pVHL expression from the adjacent healthy tissues to the tumor's ones. The two kinds of opposite tumor profiles display significant distribution difference according to TNM stages. It could be proposed that the absence of Aurora A protein associated with a strong expression of pVHL in clear cells kidney carcinoma are of good prognosis for the disease

Aurora A overexpression and pVHL reduced expression are correlated with a bad kidney cancer prognosis.

International audienceWe investigate the expression and localization of the tumor suppressor protein pVHL as well as the oncoprotein Aurora A kinase in kidney cancer. Both Aurora A kinase and pVHL protein status were evaluated using immunohistochemistry. The Aurora A expression is correlated with the Fuhrman grade and the TNM stage, while the pVHL expression is correlated with the capsule rupture and the TNM stage. Aurora A kinase expression increases in malignant tissue comparing to the non-malignant one. And there is a decrease in pVHL expression from the adjacent healthy tissues to the tumor's ones. The two kinds of opposite tumor profiles display significant distribution difference according to TNM stages. It could be proposed that the absence of Aurora A protein associated with a strong expression of pVHL in clear cells kidney carcinoma are of good prognosis for the disease