A Method for Analyzing the Ubiquitination and Degradation of Aurora-A

The cell cycle machinery consists of regulatory proteins that control the progression through the cell cycle ensuring that DNA replication alternates with DNA segregation in mitosis to maintain cell integrity. Some of these key regulators have to be degraded at each cell cycle to prevent cellular dysfunction. Mitotic exit requires the inactivation of cyclin dependent kinase1 (cdk1) and it is the degradation of the cyclin subunit that inactivates the kinase. Cyclin degradation has been well characterized and it was shown that it is ubiquitin proteasome pathway that leads to the elimination of cyclins. By now, many other regulatory proteins were shown to be degraded by the same pathway, among them members of the aurora kinase family, degraded many other regulatory proteins. Aurora kinases are involved in mitotic spindle formation as well as in cytokinesis. The abundance and activity of the kinase is precisely regulated during the cell cycle. To understand how proteolysis regulates transitions through the cell cycle we describe two assays for ubiquitination and degradation of xenopus aurora kinase A using extracts from xenopus eggs or somatic cell lines

Aurora kinases are expressed in medullary thyroid carcinoma (MTC) and their inhibition suppresses in vitro growth and tumorigenicity of the MTC derived cell line TT

International audienceBACKGROUND: The Aurora kinase family members, Aurora-A, -B and -C, are involved in the regulation of mitosis, and alterations in their expression are associated with cell malignant transformation. To date no information on the expression of these proteins in medullary thyroid carcinoma (MTC) are available. We here investigated the expression of the Aurora kinases in human MTC tissues and their potential use as therapeutic targets. METHODS: The expression of the Aurora kinases in 26 MTC tissues at different TNM stages was analyzed at the mRNA level by quantitative RT-PCR. We then evaluated the effects of the Aurora kinase inhibitor MK-0457 on the MTC derived TT cell line proliferation, apoptosis, soft agar colony formation, cell cycle and ploidy. RESULTS: The results showed the absence of correlation between tumor tissue levels of any Aurora kinase and tumor stage indicating the lack of prognostic value for these proteins. Treatment with MK-0457 inhibited TT cell proliferation in a time- and dose-dependent manner with IC50 = 49.8 ± 6.6 nM, as well as Aurora kinases phosphorylation of substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it. Cytofluorimetric analysis confirmed that MK-0457 induced accumulation of cells with ≥ 4N DNA content without inducing apoptosis. Finally, MK-0457 prevented the capability of the TT cells to form colonies in soft agar. CONCLUSIONS: We demonstrate that Aurora kinases inhibition hampered growth and tumorigenicity of TT cells, suggesting its potential therapeutic value for MTC treatment

Constitutive Phosphorylation of Aurora-A on Ser51 Induces Its Stabilization and Consequent Overexpression in Cancer

The serine/threonine kinase Aurora-A (Aur-A) is a proto-oncoprotein overexpressed in a wide range of human cancers. Overexpression of Aur-A is thought to be caused by gene amplification or mRNA overexpression. However, recent evidence revealed that the discrepancies between amplification of Aur-A and overexpression rates of Aur-A mRNA were observed in breast cancer, gastric cancer, hepatocellular carcinoma, and ovarian cancer. We found that aggressive head and neck cancers exhibited overexpression and stabilization of Aur-A protein without gene amplification or mRNA overexpression. Here we tested the hypothesis that aberration of the protein destruction system induces accumulation and consequently overexpression of Aur-A in cancer.Aur-A protein was ubiquitinylated by APC(Cdh1) and consequently degraded when cells exited mitosis, and phosphorylation of Aur-A on Ser51 was observed during mitosis. Phosphorylation of Aur-A on Ser51 inhibited its APC(Cdh1)-mediated ubiquitylation and consequent degradation. Interestingly, constitutive phosphorylation on Ser51 was observed in head and neck cancer cells with protein overexpression and stabilization. Indeed, phosphorylation on Ser51 was observed in head and neck cancer tissues with Aur-A protein overexpression. Moreover, an Aur-A Ser51 phospho-mimetic mutant displayed stabilization of protein during cell cycle progression and enhanced ability to cell transformation.Broadly, this study identifies a new mode of Aur-A overexpression in cancer through phosphorylation-dependent inhibition of its proteolysis in addition to gene amplification and mRNA overexpression. We suggest that the inhibition of Aur-A phosphorylation can represent a novel way to decrease Aur-A levels in cancer therapy

Fast assessment of long axis strain with standard cardiovascular magnetic resonance: a validation study of a novel parameter with reference values

Background: Assessment of longitudinal function with cardiovascular magnetic resonance (CMR) is limited to measurement of systolic excursion of the mitral annulus (MAPSE) or elaborate strain imaging modalities. The aim of this study was to develop a fast assessable parameter for the measurement of long axis strain (LAS) with CMR. Methods: 40 healthy volunteers and 125 patients with different forms of cardiomyopathy were retrospectively analyzed. Four different approaches for the assessment of LAS with CMR measuring the distance between the LV apex and a line connecting the origins of the mitral valve leaflets in enddiastole and endsystole were evaluated. Values for LAS were calculated according to the strain formula. Results: LAS derived from the distance of the epicardial apical border to the midpoint of the line connecting the mitral valve insertion points (LAS-epi/mid) proved to be the most reliable parameter for the assessment of LAS among the different approaches. LAS-epi/mid displayed the highest sensitivity (81.6 %) and specificity (97.5 %), furthermore showing the best correlation with feature tracking (FTI) derived transmural longitudinal strain (r = 0.85). Moreover, LAS-epi/mid was non-inferior to FTI in discriminating controls from patients (Area under the curve (AUC) = 0.95 vs. 0.94, p = NS). The time required for analysis of LAS-epi/mid was significantly shorter than for FTI (67 ± 8 s vs. 180 ± 14 s, p < 0.0001). Additionally, LAS-epi/mid performed significantly better than MAPSE (Delta AUC = 0.09; p < 0.005) and the ejection fraction (Delta AUC = 0.11; p = 0.0002). Reference values were derived from 234 selected healthy volunteers. Mean value for LAS-epi/mid was −17.1 ± 2.3 %. Mean values for men were significantly lower compared to women (−16.5 ± 2.2 vs. -17.9 ± 2.1 %; p < 0.0001), while LAS decreased with age. Conclusions: LAS-epi/mid is a novel and fast assessable parameter for the analysis of global longitudinal function with non-inferiority compared to transmural longitudinal strain

Tectonic structure, evolution, and the nature of oceanic core complexes and their detachment fault zones (13°20′N and 13°30′N, Mid Atlantic Ridge)

Microbathymetry data, in situ observations, and sampling along the 138200N and 138200N oceanic core complexes (OCCs) reveal mechanisms of detachment fault denudation at the seafloor, links between tectonic extension and mass wasting, and expose the nature of corrugations, ubiquitous at OCCs. In the initial stages of detachment faulting and high-angle fault, scarps show extensive mass wasting that reduces their slope. Flexural rotation further lowers scarp slope, hinders mass wasting, resulting in morphologically complex chaotic terrain between the breakaway and the denuded corrugated surface. Extension and drag along the fault plane uplifts a wedge of hangingwall material (apron). The detachment surface emerges along a continuous moat that sheds rocks and covers it with unconsolidated rubble, while local slumping emplaces rubble ridges overlying corrugations. The detachment fault zone is a set of anostomosed slip planes, elongated in the alongextension direction. Slip planes bind fault rock bodies defining the corrugations observed in microbathymetry and sonar. Fault planes with extension-parallel stria are exposed along corrugation flanks, where the rubble cover is shed. Detachment fault rocks are primarily basalt fault breccia at 138200N OCC, and gabbro and peridotite at 138300N, demonstrating that brittle strain localization in shallow lithosphere form corrugations, regardless of lithologies in the detachment zone. Finally, faulting and volcanism dismember the 138300N OCC, with widespread present and past hydrothermal activity (Semenov fields), while the Irinovskoe hydrothermal field at the 138200N core complex suggests a magmatic source within the footwall. These results confirm the ubiquitous relationship between hydrothermal activity and oceanic detachment formation and evolution

Publisher Correction: A PRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients

The original version of this Article contained an error in the title, which was incorrectly given as 'APRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients'. This has now been corrected in both the PDF and HTML versions of the Article to read 'A PRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients'