Search

Aim. To verify CoAlation of ribosomal protein S6 kinase 1 (S6K1) in the HEK293/Pank1b cells exposed to oxidative stress using proximity ligation assay (PLA). Methods. In situ proximity ligation assay. Results. S6 kinase 1 undergoes CoA covalent modification in cellular response to oxidative stress. Conclusions. The previously developed mass spectrometry-based methodology allowed us to identify over 2100 CoAlated proteins in the cells exposed to oxidative or metabolic stress. Many protein kinases were found to be CoAlated, including S6K1. In this study, the proximity ligation assay was used to demonstrate a significant increase of S6K1 CoAlation under oxidative stress compared to untreated cells. [ABSTRACT FROM AUTHOR]

Chordomas are radio- and chemo-resistant tumours and metastasise in as many as 40% of patients. The aim of this study was to identify potential molecular targets for the treatment of chordoma. In view of the reported association of chordoma and tuberous sclerosis complex syndrome, and the available therapeutic agents against molecules in the PI3K/AKT/TSC1/TSC2/mTOR pathway, a tissue microarray of 50 chordoma cases was analysed for expression of active molecules involved in this signalling pathway by immunohistochemistry and a selected number by western blot analysis. Chordomas were positive for p-AKT (92%), p-TSC2 (96%), p-mTOR (27%), total mTOR (75%), p-p70S6K (62%), p-RPS6 (22%), p-4E-BP1 (96%) and eIF-4E (98%). Phosphatase and tensin homologue deleted on chromosome 10 expression was lost in 16% of cases. Mutations failed to be identified in PI3KCA and RHEB1 in the 23 cases for which genomic DNA was available. Fluorescence in situ hybridisation analysis for mTOR and RPS6 loci showed that 11 of 33 and 21 of 44 tumours had loss of one copy of the respective genes, results which correlated with the loss of the relevant total proteins. Fluorescence in situ hybridisation analysis for loci containing TSC1 and TSC2 revealed that all cases analysed harboured two copies of the respective genes. On the basis of p-mTOR and or p-p70S6K expression there is evidence indicating that 65% of the chordomas studied may be responsive to mTOR inhibitors, rapamycin or its analogues, and that patients may benefit from combined therapy including drugs that inhibit AKT. [ABSTRACT FROM AUTHOR]

The structural and functional organization of the adaptor protein Ruk1 is characterized by the presence of three SH3-domains at the N-terminus followed by Pro- and Ser-rich sequences and a C-terminal coiled-coil region. Multiple modules in the Ruk1 structure involved in protein–protein interactions can provide for formation of ligand clusters with varied properties and subcellular location. To study the nature and biological role of such complexes, the recombinant protein Ruk1 with a Glu-epitope at the C-terminus (Ruk1 Glu-tagged) was purified from transfected HEK293 cells by affinity chromatography on protein G-Sepharose with covalently conjugated anti-Glu-tag antibodies. By SDS polyacrylamide gel electrophoresis with subsequent staining with silver, a set of minor bands in addition to the 85-kD Ruk1 Glu-tagged was detected in the purified preparation of the recombinant protein. Proteins with affinity for nucleic acids were also revealed in the Ruk1 Glu-tagged preparation by retardation of electrophoretic mobility of 32P-labeled oligodeoxyribonucleotides in gel. The Ruk1 Glu-tagged preparation was also shown to hydrolyze both deoxyribonucleotides and plasmid DNA. ZnCl2 and heparin inhibited the DNAse activity. These findings suggest the presence of DNases associated with the Ruk1 protein in HEK293 cells. Such complexes were isolated from lysates of HEK293 cells by chromatography on heparin-Sepharose. By elution with 0.5 and 1.0 M NaCl, two fractions with DNase activity and containing proteins with molecular weights of 83, 80 and 72 kD were obtained. The reaction was inhibited by ZnCl2 and heparin, and previous precipitation of Ruk-related proteins with anti-Ruk antibodies resulted in the exhaustion of nuclease activity. By immunoblotting with anti-Ruk antibodies, 83-kD protein immunologically related to the Ruk1 protein was identified in the fractions. It was concluded that the adaptor protein Ruk1 forms complexes with endonucleases in HEK293 cells. [ABSTRACT FROM AUTHOR]

Aim. To generate monoclonal antibodies to Coenzyme A (CoA) with antigenic epitope specificity different to previously developed anti-CoA mAb (1F10) by introducing alternative approach for antigen preparation animal immunization. Methods. Cross-linking of CoA to Keyhole Limpet Hemocyanin (KLH) and to Bovine serum albumin (BSA) using reversible chemical crosslinker SPDP. Hybridoma technique. Western blot analysis. Immunoprecipitation. ELISA. Results. Monoclonal antibody (A11) that specifically recognizes CoA in various immunoassays and has the same antigenic epitope specificity as existing anti-CoA mAb (1F10) has been generated. Conclusions. Application of different ways for CoA conjugation to the carrier proteins to create an antigen for immunization does not affect the antigenic epitope specificity of generated anti-CoA antibodies most probably due to the existence of only one immunogenic epitope in CoA molecule. [ABSTRACT FROM AUTHOR]

Abstract: Ribosomal protein S6 kinase plays a critical role in the regulation of cell growth and energy metabolism. S6K belongs to the AGC family of serine/threonine kinases and is a downstream effector of the mTOR and PI3K signalling pathways. The activity and subcellular localisation of S6K are tightly controlled by phosphorylation/dephosphorylation events. We have recently demonstrated that steady-state levels of S6K isoforms, S6K1 and S6K2, are regulated by ubiquitination-mediated proteasomal degradation. In this study, we report for the first time that the ubiquitination status of S6K isoforms is coordinated by signalling pathways induced by mitogenic stimuli and extracellular stresses. The induction of signal transduction by serum and growth factors significantly increases the level of S6K ubiquitination, while the treatment of cells with UV and staurosporine has the opposite effect. Furthermore, we found that the phosphorylation/activation of S6Ks does not correlate directly with the induction of their ubiquitination in response to diverse cellular stimuli. This study suggests that the ubiquitination and subsequent proteasomal degradation of S6K are controlled by signalling pathways, which could possibly facilitate their association with the components of the ubiquitination machinery. [Copyright &y& Elsevier]

Aim. Generation of monoclonal antibodies specific to Coenzyme A. Methods. Hybridoma technique. KLH carrier protein conjugated with CoA was used for immunization. Screening of positive clones was performed with BSA conjugated to CoA. Results. Monoclonal antibody that specifically recognizes CoA and CoA derivatives, but not its precursors ATP and cysteine has been generated. Conclusion. In this study, we describe for the first time the production and characterization of monoclonal antibodies against CoA. The monoclonal antibody 1F10 was shown to recognize specifically CoA in Western blotting, ELISA and immunoprecipitation. These properties make this antiboby a particularly valuable reagent for elucidating CoA function in health and disease. [ABSTRACT FROM AUTHOR]