Tuberous sclerosis (TSC) is a tumour disease caused by mutations in Tsc1 or Tsc2 genes. Both protein products of Tsc1 and Tsc2 form an intracellular complex possessing GTPase-activating (GAP) activity towards a small GTP binding protein Rheb. The activity of TSC1/2 complex is regulated by multiple phosphorylations of TSC2 mediated by several kinases, such as PKB/Akt, AMP-activated kinase (AMPK), ERK, MK2 and RSK1. So far, very little is known about the molecular mechanisms of TSC2 dephosphorylation. In the yeast two-hybrid screening, we have identified a number of potential TSC2 binding partners including protein phosphatase 5 (PP5). In this study, we provide the evidence that the interaction between TSC2 and PP5 also occurs in mammalian cells. Using TSC2 +/+ , p53-/mouse embryo fibroblasts (MEFs) transiently overexpressing myc-PP5, we showed in the immunoprecipitation assay that TSC2 specifically associates with myc-PP5 in exponentially growing cells. The physiological relevance of identified interaction, especially the involvement of PP5 in the dephosphorylation of major regulatory sites is currently under investigation.
TSC1 and TSC2 are two recently identified tumor suppressor genes encoding hamartin and tuberin, respectively. They have been implicated in the pathogenesis of tuberous sclerosis, a neurological disorder linked with the development of hamartomas in numerous organs, including the brain, kidneys, heart, and liver. Both protein products of TSC1 and TSC2 form an intracellular complex exerting GTPase-activating (GAP) activity towards a small G protein Rheb (Ras homologue enriched in brain). Inhibition of Rheb is important for the positive regulation of mTOR pathway, while mutations of hamartin or tuberin result in uncontrolled cell cycle progression. Although the precise role for the TSC1/2 complex in tumor suppression is not clear, many studies have established a link with the regulation of transcription and protein biosynthesis, increasing susceptibility to apoptosis, cell differentiation, and cell cycle control. We describe the development of a monoclonal antibody specific towards TSC2/tuberin and characterize the suitability for Western blotting, immunoprecipitation, and immunofluorescent applications. The C-terminal region of TSC2 was expressed as a His-tag fusion protein in bacteria, affinity purified and used as an immunogen. Hybrid myelomas were produced from the spleenocytes of immunized mice and SP2/0 myeloma cells. Testing the specificity of cell culture supernatants from generated hybridomas towards recombinant His-TSC2C in ELISA assay allowed us to isolate a panel of positive clones. Further analysis of selected clones by Western blotting and immunoprecipitation revealed one clone, termed D6, which specifically recognized recombinant and endogenous TSC2. The specificity of generated antibody was also confirmed in TSC2(/) and TSC2(+/+) mouse embryo fibroblasts. In summary, the produced antibody is a useful tool in our research program and will be available for researchers investigating signal transduction pathways involving TSC1/2 signaling under physiological conditions and in human pathologies.
Вивчено вплив різних концентрацій тейхоєвої кислоти (IK) Staphylococcus aureus Wood 40 на опосередкований природними кілерними клітинами (ПКК) лізис пухлинних клітин раку Ерліха. Показано, що низькі і середні концентрації ТК стимулюють ПКК-активність, а високісупресують 11 ПК-опосередкованый цитоліз пухлинних клітин. Встановлено, що одночлене засто сування ТК і форболміристатщетату мас дозозалежну пригнічуючу дію із зворотною кореляцією.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.