ABSTRACT
Overexpression of the nuclear oncoprotein prothymosin a enhanced and, in a reciprocal experiment, down-regulation of endogenous prothymosin alpha by RNA interference approach inhibited transcriptional activity of the p53 tumor suppressor in the reporter gene assay. Ectopic expression of prothymosin alpha enhanced not only p53-dependent transcription, but also intracellular level of p53 in HeLa (but not HCT116) cells. Ability to stimulate p53-dependent transcription was lost by C-terminal mutants of prothymosin alpha with impaired nuclear accumulation, but not by N-terminal deletion mutants and by the double mutant of prothymosin alpha with impaired ability to bind Keap1, suggesting that prothymosinalpha-Keap1 interaction is dispensable for p53 response. Our data suggest that the central "acidic" region of prothymosin alpha together with intact nuclear localization signal is responsible for stimulation of p53-dependent transcription. This conclusion was confirmed by the fact that another protein containing long "acidic" region and nuclear localization signal, parathymosin, was able to stimulate transcription of p53-responsive reporter gene.
Subject(s)
Cell Nucleus/metabolism , Mutation , Protein Precursors/metabolism , Thymosin/analogs & derivatives , Transcription, Genetic/physiology , Tumor Suppressor Protein p53/metabolism , Active Transport, Cell Nucleus/physiology , Cell Nucleus/genetics , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Kelch-Like ECH-Associated Protein 1 , Nuclear Localization Signals/genetics , Nuclear Localization Signals/metabolism , Protein Precursors/genetics , Thymosin/genetics , Thymosin/metabolism , Tumor Suppressor Protein p53/geneticsABSTRACT
Prothymosin alpha (ProTalpha) is a highly conserved protein in vertebrates that possesses a number of biological functions. One of these functions of ProTalpha is the ability to enhance antioxidant defence system of a cell via its interaction with Keap1 protein. Keap1 is a repressor of Nrf2, a transcription factor responsible for activation of genes that code for defensive proteins. While bound to Nrf2, Keap1 exports Nrf2 from the nucleus to the cytoplasm and, being adaptor protein for ubiquitin ligase, promotes ubiquitination of Nrf2 and its subsequent degradation by 26S proteasome. ProTalpha and Nrf2 compete for interaction with Keap1, therefore ProTalpha is able to liberate Nrf2 from complex with Keap1 and hence contribute to Nrf2-dependent transcription. Here we were interested in elucidating possible consequences for ProTalpha of its interaction with Keap1. We have shown that, despite ProTalpha interaction with Keap1, ProTalpha is a stable protein. In contrast to Nrf2 ProTalpha was not subjected to Keap-dependent ubiquitination, degradation and export from the nucleus. Furthermore, ubiquitination of ProTalpha was undetectable even when Keap1 and ubiquitin were overexpressed. It appears that ProTalpha contribution to Nrf2-dependent transcription is accomplished via the increase of free Nrf2 rather then the increase of total intracellular amount of Nrf2.
Subject(s)
Cell Nucleus/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , NF-E2-Related Factor 2/metabolism , Protein Precursors/metabolism , Thymosin/analogs & derivatives , Transcription, Genetic/physiology , Active Transport, Cell Nucleus/physiology , Cell Nucleus/genetics , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Kelch-Like ECH-Associated Protein 1 , NF-E2-Related Factor 2/genetics , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Precursors/genetics , Thymosin/genetics , Thymosin/metabolism , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitination/physiologyABSTRACT
Several novel functions of the well-known and intensively studied protein prothymosin alpha have recently been revealed. In addition to such traditional functions of this protein as immunomodulatory activity and stimulation of cellular proliferation, prothymosin alpha was shown to be involved in protection of cells against apoptosis and in regulation of expression of the oxidative stress-protective genes. Methods and approaches used for revelation of prothymosin alpha novel functions are described in this review.