Ribosomal protein S6 kinase 1 interacts with and is ubiquitinated by ubiquitin ligase ROC1
Section snippets
Materials and methods
Yeast two-hybrid screening. The full-length sequence of rat S6K1 (p70 S6K1) was amplified by PCR from pcDNA3.1-EE-S6K1 and cloned into pEG202 plasmid in-frame with LexA located at the C-terminus (Origene Technologies Inc.). The T389D mutant of S6K1 was generated using a QuikChange site-directed mutagenesis kit (Stratagene). EGY48 cells containing pEG202/S6K1 T389D and pSH18–34 plasmids were further transformed with a human HeLa cDNA library (Origene Technologies, Inc.) as described previously
Identification of ROC1 as S6K1 interacting protein in a yeast two-hybrid screen
To identify cellular proteins which have the potential to interact specifically with the activated form of S6K1 (p70S6K1), we carried out the yeast two-hybrid screening of HeLa cell line cDNA library with S6K1 T389D mutant. Transient overexpression studies in mammalian cells showed that the activity of the S6K1 T389D mutant toward ribosomal protein S6 is 2–3-folds higher when compared to the wild-type kinase [12]. Screening of 1.2 × 107 independent clones resulted in selection of 70 positive
Acknowledgments
The work on this project was supported by grants from the National Academy of Sciences of Ukraine and INTAS. Ganna Panasyuk was supported by the Royal Society International Incoming Short Visit Grant. The authors thank Prof. K. Vousden for providing pcDNA3.1/Ubiquitin-HA plasmid and Prof. Y. Xiong for anti-ROC1 antibodies. We thank Nadeem Shaikh for critical reading of the manuscript and helpful suggestions.
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2022, Cancer LettersCitation Excerpt :Fenton et al. [17] showed that p70S6K could be acetylated by p300 and PCAF (p300/CBP associated factor) to enhance its protein stability, whereas sirtuin deacetylases could be involved in its deacetylation. Poly-ubiquitination by the ubiquitin ligase ROC1 could also destabilize p70S6K by targeting it to proteasomal degradation [18]. However, the cellular functional consequences of acetylation and ubiquitinylation are yet to be investigated.
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Functions and regulation of the 70 kDa ribosomal S6 kinases
2011, International Journal of Biochemistry and Cell BiologyCitation Excerpt :In this regard, we have recently observed the ubiquitination and proteasomal degradation of S6Ks, although the physiological conditions under which S6K degradation is triggered are currently unknown (Gwalter et al., 2009; Wang et al., 2008). While little is known regarding the pathways leading to S6K destabilization, the finding that the Roc1 ubiquitin ligase specifically interacts with and ubiquitinates S6K1 implies that S6K1 and S6K2 are subject to degradation via distinct ubiquitin ligases, thus providing a further mechanism for the differential regulation of these kinases (Panasyuk et al., 2008). Furthermore, the finding that S6Ks are also acetylated and that treatment of cells with deacetylase inhibitors leads to a stabilization of S6K2 suggests that, as with other proteins such as p53, the ubiquitination and degradation of S6Ks may be opposed by lysine acetylation (Fenton et al., 2010b).
Inactivation of NF-ΚB p50 leads to insulin sensitization in liver through post-translational inhibition of p70S6K
2009, Journal of Biological ChemistryCitation Excerpt :Regulation of S6K protein by degradation is largely unknown. Two recent studies suggest that a recombinant S6K protein can be ubiquitinated and degraded in a proteasome-dependent manner (45, 46). However, the full-length S6K protein was not used in the studies.
The ubiquitination of ribosomal S6 kinases is independent from the mitogen-induced phosphorylation/activation of the kinase
2009, International Journal of Biochemistry and Cell Biology