Ribosomal protein S6 kinase 1 interacts with and is ubiquitinated by ubiquitin ligase ROC1

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Abstract

Ribosomal protein S6 kinase (S6K) is involved in the regulation of cell growth and cellular metabolism. The activation of S6K in response to diverse extracellular stimuli is mediated by multiple phosphorylations coordinated by the mTOR and PI3K signaling pathways. We have recently found that both forms of S6K are modified by ubiquitination. Following these findings, we demonstrate here for the first time that S6K1 associates specifically with ubiquitin ligase ROC1 in vitro and in vivo. The interaction was initially identified in the yeast two-hybrid screening and further confirmed by pull-down and co-immunoprecipitation assays. Furthermore, the overexpression of ROC1 leads to an increase in S6K1 ubiquitination. Consistent with this observation, we showed that the steady-state level of S6K1 is regulated by ROC1, since downregulation of ROC1 by specific siRNA promotes stabilization of S6K1 protein. The results suggest the involvement of ROC1 in S6K1 ubiquitination and subsequent proteasomal degradation.

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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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