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Related Experiment Video

Updated: Mar 13, 2026

Chromatographic Purification of Highly Active Yeast Ribosomes
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Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2).

Darkhan Utepbergenov1, Paulina M Hennig1,2, Urszula Derewenda1

  • 1Department of Molecular Physiology and Biological Physics, University of Virginia, School of Medicine, Charlottesville, Virginia, United States of America.

Plos One
|October 13, 2016
PubMed
Summary

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Bacterially expressed Ribosomal S6 kinase 2 (RSK2) can be purified and activated in vitro. This method provides a cost-effective alternative for studying RSK2, crucial in cancer and Coffin-Lowry syndrome research.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Biochemistry

Background:

  • Ribosomal S6 kinases (RSK) are key regulators in the mitogen-activated protein kinase (MAPK) pathway.
  • RSK isoforms, including RSK2, are implicated in cancer and Coffin-Lowry syndrome.
  • RSK proteins possess two kinase domains and regulatory phosphorylation sites.

Purpose of the Study:

  • To establish a method for overexpressing and purifying full-length RSK2 in Escherichia coli.
  • To characterize the activity and inhibition of bacterially produced RSK2.
  • To investigate novel activation mechanisms of RSK2.

Main Methods:

  • Overexpression of full-length RSK2 as a maltose-binding protein (MBP) fusion in E. coli.
  • Purification using affinity and size-exclusion chromatography after MBP removal.

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  • In vitro activation by ERK2 and PDK1, followed by catalytic activity and inhibitor sensitivity assays.

    • Main Results:

    • High-yield purification of active, full-length RSK2 from E. coli.
    • Bacterially produced RSK2 exhibits comparable activity and inhibitor sensitivity to insect cell-derived RSK2.
    • Autocatalytic phosphorylation at Ser386 was observed, independent of ERK, suggesting potential PDK1-mediated activation.

    Conclusions:

    • Bacterial expression offers a viable and efficient system for producing active RSK2.
    • RSK2 activation can potentially occur via PDK1, bypassing the need for ERK in certain contexts.
    • This research provides a valuable tool for further investigation into RSK2 function and its role in disease.