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Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2)
Ribosomal S6 kinases (RSK) play important roles in cell signaling through the mitogen-activated protein kinase (MAPK) pathway. Each of the four RSK isoforms (RSK1-4) is a single polypeptide chain containing two kinase domains connected by a linker sequence with regulatory phosphorylation sites. Here...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061434/ https://www.ncbi.nlm.nih.gov/pubmed/27732676 http://dx.doi.org/10.1371/journal.pone.0164343 |
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author | Utepbergenov, Darkhan Hennig, Paulina M. Derewenda, Urszula Artamonov, Mykhaylo V. Somlyo, Avril V. Derewenda, Zygmunt S. |
author_facet | Utepbergenov, Darkhan Hennig, Paulina M. Derewenda, Urszula Artamonov, Mykhaylo V. Somlyo, Avril V. Derewenda, Zygmunt S. |
author_sort | Utepbergenov, Darkhan |
collection | PubMed |
description | Ribosomal S6 kinases (RSK) play important roles in cell signaling through the mitogen-activated protein kinase (MAPK) pathway. Each of the four RSK isoforms (RSK1-4) is a single polypeptide chain containing two kinase domains connected by a linker sequence with regulatory phosphorylation sites. Here, we demonstrate that full-length RSK2—which is implicated in several types of cancer, and which is linked to the genetic Coffin-Lowry syndrome—can be overexpressed with high yields in Escherichia coli as a fusion with maltose binding protein (MBP), and can be purified to homogeneity after proteolytic removal of MBP by affinity and size-exclusion chromatography. The purified protein can be fully activated in vitro by phosphorylation with protein kinases ERK2 and PDK1. Compared to full-length RSK2 purified from insect host cells, the bacterially expressed and phosphorylated murine RSK2 shows the same levels of catalytic activity after phosphorylation, and sensitivity to inhibition by RSK-specific inhibitor SL0101. Interestingly, we detect low levels of phosphorylation in the nascent RSK2 on Ser386, owing to autocatalysis by the C-terminal domain, independent of ERK. This observation has implications for in vivo signaling, as it suggests that full activation of RSK2 by PDK1 alone is possible, circumventing at least in some cases the requirement for ERK. |
format | Online Article Text |
id | pubmed-5061434 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-50614342016-10-27 Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2) Utepbergenov, Darkhan Hennig, Paulina M. Derewenda, Urszula Artamonov, Mykhaylo V. Somlyo, Avril V. Derewenda, Zygmunt S. PLoS One Research Article Ribosomal S6 kinases (RSK) play important roles in cell signaling through the mitogen-activated protein kinase (MAPK) pathway. Each of the four RSK isoforms (RSK1-4) is a single polypeptide chain containing two kinase domains connected by a linker sequence with regulatory phosphorylation sites. Here, we demonstrate that full-length RSK2—which is implicated in several types of cancer, and which is linked to the genetic Coffin-Lowry syndrome—can be overexpressed with high yields in Escherichia coli as a fusion with maltose binding protein (MBP), and can be purified to homogeneity after proteolytic removal of MBP by affinity and size-exclusion chromatography. The purified protein can be fully activated in vitro by phosphorylation with protein kinases ERK2 and PDK1. Compared to full-length RSK2 purified from insect host cells, the bacterially expressed and phosphorylated murine RSK2 shows the same levels of catalytic activity after phosphorylation, and sensitivity to inhibition by RSK-specific inhibitor SL0101. Interestingly, we detect low levels of phosphorylation in the nascent RSK2 on Ser386, owing to autocatalysis by the C-terminal domain, independent of ERK. This observation has implications for in vivo signaling, as it suggests that full activation of RSK2 by PDK1 alone is possible, circumventing at least in some cases the requirement for ERK. Public Library of Science 2016-10-12 /pmc/articles/PMC5061434/ /pubmed/27732676 http://dx.doi.org/10.1371/journal.pone.0164343 Text en © 2016 Utepbergenov et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Utepbergenov, Darkhan Hennig, Paulina M. Derewenda, Urszula Artamonov, Mykhaylo V. Somlyo, Avril V. Derewenda, Zygmunt S. Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2) |
title | Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2) |
title_full | Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2) |
title_fullStr | Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2) |
title_full_unstemmed | Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2) |
title_short | Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2) |
title_sort | bacterial expression, purification and in vitro phosphorylation of full-length ribosomal s6 kinase 2 (rsk2) |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061434/ https://www.ncbi.nlm.nih.gov/pubmed/27732676 http://dx.doi.org/10.1371/journal.pone.0164343 |
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