title: Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2).
abstract: 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.
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, School of Medicine, Charlottesville, Virginia, United States of America.
authors.forename: Darkhan
authors.initials: D
authors.lastname: Utepbergenov
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, School of Medicine, Charlottesville, Virginia, United States of America.|Department of Molecular Genetics, University of Lodz, Lodz, Poland.
authors.forename: Paulina M
authors.identifier: http://orcid.org/0000-0003-2633-452X
authors.initials: PM
authors.lastname: Hennig
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, School of Medicine, Charlottesville, Virginia, United States of America.
authors.forename: Urszula
authors.initials: U
authors.lastname: Derewenda
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, School of Medicine, Charlottesville, Virginia, United States of America.
authors.forename: Mykhaylo V
authors.initials: MV
authors.lastname: Artamonov
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, School of Medicine, Charlottesville, Virginia, United States of America.
authors.forename: Avril V
authors.initials: AV
authors.lastname: Somlyo
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, School of Medicine, Charlottesville, Virginia, United States of America.
authors.forename: Zygmunt S
authors.initials: ZS
authors.lastname: Derewenda
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D064413:3-Phosphoinositide-Dependent Protein Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3; D019950:Mitogen-Activated Protein Kinase 1