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.
Highlights
The four isoforms of the ribosomal S6 p90 protein kinase (RSK1-4), along with the two closely related isoforms of the mitogen- and stress-activated protein kinase (MSK1-2), constitute a unique family of Ser/Thr kinases, which are made up of single polypeptide chains harboring two Ser/Thr kinase catalytic domains in tandem [1,2,3,4,5]
There are two catalytic domains: the N-terminal kinase domain (NTKD), which belongs to the AGC family and which is the biologically active module that phosphorylates downstream protein targets; and the C-terminal kinase domain (CTKD), with homology to the calmodulin-dependent family [1, 2, 4], involved in autoregulation of the enzyme
The activated CTKD phosphorylates a serine within the so-called hydrophobic motif (Ser386 in RSK2), creating a docking site for the phosphoinositide-dependent kinase 1 (PDK1)
Summary
The four isoforms of the ribosomal S6 p90 protein kinase (RSK1-4), along with the two closely related isoforms of the mitogen- and stress-activated protein kinase (MSK1-2), constitute a unique family of Ser/Thr kinases, which are made up of single polypeptide chains harboring two Ser/Thr kinase catalytic domains in tandem [1,2,3,4,5] All these enzymes mediate signaling downstream of the mitogen-activated protein kinases (MAPKs) which include, among others, the ERK, JNK and p38 kinases, and regulate cell proliferation, gene expression, mitosis, PLOS ONE | DOI:10.1371/journal.pone.0164343. The activated CTKD phosphorylates a serine within the so-called hydrophobic motif (Ser386 in RSK2), creating a docking site for the phosphoinositide-dependent kinase 1 (PDK1) The latter phosphorylates the activation loop in NTKD (Ser227 in RSK2) conferring full biological activity on RSK
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