Conformations of p90 Ribosomal S6 Kinase (RSK) Activation.

Protein kinase have evolved to be dynamic macromolecular switches that alternate between inactive and active conformations. Although most kinase are typically phosphorylated and activated by other kinases, relatively little is known about the detailed mechanistic steps required for one kinase to activate another. The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie downstream of the Ras-MAPK pathway and regulate cell proliferation, cell survival, cell growth, and cell motility. RSK is an interesting model system to study kinase activation because it contains two distinct kinase domains, an N-terminal kinase (NTK), and a C-terminal kinase (CTK) in the same polypeptide chain. The NTK is a member of the AGC kinase superfamily, and the CTK is a member of the CAMK family. A flexible linker bridges the NTK and CTK, and embedded within the linker is the highly conserved tail of AGC kinases (AGC tail) which contains the conserved turn and hydrophobic motifs (HF). RSK has a complex activation mechanism that includes sequential phosphorylation events and requires two additional kinases: extracellular signal-regulated kinase (ERK2) and 3-phosphoinositide-dependent protein kinase 1 (PDK1). ERK2 phosphorylates the CTK and the turn motif. The phosphorylated CTK is then able to phosphorylate the linker at the HF, this enables PDK1 to bind the linker and then phosphorylate and activate the NTK2. The precise structural details of RSK kinase activation are not fully understood and a more rigorous biochemical characterization could provide valuable insight into how kinase complexes assemble and how the linker facilitates multiple binding and phosphorylation events. To gain a better understanding of the FL RSK2-ERK2 signaling complex we have used cryoEM and hydrogen deuterium exchange mass spectrometry (HDX) to gain insight into the overall shape and solvent accessibility of the overall complex. We have determined a low-resolution density map of the RSK2-ERK2 complex, revealing a compact shape. Additionally, all three kinase domains are relatively well protected from solvent, with the exception of the activation loops of the CTK, and the aC helix of the NTK. While the tails of RSK2 and linker (AGC tail) are highly solvent exposed. We hypothesize that the linker region needs such solvent accessibility in order to enable the various interactions and phosphorylation events necessary for RSK activation. We have also utilized peptide array binding experiments to determine the residues within the linker that are necessary to enable ERK2 phosphorylation of the turn motif, CTK phosphorylation of the HF, and PDK1 binding and subsequent phosphorylation of the NTK activation loop. ERK2 appears to bind to the linker and NTK in a multivalent fashion via the C-lobe (300-320) and the N-lobe. The CTK seems to primarily interact with HF in the linker. And PDK1 binds to both the HF and a conserved FDX1-2Y/F motif in the linker.