The activation, operation and regulation of the mitotic kinase RSK1

Abstract

together by the adenine moiety of the bound cofactor. In contrast, the regulatory spine, or R-spine, is highly dynamic, and its assembly is required for catalytic activity (Kim et al., 2017). The R-spine includes a residue from the C-helix of the N-lobe, which serves as an allosteric hotspot for most protein kinases. This helix is also responsible for stabilizing a salt bridge between the bound nucleotide substrate and a Lys from β3. In inactive kinases the C-helix is often in an "out" or inactive conformation or disordered. (From now on, I will refer to the "nucleotide substrate" as a "cofactor" for simplification.) Embedded within the kinase fold is a functional intrinsically disordered region (IDR), the activation loop. The activation segment starts with the DFG motif and ends with the APE motif.These two flexible sequence motifs are conserved among kinases and have unique functions. The DFG motif lies between the two lobes, right below the C-helix and the Glycine-rich loop (G-loop).Embedded in the DFG motif is another R-spine residue, which couples directly with the C-Helix Rspine residue and the catalytic loop R-spine residue. Many kinases display two conformations: DFG-in and DFG-out (Hari et al., 2013). In the DFG-out conformation the R-spine is disassembled and in many cases the Phe side chain of this motif faces towards the G-loop instead of the C-helix.The DFG-out conformation is thus incompatible with ATP cofactor binding. Interplay between the DFG motif and the C-Helix in/out is highly regulated and defines the switch mechanism that leads to kinase activation. Both motifs are flexible and must be aligned.The APE motif precedes the F-helix in the C-lobe. Although it serves as a stable anchor for the flexible activation loop (AL), multiple crystal structures captured inter-molecularly bound APE motifs in kinase homodimers (Marcotte et al., 2017). In extreme cases, the APE motif can be disordered (Malakhova et al., 2008). These findings suggest that the APE motif is dynamically anchored to the C-lobe. The activation loop between the DFG and the APE motifs is usually 20-30 residues long and is often partially disordered in inactive structures. Although the activation loop is highly divergent, it usually contains at least one phosphorylation site, which orders and stabilizes the activation loop and is cooperatively coordinated by an Arg from the catalytic HRD motif (from the C-lobe), a basic residue in the activation loop, and a basic residue from the C-helix (from the Nlobe).Different kinase families developed orthogonal strategies to regulate the core kinase domain.This thesis focuses on RSK1, a tandem kinase, which includes an AGC-and a CaMK-type domains.Apparently, these two families are probably the most complicatedly regulated kinase families, which mainly organized around their C-terminal extensions.