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bioRxiv · 10.1101/2022.02.18.481032

Activation and inhibition of the C-terminal kinase domain of p90 ribosomal S6 kinases

Fruergaard, M. U.·Nielsen, C. J. F.·Rosada, C.·Iversen, L.·Andersen, J. L.·Nissen, P.
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Abstract

The p90 ribosomal S6 kinases (RSKs) contain two distinct catalytic kinase domains, the N-terminal and C-terminal kinase domains (NTKD and CTKD, respectively). The activation of CTKD is regulated by phosphorylation by extracellular signal-regulated kinase (ERK1/2) and an autoinhibitory L helix. Through a mutational series in vitro of the RSK CTKDs, we found a complex mechanism lifting autoinhibition that led us to design constitutively active RSK CTKDs. These are based on a phosphomimetic mutation and a C-terminal truncation (e.g. RSK2 T577E D694*) where a high activity in absence of ERK phosphorylation is obtained. Using these constructs, we characterize IC50 values of ATP-competitive inhibitors and provide a setup for determining specificity constants (kinact/Ki) of covalent CTKD inhibitors.

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Fruergaard, M. U., Nielsen, C. J. F., Rosada, C., Iversen, L., Andersen, J. L., Nissen, P.. 2022-02-19. Activation and inhibition of the C-terminal kinase domain of p90 ribosomal S6 kinases. https://doi.org/10.1101/2022.02.18.481032

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