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

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 alpha 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 ac-tivity in absence of ERK phosphorylation is obtained. Using these constructs, we characterize IC50 values of ATP-competitive in-hibitors and provide a setup for determining specificity constants (kinact/Ki) of covalent CTKD inhibitors.

Automated summary

The p90 ribosomal S6 kinases (RSKs) consist of N-terminal and C-terminal kinase domains (NTKD and CTKD). CTKD activation is regulated by phosphorylation by extracellular signal-regulated kinase (ERK1/2) and an autoinhibitory alpha L helix. Through mutational experiments, a mechanism of lifting autoinhibition was discovered, leading to the design of constitutively active RSK CTKDs. These findings provide insights into the characterization of inhibitors and determination of specificity constants of covalent CTKD inhibitors.