Activation of RSK by phosphomimetic substitution in the activation loop is prevented by structural constraints

Desiana Somale, Giovanna Di Nardo, Laura di Blasio, Alberto Puliafito, Marianela Vara-Messler, Giulia Chiaverina, Miriam Palmiero, Valentina Monica, Gianfranco Gilardi, Luca Primo, Paolo Armando Gagliardi

Research output: Contribution to journalArticlepeer-review


The activation of the majority of AGC kinases is regulated by two phosphorylation events on two conserved serine/threonine residues located on the activation loop and on the hydrophobic motif, respectively. In AGC kinase family, phosphomimetic substitutions with aspartate or glutamate, leading to constitutive activation, have frequently occurred at the hydrophobic motif site. On the contrary, phosphomimetic substitutions in the activation loop are absent across the evolution of AGC kinases. This observation is explained by the failure of aspartate and glutamate to mimic phosphorylatable serine/threonine in this regulatory site. By detailed 3D structural simulations of RSK2 and further biochemical evaluation in cells, we show that the phosphomimetic residue on the activation loop fails to form a critical salt bridge with R114, necessary to reorient the αC-helix and to activate the protein. By a phylogenetic analysis, we point at a possible coevolution of a phosphorylatable activation loop and the presence of a conserved positively charged amino acid on the αC-helix. In sum, our analysis leads to the unfeasibility of phosphomimetic substitution in the activation loop of RSK and, at the same time, highlights the peculiar structural role of activation loop phosphorylation.

Original languageEnglish
Pages (from-to)591
JournalSci. Rep.
Issue number1
Publication statusPublished - Jan 17 2020


  • Amino Acid Motifs
  • Amino Acid Substitution
  • Enzyme Activation
  • Evolution, Molecular
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Molecular Mimicry
  • Phosphorylation
  • Phylogeny
  • Protein Structure, Secondary
  • Ribosomal Protein S6 Kinases, 90-kDa/chemistry


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