Abstract
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.
Highlights
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
Phosphorylation on Ser or Thr in the activation loop (AL) and in the hydrophobic motif (HM) of AGC kinases represents a critical event leading to their activation
To investigate whether these key serine/threonine residues have been replaced by phosphomimetic amino acids (Asp or Glu) during protein kinase evolution, we aligned the protein sequences of all human AGC kinases. 60 out of 61 AGC kinases show a conserved AL sequence and 53 out of 61 AGC kinases show a HM sequence, in some kinases, including PKA, there is only a truncated form of it (Fig. 1A)
Summary
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. The phosphate group added on the AL form a complex set of salt bridges with basic amino acid groups, that in PKA are respectively: R165 in the catalytic loop, H87 in the αC-helix and K189 in the AL, just after the DFG motif[7,8] By connecting these residues, the phosphorylation of the AL promotes the transition in a more ordered conformation, the stabilization of the two lobes in the closed/active conformation and the assembly of a key hydrophobic core, defined R-spine[9,10,11]. Phosphorylation can affect the activity and the function of proteins in different ways: (1) by favoring the disordered-ordered transitions; (2) by allosteric regulation at the level of tertiary and quaternary structures; (3) by changing the recognition properties of protein binding sites; (4) by regulating post-translational modifications[19]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
