C-spine mutations of protein kinase C and Akt as a novel generalizable approach to create stable pseudokinases

Abstract

Protein kinases function not only through their catalytic phospho-transfer activity but also by non-catalytic scaffold mechanisms. Introduction of mutations to inactivate catalysis provides a tool to differentiate between the two; however, kinase-inactivating mutations may alter the structure of the kinase domain and perturb scaffold functions. Here, we developed a strategy that prevents ATP binding, thereby preventing catalysis, while stabilizing the active conformation of the kinase domain. This approach leverages the structural role of ATP in assembling the catalytic spine (C-spine), a hydrophobic core essential for the active conformation. Specifically, we substituted Val or Ala residues proximal to the binding position of the adenosine ring of ATP with Phe in three protein kinase C isozymes (PKCβII, γ, θ) and Akt1. Structural modeling suggests that Phe substitutions at these positions are a surrogate for the adenosine ring of ATP to assemble the C-spine. Live-cell imaging using genetically encoded PKC and Akt activity reporters reveals that C-spine mutations abolish kinase activity. Furthermore, phosphorylation of the hydrophobic motif, an autophosphorylation site, is abolished in C-spine mutants of PKC family members and reduced in C-spine mutants of Akt1, independent of epidermal growth factor (EGF) stimulation. In PKCβII, these C-spine mutations accelerate plasma membrane translocation, consistent with impaired autoinhibition due to the lack of hydrophobic motif phosphorylation. Despite adopting reduced autoinhibition, turnover experiments with PKCθ reveal C-spine mutants do not impair the stability of the full-length PKC. The generation of pseudokinases by C-spine mutations provides a generalizable strategy for elucidating non-catalytic kinase functions.

Keywords: Akt; C-spine; kinase-dead; protein kinase C; pseudokinase.