Activation of Protein Kinase PKR Requires Dimerization-induced cis-phosphorylation within the Activation Loop

Abstract Protein kinase R (PKR) functions in a plethora of cellular processes, including viral and cellular stress responses, by phosphorylating the translation initiation factor eIF2α. The minimum requirements for PKR function are homo-dimerization of its kinase and RNA-binding domains, and autophosphorylation at the residue T446 in a flexible loop called the activation loop. We investigated the inter-dependence between dimerization and T446-autophosphorylation using the yeast Saccharomyces cerevisiae model system. We showed that an engineered PKR that bypassed the need for T446-autophopshorylation (PKRT446~P-bypass mutant) could function without a key residue (Asp-266 or Tyr-323) that is essential for the PKR dimerization, suggesting that dimerization precedes and stimulates activation loop autophosphorylation. We also showed that the PKRT446~P-bypass mutant was able to phosphorylate eIF2α even without its RNA-binding domains. These two significant findings reveal that the PKR dimerization and activation loop autophosphorylation are mutually exclusive yet interdependent process. Also, we provide evidence that T446-autophosphorylation during PKR activation occurs in a cis mechanism following dimerization.