title: 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 homodimerization of its kinase and RNA-binding domains, and autophosphorylation at the residue Thr-446 in a flexible loop called the activation loop. We investigated the interdependence between dimerization and Thr-446 autophosphorylation using the yeast Saccharomyces cerevisiae model system. We showed that an engineered PKR that bypassed the need for Thr-446 autophosphorylation (PKR(T446∼P)-bypass mutant) could function without a key residue (Asp-266 or Tyr-323) that is essential for PKR dimerization, suggesting that dimerization precedes and stimulates activation loop autophosphorylation. We also showed that the PKR(T446∼P)-bypass mutant was able to phosphorylate eIF2α even without its RNA-binding domains. These two significant findings reveal that PKR dimerization and activation loop autophosphorylation are mutually exclusive yet interdependent processes. Also, we provide evidence that Thr-446 autophosphorylation during PKR activation occurs in a cis mechanism following dimerization.
authors: From the Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211.
authors: Madhusudan
authors: M
authors: Dey
authors: Brian Rick
authors: BR
authors: Mann
authors: Ashish
authors: A
authors: Anshu
authors: M Amin-ul
authors: MA
authors: Mannan
mesh_terms: D019943:Amino Acid Substitution; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010766:Phosphorylation; D055503:Protein Multimerization / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015852:Eukaryotic Initiation Factor-2; D029701:Saccharomyces cerevisiae Proteins; D019892:eIF-2 Kinase
keywords: Activation Loop; Phosphorylation; Protein Kinase RNA (PKR); Protein Kinases; Protein Phosphorylation; Stress Response; eIF2α