Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 289, Issue 9, Pages 5747-5757Publisher
ELSEVIER
DOI: 10.1074/jbc.M113.527796
Keywords
Phosphorylation; Protein Kinase RNA (PKR); Protein Kinases; Protein Phosphorylation; Stress Response; Activation Loop; eIF2
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Funding
- National Institutes of Health [1R15GM101575]
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Background: PKR regulates many biological processes including stress response. Results: An activation-loop-phosphorylation bypass mutant of PKR functions without a key dimer-interface residue. The PKR kinase domain together with a kinase-dead partner, but not alone, activates the catalytic function. Conclusion: Activation loop phosphorylation occurs in cis following dimerization. Significance: PKR autophosphorylates in the activation loop in cis to initiate the antiviral signaling cascade. Protein kinase R (PKR) functions in a plethora of cellular processes, including viral and cellular stress responses, by phosphorylating the translation initiation factor eIF2. 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 (PKRT446 approximate to 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 PKRT446 approximate to P-bypass mutant was able to phosphorylate eIF2 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.
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