Activation of Protein Kinase PKR Requires Dimerization-induced cis-Phosphorylation within the Activation Loop
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Authors: | Madhusudan Dey Brian Rick Mann Ashish Anshu M. Amin-ul Mannan |
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Affiliation: | From the Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211 |
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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 (PKRT446∼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∼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. |
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Keywords: | Phosphorylation, Protein Kinase RNA (PKR), Protein Kinases, Protein Phosphorylation, Stress Response, Activation Loop, eIF2α |
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