trans-Autophosphorylation by the Isolated Kinase Domain Is Not Sufficient for Dimerization or Activation of the dsRNA-Activated Protein Kinase PKR
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- 作者：Shiyong Wu and Randal J. Kaufman
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：August 31, 2004
- 年：2004
- 卷：43
- 期：34
- 页码：11027 - 11034
- 全文大小：161K
- 年卷期：v.43,no.34(August 31, 2004)
- ISSN：1520-4995
文摘
The double-stranded (ds) RNA-activated protein kinase PKR phosphorylates the 
-subunit ofthe eukaryotic initiation factor 2 (eIF2) and inhibits translation initiation. PKR contains two dsRNAbinding domains in its amino terminus and a kinase domain in its carboxy terminus. dsRNA bindingactivates PKR from a latent state by inducing dimerization and trans-autophosphorylation. Recent studiesshow that PKR is also activated by caspase cleavage to remove the inhibitory dsRNA binding domains.In this report, we show that the isolated kinase domain of PKR is a constitutively active monomerickinase that has an activity similar to that of wild-type PKR. We used a solid-phase kinase assay systemto show that PKR does not transfer its own phosphate to either PKR or eIF2 but rather uses the
-phosphate from ATP. In addition, the isolated autophosphorylated kinase domain of PKR phosphorylatedintact monomeric PKR in trans in a reaction that did not require dsRNA binding. However, this trans-phosphorylation did not occur at the critical Thr446/451 sites and was not sufficient to induce dimerizationand/or activation of PKR. The results show that dsRNA binding domains of PKR are not only requiredfor dimerization of PKR but also required for phosphorylation of Thr446/451 sites of PKR. The resultsimply that even though the isolated kinase domain of PKR phosphorylates intact PKR and eIF2, it isunable to activate PKR.
-subunit ofthe eukaryotic initiation factor 2 (eIF2) and inhibits translation initiation. PKR contains two dsRNAbinding domains in its amino terminus and a kinase domain in its carboxy terminus. dsRNA bindingactivates PKR from a latent state by inducing dimerization and trans-autophosphorylation. Recent studiesshow that PKR is also activated by caspase cleavage to remove the inhibitory dsRNA binding domains.In this report, we show that the isolated kinase domain of PKR is a constitutively active monomerickinase that has an activity similar to that of wild-type PKR. We used a solid-phase kinase assay systemto show that PKR does not transfer its own phosphate to either PKR or eIF2 but rather uses the-phosphate from ATP. In addition, the isolated autophosphorylated kinase domain of PKR phosphorylatedintact monomeric PKR in trans in a reaction that did not require dsRNA binding. However, this trans-phosphorylation did not occur at the critical Thr446/451 sites and was not sufficient to induce dimerizationand/or activation of PKR. The results show that dsRNA binding domains of PKR are not only requiredfor dimerization of PKR but also required for phosphorylation of Thr446/451 sites of PKR. The resultsimply that even though the isolated kinase domain of PKR phosphorylates intact PKR and eIF2, it isunable to activate PKR.