Fig. 5: PACT’s inhibition of PKR involves weak but direct and specific protein-protein interactions.
From: PACT prevents aberrant activation of PKR by endogenous dsRNA without sequestration
A In vitro PKR kinase assay using 112 bp dsRNA (0.25 ng/μl) in the presence of 0, 20, 50, 100, 250 nM PACT, TRBP, RIG-I ∆CARDs, ADAR1 DRBDs. Right: SDS-PAGE showing purity of the proteins. The results were reproduced in 3 independent replicates. B dsRNA binding curve of PACT, TRBP, RIG-I ∆CARDs, ADAR1 DRBDs, derived from the native gel-shift assay in Supplementary Fig. 10A monitoring 112 bp dsRNA binding. For curve fitting, Hill equation was used with the assumption of 8 protein binding sites per 112 bp dsRNA (see Methods). C Pulldown of PACT using PKR-GST or GST. All proteins were purified and treated with benzonase to remove potential nucleic acid contaminants. Catalytically dead K296R PKR was used. The results were reproduced in 3 independent replicates. D In vitro PKR kinase assay using an increasing concentration of PKR in the absence of dsRNA. Benzonase was added during the reaction (in addition to during purification) to ensure that the observed PKR activity is RNA-independent. The results were reproduced in 2 independent replicates. E RNA-independent PKR kinase assay with 1 μM PKR in the presence of 0, 1.25, 2.5, 5 μM PACT, GST, MBP. The results were reproduced in 3 independent replicates. F RNA-independent PKR kinase assay with 1 μM PKR in the presence of 0, 1.25, 2.5, 5 μM PACT wild type, ∆D1, ∆D2; and 0, 2.5, 5 μM ∆D3. G Correlation between the levels of PKR inhibition and PACT phosphorylation from F. Values are mean ( ± SD) of 2 biological replicates. H Pulldown of PKR or PKR∆DRBDs using PACT D1D2-GST or GST. Catalytically dead K296R mutant was used for both PKR and PKR∆DRBDs. The results were reproduced in 3 independent replicates. I RNA-independent PKR kinase assay with 1 μM PKR or 2 μM PKR∆DRBDs in the presence of 0, 1.25, 2.5, 5 μM PACT. Higher concentration of PKR∆DRBDs was due to its lower activity. The results were reproduced in 3 independent replicates. Source data are provided as a Source Data file.
