FIGURE 3.
The Thr-446 phosphorylation does not occur in trans mechanism. A, in vitro analysis of PKR autophosphorylation. Purified PKR-K296R and GST-PKRKD fusion proteins were incubated in a kinase reaction buffer. The reaction products were then separated by SDS-PAGE and subjected to Western blot analyses using phosphospecific antibody of Thr-446 followed by a polyclonal antibody of PKR. B, analysis of Thr-446 phosphorylation. GST-PKRKD co-expressed with WT PKR or PKR-K296R mutant in yeast. Whole cell extracts were prepared from these cells and subjected to Western blot analysis using a Thr-446 phosphospecific antibody of PKR (T446∼P). The membrane was stripped and re-probed with a polyclonal antibody of PKR.