Purification and characterization of the functional catalytic domain of PKR-like endoplasmic reticulum kinase expressed in escherichia coli

PKR-like endoplasmic reticulum (ER) kinase (PERK) is a type I transmembrane ER-resident protein containing a cytoplasmic catalytic domain with a Ser/Thr kinase activity, which is most closely related to the eukaryotic translation initiation factor-α (eIF2α) kinase PKR involved in the antiviral defense pathway by interferon. We cloned and expressed the PERK C-terminal kinase domain (cPERK) in Escherichia coli. Like PERK activation in cells under ER stress, wild-type cPERK underwent autophosphorylation when overexpressed in E. coli, whereas the cPERK(K621M) with a methionine substitution for the lysine at amino acid 621 lost the autophosphorylation activity. The activated form cPERK which was purified to near homogeneity, formed an oligomer and was able to trans-phosphorylate specifically its cellular substrate eIF2α. Two-dimensional phosphoamino acids analysis revealed that phosphorylation of cPERK occurs at the Ser and Thr residues. The ftinctionally active recombinant cPERK, and its inactive mutant should be useful for the analysis of biochemical functions of PERK and for the determination of their three-dimensional structures.