The double-stranded RNA-activated protein kinase PKR is dispensable for regulation of translation initiation in response to either calcium mobilization from the endoplasmic reticulum or essential amino acid starvation

The alpha -subunit of eukaryotic initiation factor eIF2 is a preferred subs trate for the double-stranded RNA-activated protein kinase, PKR. Phosphoryl ation of eIF2 alpha converts the factor from a substrate into a competitive inhibitor of the guanine nucleotide exchange factor, eIF2B, leading to a d ecline in mRNA translation. Early studies provided evidence implicating PKR as the kinase that phosphorylates eIF2 alpha under conditions of cell stre ss such as the accumulation of misfolded proteins in the lumen of the endop lasmic reticulum, i.e., the unfolded protein response (UPR). However, the r ecent identification of a trans-microsomal membrane eIF2 alpha kinase, term ed PEK or PERK, suggests that this kinase, and not PKR, might be the kinase that is activated by misfolded protein accumulation. Similarly, genetic st udies in yeast provide compelling evidence that a kinase termed GCN2 phosph orylates eIF2 alpha in response to amino acid deprivation. However, no dire ct evidence showing activation of the mammalian homologue of GCN2 by amino acid deprivation has been reported. In the present study, we find that in f ibroblasts treated with agents that promote the UPR, protein synthesis is i nhibited as a result of a decrease in eIF2B activity. Furthermore, the redu ction in eIF2B activity is associated with enhanced phosphorylation of eIF2 alpha. Importantly, the magnitude of the change in each parameter is ident ical in wildtype cells and in fibroblasts containing a chromosomal deletion in the PHR gene (PKR-KO cells). In a similar manner, we find that during a mino acid deprivation the inhibition of protein synthesis and extent of inc rease in eIF2 alpha phosphorylation are identical in wildtype and PKR-KO ce lls. Overall, the results show that PKR is not required for increased eIF2 alpha phosphorylation or inhibition of protein synthesis under conditions p romoting the UPR or in response to amino acid deprivation. (C) 2001 Academi c Press.