Abstract
Phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2) is a well characterized mechanism regulating protein synthesis. Viral and cellular proteins have been identified that regulate the activity of the eIF-2alpha kinases. The regulatory protein, K3L, from vaccinia virus is homologous to the amino terminus of eIF-2alpha and is thought to inhibit the activity of the double-stranded RNA-dependent kinase suppressing the antiviral mechanism mediated by this kinase. We investigated whether K3L can inhibit the activity of the yeast eIF-2alpha kinase GCN2. Expression of K3L protein in yeast reduced the level of eIF-2alpha phosphorylation by GCN2 and blocked the stimulation of the general amino acid control pathway in response to starvation conditions. Accompanying in vitro studies showed that recombinant K3L protein reduced GCN2 autophosphorylation and phosphorylation eIF-2alpha. In agreement with the hypothesis that K3L inhibits eIF-2alpha kinases by functioning as a pseudosubstrate, we observed that K3L directly interacted with the kinase catalytic domain of GCN2. Together, these results indicate that K3L is a specific inhibitor of eIF-2alpha kinases from mammals and yeast and suggest that the kinases contain common structural features important for recognition of their substrate eIF-2alpha.
Highlights
The eukaryotic initiation factor-2 (eIF-2)␣ kinases are members of a distinct family of eukaryotic protein kinases
While GCN2 autophosphorylation was readily detected in the sample with wild-type GCN2, phosphorylated GCN2 was greatly reduced in the gcn2-K559R reaction and completely absent in the ⌬gcn2 sample
In this report we show that K3L protein expressed in yeast can reduce the level of GCN2 phosphorylation of eIF-2␣, resulting in an inability of the cell to stimulate the general control pathway in response to starvation for amino acids
Summary
The eIF-2␣ kinases are members of a distinct family of eukaryotic protein kinases. Pairwise sequence comparisons beinteracted with the kinase catalytic domain of GCN2. tween the kinase domains of GCN2, PKR, and HRI revealed. Tween the kinase domains of GCN2, PKR, and HRI revealed. There do not appear to be any sequence similarities among the eIF-2␣ kinases Both PKR and GCN2 kinases are thought to be regulated by RNA molecules. Composed of three nonidentical subunits, eIF-2 complexes with Met-tRNAiMet and GTP, and participates in the ribosomal recognition of the start codon [1,2,3,4]. During this initiation process, the GTP complexed with eIF-2 is hydrolyzed to GDP, and eIF-21⁄7GDP is released from the ribosome. Three protein kinases that phosphorylate the regulated site of eIF-2␣ have been characterized and their genes cloned [5, 6]
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