Protein kinase GCN2 is a multidomain protein that contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic moiety. Previous studies have shown that in response to histidine starvation, GCN2 phosphorylates eukaryotic initiation factor 2 (eIF-2), to induce the translational expression of GCN4, a transcriptional activator of genes subject to the general amino acid control. It was proposed that the synthetase-related sequences of GCN2 stimulate the activity of the kinase by interacting directly with uncharged tRNA that accumulates during amino acid limitation. In addition to histidine starvation, expression of GCN4 is also regulated by a number of other amino acid limitations. Questions that we posed in this report are whether uncharged tRNA is the most direct regulator of GCN2 and whether the function of this kinase is required to recognize each of the different amino acid starvation signals. We show that GCN2 phosphorylation of eIF-2, and the resulting general amino acid control pathway, is stimulated in response to starvation for each of several different amino acids, in addition to histidine limitation. Cells containing a defective aminoacyltRNA synthetase also stimulated GCN2 phosphorylation of eIF-2 in the absence of amino acid starvation, indicating that uncharged tRNA levels are the most direct regulator of GCN2 kinase. Using a Northwestern blot (RNA binding) assay, we show that uncharged tRNA can bind to the synthetase-related domain of GCN2. Mutations in the motif 2 sequence conserved among class II synthetases, including histidyl-tRNA synthetases, impair the ability of this synthetase-related domain to bind tRNA and abolish GCN2 phosphorylation of eIF-2 required to stimulate the general amino acid control response. These in vivo and in vitro experiments indicate that synthetase-related sequences regulate GCN2 kinase function by monitoring the levels of multiple uncharged tRNAs that accumulate during amino acid limitations.

In yeast, starvation for amino acids stimulates GCN2 phosphorylation of the ␣ subunit of eukaryotic initiation factor-2 (eIF-2). Phosphorylation of eIF-2␣ induces the translational expression of GCN4, a transcriptional activator of the general amino acid control pathway. It has been proposed that GCN2 sequences containing homology to histidyl-tRNA synthetases (HisRS) bind uncharged tRNA that accumulate during amino acid limitation and stimulate the activity of GCN2 kinase. In this report we address whether the HisRS-related sequences are required for GCN2 phosphorylation of eIF-2␣ in an in vitro assay. To measure the activity of GCN2 kinase in cellular extracts, we expressed and purified a truncated form of yeast eIF-2␣. Phosphorylation of the recombinant eIF-2␣ substrate was dependent on both GCN2 kinase activity and the eIF-2␣ phosphorylation site, serine 51. Mutations in the HisRS-related domain of GCN2, which have been shown to block phosphorylation of eIF-2␣ in vivo and the subsequent stimulation of the general control pathway, also greatly reduced eIF-2␣ phosphorylation in the in vitro assay. These results indicate that the HisRS-related sequences are required for activation of GCN2 kinase function.A family of serine/threonine protein kinases regulate protein synthesis by phosphorylation of the ␣ subunit of eukaryotic initiation factor-2 (eIF-2) 1 (1-4). In mammals, two different protein kinases are known to phosphorylate the regulated site in eIF-2␣, serine 51. One is the RNA-dependent protein kinase (PKR), which is a component of the antiviral defense mechanism mediated by interferon (3-5) and is thought to function as a suppressor of cell proliferation and tumorigenesis (6 -8). The second protein kinase, heme-regulated inhibitor, is found predominately in reticulocytes and bone marrow and is activated by reduced heme levels or heat shock (9). Phosphorylation of mammalian eIF-2␣ inhibits general protein synthesis by impairing the conversion of eIF-2-GDP to eIF-2-GTP, a nucleotide exchange that is required for each round of translation initiation (1, 2, 10). In yeast Saccharomyces cerevisiae, a third eIF-2␣ kinase, GCN2 (general control nonderepressible), participates in the regulation of gene-specific translation (4, 11).In yeast, starvation for any one of several different amino acids enhances the translation of GCN4, a transcriptional activator of more than 30 genes involved in the biosynthesis of amino acids (12). Control of GCN4 translation involves four short upstream open reading frames located in the 5Ј-untranslated region of the GCN4 mRNA. In cells not limiting for amino acids, the upstream open reading frames block translational initiation of the GCN4 coding sequences. During amino acid starvation, GCN2 phosphorylation of eIF-2␣ is thought to lead to lowered eIF-2-GTP levels, thus reducing the inhibitory effects of the upstream open reading frames and allowing for increased GCN4 translation (11,13,14). How does amino acid starvation stimulate the kinase function of GCN2? The carboxyl-terminal po...

Protein kinase PKR is activated in mammalian cells during viral infection, leading to phosphorylation of the ␣ subunit of eukaryotic initiation factor-2 (eIF-2␣) and inhibition of protein synthesis. This antiviral response is thought to be mediated by association of doublestranded RNA (ds-RNA), a by-product of viral replication, with two ds-RNA-binding domains (DRBDs) located in the amino terminus of PKR. Recent studies have observed that expression of mammalian PKR in yeast leads to a slow growth phenotype due to hyperphosphorylation of eIF-2␣. In this report, we observed that while DRBD sequences are required for PKR to function in the yeast model system, these sequences are not required for in vitro phosphorylation of eIF-2␣. To explain this apparent contradiction, we proposed that these sequences are required to target the kinase to the translation machinery. Using sucrose gradient sedimentation, we found that wild-type PKR was associated with ribosomes, specifically with 40 S particles. Deletions or residue substitutions in the DRBD sequences blocked kinase interaction with ribosomes. These results indicate that in addition to mediating ds-RNA control of PKR, the DRBD sequences facilitate PKR association with ribosomes. Targeting to ribosomes may enhance in vivo phosphorylation of eIF-2␣, by providing PKR access to its substrate.

A family of protein kinases regulate translation initiation in response to cellular stresses by phosphorylation of eukaryotic initiation factor-2 (eIF-2). One family member from yeast, GCN2, contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic domain. It is thought that uncharged tRNA accumulating during amino acid starvation binds to the synthetase-related sequences and stimulates phosphorylation of the ␣ subunit of eIF-2. In this report, we define another domain in GCN2 that functions to target the kinase to ribosomes. A truncated version of GCN2 containing only amino acid residues 1467 to 1590 can independently associate with the translational machinery. Interestingly, this region of GCN2 shares sequence similarities with the core of the double-stranded RNA-binding domain (DRBD). Substitutions of the lysine residues conserved among DRBD sequences block association of GCN2 with ribosomes and impaired the ability of the kinase to stimulate translational control in response to amino acid limitation. Additionally, as found for other DRBD sequences, recombinant protein containing GCN2 residues 1467-1590 can bind doublestranded RNA in vitro, suggesting that interaction with rRNA mediates ribosome targeting. These results indicate that appropriate ribosome localization of the kinase is an obligate step in the mechanism leading to translational control by GCN2.

Background: Thrombus formation involves coagulation proteins and platelets. The latter, referred to as platelet-mediated thrombogenesis, is predominant in arterial circulation. Platelet thrombogenesis follows vascular injury when extracellular von Willebrand factor (VWF) binds via its A3 domain to exposed collagen, and the free VWF A1 domain binds to platelet glycoprotein Ib (GPIb). Objectives:To characterize the antiplatelet/antithrombotic activity of the pegylated VWF antagonist aptamer BT200 and identify the aptamer VWF binding site.Methods: BT100 is an optimized aptamer synthesized by solid-phase chemistry and pegylated (BT200) by standard conjugation chemistry. The affinity of BT200 for purified human VWF was evaluated as was VWF inhibition in monkey and human plasma.Efficacy of BT200 was assessed in the monkey FeCl 3 femoral artery thrombosis model. Results: BT200 bound human VWF at an EC 50 of 5.0 nmol/L and inhibited VWF A1 domain activity in monkey and human plasma with mean IC 50 values of 183 and 70 nmol/L. BT200 administration to cynomolgus monkeys caused a time-dependent and dose-dependent effect on VWF A1 domain activity and inhibited platelet function as measured by collagen adenosine diphosphate closure time in the platelet function analyzer. BT200 demonstrated a bioavailability of ≥77% and exhibited a half-life of >100 hours after subcutaneous injection. The treatment effectively prevented arterial occlusion in an FeCl 3 -induced thrombosis model in monkeys. Conclusions: BT200 has shown promising inhibition of human VWF in vitro and prevented arterial occlusion in non-human primates. These data including a long half-life after subcutaneous injections provide a strong rationale for ongoing clinical development of BT200.