HISRS-DOMAIN AND REGULATING GCN2 PROTEIN KINASE

Project: Research project

Description

The goal of the proposed research is to understand the mechanisms by
which eukaryotic cells regulate their metabolic pathways in response to
changes in growth conditions. In the model system Saccharomyces
cerevisiae, starvation for amino acids induces the GCN2 protein kinase
to phosphorylate the alpha-subunit of eukaryotic translation initiation
factor-2 (eIF-2). Phosphorylation of this factor stimulates the
translation of GCN4, a transcriptional activator of amino acid
biosynthetic genes. Previously, it was shown that the sequence of the
C-terminal region of GCN2 is homologous to the entire sequence of
histidyl-tRNA synthetase (HisRS) from S. cerevisiae and humans. Given
that aminoacyl-tRNA synthetases bind uncharged tRNA as a substrate and
distinguish between charged and uncharged tRNA, it was proposed that the
HisRS-related domain in GCN2 monitors the concentration of uncharged tRNA
in the cell and activates the adjacent protein kinase moiety under
starvation conditions when uncharged tRNA accumulates. A primary
objective of this proposal is to test in a purified system whether the
levels of uncharged tRNA regulate GCN2 phosphorylation of eIF-2-alpha. To test this model the following line of investigation is proposed.
First, which amino acids are monitored in vivo by GCN2. It is known that
GCN4 translation is stimulated in response to starvation for any one of
at least ten different amino acids. Is GCN2 required for each
limitation? Secondly, the interaction between different species of
uncharged tRNA and GCN2 protein kinase will be measured by in vitro
binding assays and the tRNAs shown to complex with GCN2 will be examined
further to determine if they kinetically enhance the phosphorylation of
eIF-2-alpha by GCN2. The final proposed experiments use genetic tools
available in this system to isolate and characterize mutations in GCN2
with altered substrate specificity. A better understanding of the regulation and substrate affinity of GCN2
will provide useful information about stress responses in mammalian
cells. It is known that eIF-2-alpha kinases related to GCN2 inhibit
protein synthesis in response to viral infection (double-stranded RNA
leads to activation of DAI kinase) and heme deprivation in reticulocytes
(activates HCR protein kinase).
StatusActive
Effective start/end date4/1/934/30/20

Funding

  • National Institutes of Health: $353,588.00
  • National Institutes of Health: $337,885.00
  • National Institutes of Health: $233,488.00
  • National Institutes of Health: $95,445.00
  • National Institutes of Health: $104,196.00
  • National Institutes of Health: $337,703.00
  • National Institutes of Health: $110,859.00
  • National Institutes of Health: $343,953.00
  • National Institutes of Health: $337,795.00
  • National Institutes of Health: $279,048.00
  • National Institutes of Health: $276,371.00
  • National Institutes of Health: $276,647.00
  • National Institutes of Health
  • National Institutes of Health: $116,099.00
  • National Institutes of Health: $243,485.00
  • National Institutes of Health: $388,795.00
  • National Institutes of Health: $343,929.00
  • National Institutes of Health: $240,469.00
  • National Institutes of Health: $334,232.00
  • National Institutes of Health: $279,147.00
  • National Institutes of Health
  • National Institutes of Health: $99,366.00
  • National Institutes of Health: $331,903.00
  • National Institutes of Health

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Physiological Stress
Phosphotransferases
Eukaryotic Initiation Factor-2
Protein Kinases
Phosphorylation
Transfer RNA
Starvation
Yeasts
Amino Acids
Gene Expression
Proteins
Genes
Histidine-tRNA Ligase
Heat-Shock Proteins
Peptide Initiation Factors
Neurodegenerative Diseases
Disease Progression
Amino Acyl-tRNA Synthetases
Neoplasms
Eukaryotic Initiation Factors

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)