Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2 alpha kinase

In eukaryotic cells, protein synthesis is regulated in response to various environmental stresses by phosphorylating the a subunit of the eukaryotic i nitiation factor 2 (eIF2 alpha), Three different eIF2 alpha kinases have be en identified in mammalian cells, the heme-regulated inhibitor (HRI), the i nterferon-inducible RNA-dependent kinase (PKR) and the endoplasmic reticulu m-resident kinase (PERK. A fourth eIF2 alpha kinase, termed GCN2, was previ ously characterized from Saccharomyces cerevisiae, Drosophila melanogaster and Neurospora crassa. Here we describe the cloning of a mouse GCN2 cDNA (M GCN2), which represents the first mammalian GCN2 homolog. MGCN2 has a conse rved motif, N-terminal to the kinase subdomain V, and a large insert of 139 amino acids located between subdomains IV and V that are characteristic of the known eIF2 alpha kinases. Furthermore, MGCN2 contains a class II amino acyl-tRNA synthetase domain and a degenerate kinase segment, downstream and upstream of the eIF2 alpha kinase domain, respectively, and both are singu lar features of GCN2 protein kinases. MGCN2 mRNA is expressed as a single m essage of approximate to 5.5 kb in a wide range of different tissues, with the highest levels in the liver and the brain. Specific polyclonal anti-(MG CN2) immunoprecipitated an eIF2 alpha kinase activity and recognized a 190 kDa phosphoprotein in Western blots from either mouse liver or MGCN2-transf ected 293 cell extracts. Interestingly, serum starvation increased eIF2 alp ha phosphorylation in MGCN2- transfected human 293T cells. This finding pro vides evidence that GCN2 is the unique eIF2 alpha kinase present in all euk aryotes from yeast to mammals and underscores the role of MGCN2 kinase in t ranslational control and its potential physiological significance.