Chen 9405781 The objective of this proposal is to elucidate the mechanisms of regulation of heme-regulated eIF-2a kinase (HRI) at the molecular structural level. HRI is a potent inhibitor of protein synthesis. It is activated in heme-deficiency of reticulocytes. HRI is expressed in erythroid cells and the physiological role of HRI is to maintain the balanced synthesis of heme and globin chains. The specific aims are (1) Regulation of HRI by phosphorylation; (2) Molecular mechanisms in the regulation of HRI by heme; (3) Determination of HRI structure by X-ray crystallography. The methods to be employed are recombinant DNA techniques, polymerase chain reaction, synthesis of oligodeoxynucleodtides, high pressure liquid chromatography, amino acid microsequencing, in vitro transcription and translation, protein kinase assays, gel electrophoresis, deoxynucleotide sequencing, expression of HRI and other proteins using recombinant baculovirus, immunoprecipitation, Western Blot analysis, and x-ray crstallography. HRI is a key regulator of protein synthesis, a process which is essential for the regulation of cellular processes. %%% Protein synthesis is vital for cell growth, differentiation, viral infection, and stress responses. Regulation of protein synthesis by the phosphorylation of the a-subunit of eukaryotic initiation factor 2 (eIF-2a) is employed by a variety of cells under these conditions. Phosphorylation of eIF-2a results in the inhibition of protein synthesis globally. Two distinct eIF-2a kinases, which phosphorylate specifically the eIF-2a in mammalian cells, have been identified and molecularly cloned. The double-stranded RNA dependent eIF-2a kinase is induced by interferon and its activity is regulated by double-stranded RNA which is often introduced by viral infections. The heme-regulated eIF-2a kinase (HRI) is activated in heme-deficiency of immature erythroid cells and its activity is inhibited by heme. Our recent studies indicate that HRI is p resent in erythroid cells only. Since heme is the prothetic group of hemoglobin which is the principal protein in erythroid cells, the major physiological role of HRI is in the regulation of balanced synthesis of heme and hemoglobin which carries oxygens in our blood stream. This research project is focused on the structure and function study of HRI. We will study the molecular mechanism of regulation of HRI by heme and identify the heme-binding domain of HRI. Since activation of HRI is accompanied by the phosphorylation of HRI, regulation of HRI by phosphorylation will also be studied. For a more detailed understanding of the regulation of HRI, it is essential to obtain the overall structure of this kinase by X-ray crystallography. The crystal structure of HRI is not only important for our understanding of protein kinases, but also important for the understanding of heme-binding to enzymes. Since regulation of protein synthesis is closely related to oncogenic transformation, further structure-function study of HRI should advance our understanding of protein phosphorylation in the regulation of cellular processes, in particular cell growth and differentiation.
