Translational control of protein synthesis is recognized as an important aspect of cellular regulation. The purpose of this project is to investigate a recently-discovered process whereby the translational apparatus may be regulated by intracellular Ca. The system consists of the enzyme calmodulin-dependent protein kinase III (CaM-PK III) and its specific substrate elongation factor-2 (EF-2). Phosphorylation of EF-2 at a single threonine residue abolishes the ability of the factor to catalyze peptide elongation in a in vitro system.
The specific aims of this proposal are first, to determine the function(s) of EF-2 that are affected by phosphorylation. Changes in guanine nucleotide binding and hydrolysis, ribosome-binding and participation in the translocation reaction will be measured. Secondly, the enzyme CaM- PK III, a new member of the CaM-kinase family, will be thoroughly purified and characterized. After purification of the enzyme, subunit structure, kinetic and autophosphorylation behavior will be assessed. Determinants of substrate specificity will be evaluated using synthetic peptide analogs of the known sequence around the phosphorylation site(s) of EF-2. Microsequencing of enzyme subunits will be performed in order to derive oligonucleotide probes that will be used in probing cDNA libraries for CaM-PK III cDNA's. These cDNA's will be sequenced to provide structural information on the enzyme and to compare it with other CaM-kinases. Thirdly, a study will be made of the correlation between the phosphorylation of EF-2 and the regulation of proteins synthesis in intact cells and broken-cell preparations. One system is the Ca ionophore-treated rabbit reticulocyte and Ca-treated reticulocyte lysates. The second system is the mitogen-stimulated quiescent human fibroblast. In both of these systems substantial preliminary data has been obtained suggesting that EF-2 is phosphorylated under physphorylation will be estimated by immunoprecipitation and elongation activity will be determined by measurement of ribosomal transit times and polysomal profiles. Finally, the regulation of the CaM-PK III/EF-2 system at the level of the enzyme will be analyzed. Previous data indicates that in certain cell types Cam-PK III can be """"""""down-regulated"""""""" by treatment with growth factors or agents which elevate cAMP-dependent protein kinase activity in the cells. The distribution of this phenomenon in different cell types will be investigated. The fate of CaM-PK III subunits in down-regulated cells will be followed using antibodies to the enzyme. The possibility of phophorylation or other secondary modification of the enzyme will be evaluated. The results from this study will amplify our knowledge of the regulation of protein synthesis and offer new insights into the mechanisms whereby Ca exerts such profound effects on cell behavior.
