In an effort to directly test the suspected role of cAMP dependent protein kinase (PK) in mediating the effects of cAMP derivatives on tyrosine aminotransferase (TAT) synthesis in cultured H35 cells, microinjection of PK subunits and the PK inhibitor into H35 cells will be carried out by fusion with loaded RBC hosts (RBCG). PK subunits (C and R) will be purified from beef heart and the inhibitor from skeletal muscle. The proteins will be loaded into RBCG by dialysis against hypotonic reverse PBS. Fusion is accomplished by 22% PEG and H35 cells are allowed to recover for several hr prior to harvest and assay. Microinjected PK-C has been found to increase TAT synthesis and the inhibitor blocked the effects of cAMP but not those of dexamethasone, another TAT inducer. Affinity chromatography with PK-C linked to a solid support will be used to isolate potential substrates for PK from H35 cells and rat liver. The substrates will be tested for cAMP dependent phosphorylation by PK using 2D gels for display of phosphorylated and unphosphorylated forms. The crude substrate preparation will be phosphorylated with ATP-Gamma-S and loaded into RBCG and microinjected into H35 cells. If PK mediates cAMP action on TAT synthesis, the phosphorylated substrate involved should induce TAT. This approach will be used as a bioassay for the putative substrate which controls TAT synthesis that will allow us to purify this component. Once identified, further studies on the function of this component should produce significant clues as to the molecular mechanism by which TAT synthesis is regulated by cAMP.
