Direct evidence that the protein kinase catalytic subunit mediates the effects of cAMP on tyrosine aminotransferase synthesis.
The effect of purified beef heart cAMP-dependent protein kinase catalytic subunit on tyrosine aminotransferase activity in intact cultured rat H35 hepatoma cells was directly tested by micro-injection using human red blood cell ghosts as vehicles. Although the micro-injection procedure itself produced temporary fluctuations in protein synthesis and in tyrosine aminotransferase activity in H35 cells, after a recovery period of 8-12 h, these parameters returned to normal in parallel with restoration of full inducibility of the aminotransferase by both 8-Br-cAMP and dexamethasone. Eight to sixteen hours after fusion of H35 cells with unloaded ghosts, ghosts loaded with bovine serum albumin or mock-loaded with the partially purified protein kinase catalytic subunit, no significant change in the activity of the aminotransferase was detected. In contrast, fusion with ghosts loaded with the catalytic subunit at concentrations between 0.1-2 mg/ml caused reproducible 2-3-fold increases in enzyme activity. Homogeneous preparations of the catalytic subunit exhibited even greater potency as an inducer. The effect was both time- and concentration-dependent and was abolished by inactivation of the catalytic subunit with N-ethylmaleimide prior to loading. The partially purified inhibitor of protein kinase from beef heart, while not affecting basal tyrosine aminotransferase activity, selectively inhibited the ability of 8-Br-cAMP but not that of dexamethasone to stimulate the activity of this enzyme. In addition, micro-injection of the pure regulatory subunit of the kinase blocked the response of the aminotransferase to low concentrations of 8-Br-cAMP. These results provide strong support for the proposition that the catalytic subunit of protein kinase mediates the effects of cAMP on the synthesis of tyrosine aminotransferase.
D015124:8-Bromo Cyclic Adenosine Monophosphate; D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D046911:Macromolecular Substances; D008845:Microinjections; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013997:Time Factors; D014444:Tyrosine Transaminase / Q000096:biosynthesis*
D046911:Macromolecular Substances; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003907:Dexamethasone; D000242:Cyclic AMP; D014444:Tyrosine Transaminase; D011494:Protein Kinases