In order to determine the role of cAMP dependent protein kinase in mediating the action of cAMP in tyrosine aminotransferase induction, cAMP-dependent protein kinases from cytosol fractions of rat liver and three lines of rat hepatoma cells (H-35, HTC, and MH1C1) were studied and compared. The radioactive photoaffinity label 8-azido-adenosine 3':5'[32P]monophosphate (8-N3-[32P]cAMP) was used to analyze the cAMP-binding proteins present in these cytosol preparations. The amounts of regulatory subunits (R) of the type I and type II cAMP-dependent protein kinases were determined by measuring the amount of 8-N3-[32P]cAMP incorporated into protein bands of molecular weight 47,000 and 54,000 in sodium dodecyl sulfate polyacrylamide gels, respectively. The catalytic subunit (C) of cAMP-dependent protein kinase was studied by assaying for cAMP-dependent histone kinase activity. cAMP-dependent protein kinases present in the cytosol fraction of rat liver and rat hepatoma cells were characterized by the concentrations of regulatory subunit (R) of both the type I and type II species, the concentration of catalytic subunit (C), the molar ratio of R to C, the dissociation constant (Kd) for 8-N3-[32P]cAMP as compared to the concentration of cAMP necessary to activate protein kinase (Ka) half-maximally, and the elution profile from DEAE-cellulose columns. The results of this study have demonstrated the predominance (70 to 80%) of type I cAMP-dependent protein kinase in the cytosol fraction of rat liver as compared to the predominance (90 to 95%) of type II cAMP-dependent protein kinase in cytosol fractions of all three lines of rat hepatoma cells. The type II kinase from rat liver and rat hepatoma cells appeared the same. R and C were found to exist in equimolar amounts in cytosol fractions of liver, H-35, and MH1C1 cells, but not in HTC cells. The induction of tyrosine aminotransferase by dibutyryl-cAMP (Bt2cAMP) in H-35 and MH1C1 rat hepatoma cells was studied. Addition of Bt2cAMP to the culture medium resulted in a 4- to 5-fold increase of tyrosine aminotransferase activity in H-35 hepatoma cells, but had little or no effect on tyrosine aminotransferase activity in MH1C1 hepatoma cells. Dexamethasone, a glucocorticoid, was effective in inducing tyrosine aminotransferase activity in both cell lines. It appears that the presence of cAMP dependent protein kinase is necessary but not sufficient to support the cAMP-induced increase in tyrosine aminotransferase activity.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - 1980|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology