title: Regulation of cAMP-dependent protein kinase subunit levels in Friend erythroleukemic cells. Effects of differentiation and treatment with 8-Br-cAMP and methylisobutyl xanthine.
abstract: Friend erythroleukemic cells contain both type I and type II cAMP-dependent protein kinases. The amounts of type I and type II regulatory subunits (R I and R II) were established by a combination of the equilibrium binding of cyclic [3H]AMP and specific immunoprecipitation. In control cells the total specific cAMP-binding activity was 9.8 pmol/mg of cytosolic protein and the ratio of R II to R I was 1.2. After the induction of differentiation by growth in 2% dimethyl sulfoxide, the total cAMP-binding capacity doubled, but larger and opposing changes in R II and R I levels were observed. The concentration of R II tripled (to 16.6 pmol/mg) while R I content (1.5 pmol/mg) declined to one-third of the control cell level, resulting in an R II:R I value of 11.1. Treatment of Friend cells with 0.5 mM 8-bromoadenosine 3':5'-monophosphate (8-Br-cAMP) and 0.2 mM methylisobutyl xanthine for 2 days did not stimulate cell differentiation. however, the cAMP-binding capacity nearly doubled and the cells accumulated a high level of R II while R I declined. This resulted in a distribution of R subunits closely resembling that of differentiated cells. Cyclic AMP-stimulated histone kinase activity increased in proportion to cAMP-binding activity during differentiation; no such increase was seen following 8-Br-cAMP treatment. Fractionation of cytosolic extracts by DEAE-cellulose chromatography revealed that both R I and R II were fully associated with the catalytic subunit (C) of protein kinase before and after differentiation. In extracts of 8-Br-cAMP-treated cells all of R I and 40 to 60% of R II were not associated with C. Free R II isolated from these cells was indistinguishable from RII present in holoenzyme. Thus, erythroid differentiation of Friend cells results in a coordinate accumulation of R II and C subunits as well as a decline in R I content. In contrast, chronic exposure to 8-Br-cAMP elicits similar changes in R subunit levels but disrupts the coordinate regulation of R and C content.
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology*; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000818:Animals; D002454:Cell Differentiation; D002848:Chromatography, DEAE-Cellulose; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D007525:Isoelectric Focusing; D007942:Leukemia, Experimental / Q000201:enzymology*; D046911:Macromolecular Substances; D006656:Protamine Kinase / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D013806:Theophylline / Q000031:analogs & derivatives*
chemical_list: D046911:Macromolecular Substances; D015124:8-Bromo Cyclic Adenosine Monophosphate; D013806:Theophylline; D000242:Cyclic AMP; D011494:Protein Kinases; D006656:Protamine Kinase; D015056:1-Methyl-3-isobutylxanthine