Purification and characterization of a dimer form of the cAMP-dependent protein kinase from mouse liver cytosol (original) (raw)
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Adenosine 3':5'-cyclic monophosphate-dependence of protein kinase isoenzymes from mouse liver
Biochemical Journal, 1976
Conditions influencing the cyclic AMP-dependence of protein kinase (ATP-protein phosphotransferase, EC 2.7.1.37) during the phosphorylation of histone were studied. Protein kinase from mouse liver cytosol and the two isoenzymes [PK (protein kinase) I and PK II] isolated from the cytosol by DEAE-cellulose chromatography were tested. A relation between concentration of enzyme and cyclic AMP-dependence was observed for both isoenzymes. Moderate dilution of isoenzyme PK II decreased the stimulation of the enzyme by cyclic AMP. Isoenzyme PK I could be diluted 200 times more than isoenzyme PK II before the same decrease in cyclic AMP-dependence appeared. Long-term incubation with high concentrations of histone increased the activity in the absence of cyclic AMP relative to the activity in the presence of the nucleotide. This was more pronounced for isoenzyme PK II than for isoenzyme PK I. The cyclic AMP concentration needed to give half-maximal binding of the nucleotide was the same as th...
Biology of Reproduction, 1996
We report the basal and cAMP-regulated expression of protein kinase A (PKA) subunits in a mouse Sertoli cell line (MSC-1). Of the PKA subunits expressed by these cells (Rlat, Rlla, Rll, Cao, CI3), only Rll3P was regulated by cAMP. An approximately 8-fold induction of RIIi mRNA and a 3-fold induction of RII1i protein was observed during 48 h of cAMP-stimulation. This cAMP-mediated R11l3 mRNA induction, reaching maximal levels after approximately 12 h, did not require ongoing protein synthesis. Fairly rapid decay of maximally induced RIIl mRNA was observed after removal of cAMP (t,, 2-5 h). Further, ongoing transcription and translation were necessary for rapid degradation of Rll5 mRNA. Thus, the MSC-1 cells expressed all the PKA subunits present in primary cultures of Sertoli cells and responded to cAMP with increased levels of Rl1y at both mRNA and protein levels. Although the nature of some of these responses distinguished the observations in MSC-1 cells from previously described responses in primary cultures, these cells may prove to be useful in future studies addressing cAMP-mediated gene regulation.
Molecular Endocrinology, 1988
In the present study we have examined the effect of long-term stimulation with (Bu) 2 cAMP on mRNA levels for the hormone responsive regulatory subunit (Rlli) of cAMP-dependent protein kinase in cultured rat Sertoli cells. The effects of the same treatment on two other mRNAs [androgen binding protein (ABP) and cellular retinol binding protein (cRBP)], shown to be regulated by cAMP, were examined simultaneously. The addition of (Bu) 2 cAMP (0.1 mM) to primary Sertoli cell cultures, for 14 and 24 h, caused a 50-to 60-fold stimulation in the steady state levels of mRNA for Rll^. During the same period of stimulation, we also observed a significant increase (2-to 3fold) in the mRNA levels for ABP, and a 80% decrease in the mRNA levels for cRBP. Continued stimulation for 36 and 48 h was associated with a significant time-dependent decrease in the mRNA level for Rll^, in spite of the continuous presence of (Bu) 2 cAMP (0.1 mM) in the medium. This reduced response by long term stimulation with (Bu) 2 cAMP appears to be specific for Rll^, since mRNA for ABP remained elevated and mRNA for cRBP remained depressed during the entire period of cAMP stimulation. Our data demonstrate the presence of a biphasic type of regulation at the mRNA level, specific for the regulatory subunit Rll^ of cAMP-dependent protein kinase. This response may be analogous to the desensitization mechanisms observed at other lev
Biochimica et Biophysica Acta (BBA) - General Subjects, 1977
Both cyclic AMP-binding and cyclic AMP-dependent protein kinase activities exist in Chinese hamster ovary cell extract. Competition experiments demonstrate that the binding is specific for cyclic AMP. All cellular elements including nucleus, mitochondria, plasma membrane, microsome, ribosome and cytosol contain both activities. Binding activity is highest in the cytosol and lowest in the nucleus. Each fraction contains endogenous protein kinase activity which is insensitive to cyclic AMP activation. When histone was used as a substrate, protein kinase activity in all fractions was stimulated by cyclic AMP (with the highest in cytosol and lowest in the nucleus) and inhibited by Walsh's protein kinase inhibitor.
Archives of Biochemistry and Biophysics, 2002
The Cc and Ca isoforms of the cAMP-dependent protein kinase (PKA) share 83% identity including all critical catalytic and substrate-binding residues defined to date. Compared to Ca, Cc has a different substrate specificity and a selective pseudosubstrate specificity, exhibiting inhibition by regulatory subunits, but not by the protein kinase inhibitor. In these studies, Cc-mediated gene transcription regulation was compared with that of Ca in four cell lines using transient transfection/dual luciferase assays. As compared to Cc, Ca more efficiently activated a cAMP-response element (CRE)-regulated fragment of the human a-glycoprotein hormone promoter which was coupled to a firefly luciferase reporter gene (pGHa-fluc). This occurred in Cos7, Y1, and Kin8 adrenal cells by 23-, 6.5-, and 1.4-fold, respectively. In contrast, Cc, but not Ca, activated the Sp1RE-regulated herpes simplex virus thymidine kinase promoter which was coupled to a Renilla luciferase reporter (pTK-rluc). In Sp1-deficient Sf9 cells, pGHa-fluc expression was maintained for both isoforms, but cotransfection with an Sp1 expression plasmid was necessary and sufficient for activation of pTK-rluc expression by Cc. In all cell lines, cotransfection with a PDK1 expression plasmid enhanced the transcriptional activation of both Ca and Cc (1.5-to 3-fold), while a catalytically inactive PDK1 mutant (PDKÁKD) did not. These results suggest that both Ca and Cc can activate CRE-responsive genes; however, Ca does so with better efficiency than Cc. In contrast to Ca, Cc activates transcription of genes containing pTK-like Sp1RE sites. Activation of different C subunit isoforms can provide a means to diversify cAMP-mediated transcription, possibly affecting cell phenotype.
Isolation of cDNA clones coding for the catalytic subunit of mouse cAMP-dependent protein kinase
Proceedings of the National Academy of Sciences, 1986
mRNA coding for the catalytic (C) subunit of cAMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was partially purified from bovine testis by polysome immunoadsorption and oligo(dT)-chromatography. This enriched mRNA preparation was used to prepare and differentially screen a cDNA library. One of the selected cDNA clones was shown to hybrid-select mRNA coding for a 40-kDa protein that was specifically precipitated with antibodies to the C subunit. This bovine cDNA clone was then used to isolate a series of mouse cDNA clones that are complementary to the entire mouse C subunit mRNA. The mouse clones code for a protein of 351 animino acids that shows 98% homology to the bovine C subunit and hybridize to a single mRNA of 2.4 kilobases in mouse heart and brain. Southern blot analysis of total genomic DNA suggests that there is a single mouse gene coding for the C subunit. mRNA levels for both the C subunit and the type I regulatory subunit in various mouse tissues and cell lines were quantitated and compared by using singlestranded RNA probes prepared with SP6 polymerase.
Molecular Endocrinology, 1991
The present study addresses possible mechanisms through which cAMP mediates its effects on mRNA levels for the subunits of protein kinase A (PKA) and the cellular protooncogene, c-fos. Messenger RNAs for the PKA subunits (Rla, Rlla, RII/3, and Ca) were regulated by cAMP with similar kinetics in Sertoli cells. However, effects of cAMP on the PKA mRNAs were slow compared to a well characterized cAMP responsive gene, c-fos. The magnitude of stimulation was dramatically different between the various PKA subunits, in that RII/5 mRNA increased more than 50-fold while the mRNAs for the other subunits were induced only two to four times. Separation of nuclear and cytoplasmic RNA demonstrated that mRNAs for PKA subunits were stimulated to the same extent in these two cellular compartments. The more rapid induction of c-fos mRNA by cAMP, compared to the mRNA for RII/3, was also seen at the level of transcription. Maximal transcription rate for c-fos, Rla, and Ca were observed after 30 min, whereas that for RII/9 was increasing during the 2-h period examined. Transcriptional activation of the Rla gene also appeared faster than that for RII/3. When Sertoli cells were incubated with 8-(4-chlorophenylthio) cAMP and cycloheximide, a potent inhibitor of protein synthesis, we observed a superinduction of the mRNAs for c-fos (10-fold) and Rla (2-fold). In contrast, the inductions of RII/3, Rlla, and Ca were almost completely inhibited by cycloheximide. This was also found in nuclear RNA. These results indicate that different factors are involved in cAMP-mediated induction of mRNAs for the various PKA subunits. In the case of Rla it seems to be a direct effect of cAMP on stable factors, whereas proteins with either rapid turnover or factors induced by cAMP are involved in the stimulation of Ca and the Rll subunits. (Molecular Endocrinology 5: 21-28, 1991)