Richard A. Jungmann

Cyclic AMP, cyclic AMP-dependent protein kinase, and the regulation of gene expression.

Abstract Biological responses produced by cAMP in mammalian cells are remarkably diverse and differ profoundly from one cell type and tissue to another. However, it is thought at this time that all effects of cAMP are mediated through the activation of cAMP-dependent protein kinases. The specificity of the cellular response to cAMP has been considered to result from switching on a specific built-in program of action within the specialized cell type which has been developed during development and differentiation. This review deals with the questions of cAMP control of protein synthesis at the transcriptional and translational levels. Hypotheses are suggested in an attempt to explain the data in tissue preparations and whole animals which suggest that regulation of the synthesis of at least some proteins by cAMP-dependent protein kinases does occur at the transcriptional level.

The phosphoform of the regulatory subunit RII of cyclic AMP-dependent protein kinase possesses intrinsic topoisomerase activity.

The phosphoform of the type II regulatory subunit (phospho-RII-cAMP) of cAMP-dependent protein kinase from rat liver was found to possess intrinsic topoisomerase activity towards several DNA substrates such as phi X174, pBR322, SV40, and M13. Like the type I topoisomerases from several eukaryotic cells, phospho-RII X cAMP can relax both positive and negative superhelical turns of phi X174 DNA. Topological isomers with a decreasing number of superhelical turns can be identified as transient products. Conditions under which phospho-RII X cAMP relaxes superhelical phi X174 DNA lead to transient formation of a DNA-phospho-RII X cAMP complex via DNA strand breakage and covalent attachment of the DNA to a tyrosine residue of phospho-RII X cAMP via a phospho-RII X cAMP depends on the presence of cAMP and is altered by changes in the degree of phosphorylation of RII. Both dephosphorylation and removal of cAMP from phospho-RII X cAMP abolish its topoisomerase activity.

Characterization of cyclic 3′:5′-AMP-dependent protein kinase in sarcoplasmic reticulum and cytosol of canine myocardium

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic cAMP-dependent and Ca2+ -calmodulin-dependent protein kinase (EC 2.7.1.37) activities and a common substrate, phospholamban, for these enzymes. Cyclic AMP-dependent protein kinase associated with sarcoplasmic reticulum membranes was solubilized with Triton X-100. Solubilization of the sarcoplasmic reticulum protein kinase did not affect its dependency on cAMP or its substrate specificity. The solubilized cAMP-dependent protein kinase was purified by DEAE-cellulose chromatography and was characterized as a type II enzyme on the basis of its elution at high ionic strength. DEAE-purified cAMP-dependent protein kinase exhibited no Ca2+ -calmodulin-dependent protein kinase activity. Cytosol from canine cardiac muscle cells, chromatographed on DEAE-cellulose under conditions identical to those used with sarcoplasmic reticulum, exhibited the presence of both type I and type II cAMP-dependent protein kinase isozymes. The properties of the DEAE-cellulose purified type II protein kinases from sarcoplasmic reticulum and cytosol were similar. We conclude that cardiac sarcoplasmic reticulum contains primarily type II cAMP-dependent protein kinase and this is probably the enzyme which phosphorylates sarcoplasmic reticulum in vivo and regulates Ca2+ transport.

Regulation of LDH-A gene expression by transcriptional and posttranscriptional signal transduction mechanisms

The lactate dehydrogenase-A (LDH-A) gene, whose product plays a pivotal role in normal anaerobic glycolysis and is frequently increased in human cancers, is highly regulated at the transcriptional and posttranscriptional levels. Our laboratory has carried out extensive studies concerning the regulation of LDH-A subunit expression. We have elucidated complex regulatory mechanisms by identifying multiple cis-acting promoter elements including functional sites for Sp1 and c-Myc interactions as well as sites that interact with the protein kinase A and protein kinase C substrates, CREB and AP1, respectively. Furthermore, we have reported the existence of a CRE-dependent silencer element in the LDH-A promoter. LDH-A expression is additionally regulated through the protein kinase A and C signal pathways at the posttranscriptional level, specifically mRNA stability.

Subcellular distribution of protein kinase C of GH3 cells: Quantitation and characterization by polyacrylamide gel electrophoresis☆

Quantitative estimation of cytosolic Ca2+- and phospholipid-dependent protein kinase (PKC) activity was performed by polyacrylamide gel electrophoresis under nondenaturating conditions (PAGE). With this method less than 50 micrograms of cytosol protein can be accurately quantitated for PKC activity. The amount of cytosolic PKC activity recovered after PAGE was comparable to the amount obtained by DEAE-cellulose chromatography. Homogenization of GH3 cells in the presence of 2 mM EGTA/EDTA revealed that 80% of the total cellular PKC activity resided in the cytosol. However, omission of the ion chelator during cell disruption followed by subcellular fractionation and extraction of subcellular fractions by EDTA/EGTA showed that 80% of the total PKC was found in the lysosomal-mitochondrial and microsomal extracts. Detailed analysis of PKC activities demonstrated that cytosolic PKC was identical with the PKC solubilized by EDTA/EGTA from subcellular fractions. In conclusion, GH3 cells appear to contain one species of PKC with an apparent molecular weight of 90,000 which seems to be associated with membranes via a calcium-dependent mechanism (or mechanisms).

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.

Adrenocorticotropic hormone and dibutyryl adenosine cyclic monophosphate-mediated Ca2+ uptake by rat adrenal glands

Abstract The effect of adrenocorticotropic hormone and dibutyryl cyclic AMP on the uptake of 45 Ca 2+ by the rat adrenal gland has been investigated. After injection of 45 Ca 2+ and adrenocorticotropic hormone into rats, the adrenal 45 Ca 2+ concentration was significantly enhanced 90 to 180 min following hormone administration. The rise in adrenal 45 Ca 2+ content was accompanied by a marked increase of the serum corticosterone levels. During incubation of rat adrenal glands in the presence of 45 Ca 2+ , adrenocorticotropic hormone and dibutyryl cyclic AMP caused significant accumulation of adrenal 45 Ca 2+ and increased corticosterone synthesis. The degree of stimulation of both adrenal 45 Ca 2+ uptake and corticosterone synthesis by adrenocorticotropic hormone or dibutyryl cyclic AMP was dependent upon the concentration of calcium in the incubation medium and upon the amount of adrenocorticotropic hormone or dibutyryl cyclic AMP added. Theophylline mimicked the stimulatory effect of adrenocorticotropic hormone and dibutyryl cyclic AMP and increased the uptake of 45 Ca 2+ by rat adrenal glands in vitro . Determination of calcium by atomic absorption spectroscopy showed that the adrenocorticotropic hormone-mediated adrenal 45 Ca 2+ uptake was due to a net accumulation of calcium in the tissue and not only to an increased rate of exchange of extracellular 45 Ca 2+ with the intracellular calcium pool. Adrenocorticotropic hormone-stimulated adrenal 45 Ca 2+ uptake was not observed when steroidogenesis was inhibited with elipten. Both adrenocorticotropic hormone-mediated corticosterone synthesis and adrenal 45 Ca 2+ uptake were abolished after treatment of rats with cycloheximide but not after treatment with actinomycin D, indicating that adrenal 45 Ca 2+ uptake and steroidogenesis have similar requirements for de novo protein synthesis, but not RNA synthesis.

Transcriptional regulation of the lactate dehydrogenase A subunit gene by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate

Regulation of lactate dehydrogenase (LDH) (EC 1.1.1.27) isozymes occurs through a multitude of physiological signals. Here, we show that modulation of LDH A subunit occurs via the protein kinase C pathway. Activators of protein kinase C, such as tetradecanoylphorbol acetate (TPA) and dioctanoylglycerol (DG), caused a 3-4-fold accumulation of LDH A subunit mRNA in rat C6 glioma cells. The specific protein kinase C inhibitor bisindolylmaleimide GF 109203X prevented the TPA-induced increase of LDH A subunit mRNA. To analyze the molecular basis of these effects in more detail, the transcription-modulatory effects of TPA and DG were evaluated in transient transfection assays using plasmids which contain LDH A subunit promoter fragments fused to a chloramphenicol acetyltransferase reporter gene. Both effector agents caused a marked increase of the transcriptional activity of an LDH -830/+25 bp promoter/CAT construct. In contrast, a phorbol ester which fails to activate protein kinase C, phorbol 12 beta,13 alpha-didecanoate, had no effect on the LDH promoter activity. Transient transfection analysis of LDH promoter deletion/CAT constructs, DNA/protein binding assays, including footprint and gel shift analyses, identified a TRE/AP-1 enhancer module at position -294 bp which was the target for the protein kinase C-mediated signal transduction pathway. Thus, our data demonstrate an active role of the protein kinase C signal pathway in regulating LDH A subunit gene expression which may be significant in regulating LDH isozyme patterns under various physiologic conditions.

Altered levels of cyclic nucleotides, cyclic AMP phosphodiesterase and adenylyl cyclase activities in normal, dysplastic and neoplastic human mammary tissue.

Recently, we have reported decreased cellular levels of CAMP-dependent protein kinase and CAMP-binding activity in neoplastic human mammary tissue [l] . Considering the sensitivity of the CAMP-protein kinase system to changes of CAMP, it was important to determine intracellular levels of this cyclic nucleotide and to identify changes of phosphodiesterase and adenylyl cyclase activities, the two enzyme systems which directly regulate the intracellular CAMP metabolism. Recently, increased levels of CAMP were reported in human mammary carcinomas [2] . Our results show that neoplastic mammary tissues exhibit increased specific levels but decreased cellulsr levels of CAMP and cGMP. In addition, the cellular activities of adenylyl cyclase phosphodiesterase are significantly lower in neoplastic as compared with normal human breast tissues.

Phosphorylation of calf thymus RNA polymerase II by nuclear cyclic 3′,5′-AMP-independent protein kinase

Nucleoplasmic RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from calfthymus is phosphorylated by homologous cyclic AMP-independent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). Polyacrylamide gel electrophoresis of the 32P-labeled RNA polymerase II under non-denaturing conditions revealed that both forms of the enzyme were phosphorylated. Polyacrylamide gel electrophoresis of the 32P-labeled RNA polymerase II under denaturing conditions showed that the 25 000 dalton subunit was the phosphate acceptor subunit. Partial acid hydrolysis of the 32P-labeled RNA polymerase II followed by ion-exchange chromatography revealed serine and threonine as the [32P]phosphate acceptor amino acids. Phosphorylation of the RNA polymerase II was accompanied by a stimulation of enzymatic activity and was dependent upon the presence of ATP.