Regulation of lactate dehydrogenase gene expression by cAMP-dependent protein kinase subunits.

The studies described in this report suggest a rather complex, albeit incomplete, sequence of molecular events that we believe form part of the cascade of reactions through which a series of hormones, via cAMP, regulates the expression of specific gene products. The majority of our own studies relate to cAMP-mediated induction of LDH. Some, if not all, of the molecular steps discussed in this paper may ultimately be recognized as part of a universal mechanism by which cAMP controls gene expression in higher eukaryotes. The idea of a functional role for cAMP-dependent protein kinase subunits in cAMP-mediated gene control has already had experimental support, but our identification of the regulatory subunit RII as a topoisomerase now more firmly points to a complex function for the kinase in regulating gene function at the DNA level. We look forward to the elucidation of the function of those nuclear proteins that serve as substrate for the catalytic subunit of cAMP-dependent protein kinase. Further studies related to the molecular interaction of RII with chromosomal DNA should be a fruitful area for future research.

Localization of nuclear subunits of cyclic AMP-dependent protein kinase by the immunocolloidal gold method.

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.

Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP.

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).

Cyclic AMP regulation of lactate dehydrogenase. Isoproterenol and N6,O2'-dibutyryl cyclic AMP increase the levels of lactate dehydrogenase-5 isozyme and its messenger RNA in rat C6 glioma cells.

The mechanism of isoproterenol and N6,O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cAMP) induction of lactate dehydrogenase (EC 1.1.1.27) was investigated in the C6 rat glioma cell line. [3H]Leucine-labeled lactate dehydrogenase in noninduced and induced cells was quantitatively immunoprecipitated with rabbit anti-rat lactate dehydrogenase-5 antiserum. The immunoprecipitates were analyzed for 3H-labeled lactate dehydrogenase by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and isoelectrofocusing. Using this technique, it was shown that isoproterenol + 3-isobutyl-1-methylxanthine and dibutyryl cAMP cause an increase of the [3H]leucine incorporation into glioma cell lactate dehydrogenase. Analysis of the kinetics of induction and deinduction revealed no change in the rate of degradation of lactate dehydrogenase in the presence and absence of inducing agent, indicating that the induction was due to an increase in the rate of synthesis of the enzyme. The increased rate of synthesis was prevented by actinomycin D. Isoproterenol + 3-isobutyl-1-methylxanthine increased only the specific rate of synthesis of lactate dehydrogenase-5 isozyme and of the M subunit. The mechanism was further studied by assaying the level of functional mRNA coding for lactate dehydrogenase in a reticulocyte cell-free protein-synthesizing system using glioma cell poly(A)-containing RNA isolated from either isoproterenol or dibutyryl cAMP-induced cells. Analysis of the immunoprecipitated translation product by isoelectrofocusing revealed that isoproterenol or dibutyryl cAMP produced an approximately 8-fold stimulation of the poly(A) + RNA-directed synthesis of the lactate dehydrogenase M subunit. These data demonstrate that isoproterenol and dibutyryl cAMP control the level of functionally active lactate dehydrogenase mRNA in glioma cells which, in turn, determines the extent of synthesis of the lactate dehydrogenase M subunit.

Testicular protein kinases. Characterization of multiple forms and ontogeny.

Protein phosphokinase activity from the cytosol (105,000 X g soluble fraction) of testes from sexually mature rats has been resolved be DEAE-cellulose chromatography in three forms of protein kinase, cAMP-dependent protein kinases I and II and cAMP-independent protein kinase III. Adenosine 3':5'-monophosphate-binding activity (cAMP-binding activity) was associated with protein kinases I and II but not with protein kinase III. Protein kinases I, II, and III exhibited different pH optima, cyclic nucleotide dependency, and relative substrate specificity. Protein kinases I and II were inhibited by a heat-stable protein inhibitor from rat skeletal muscle, whereas protein kinase III was not inhibited. According to previously established criteria (Traugh, J. A., Ashby, C.D., and Walsh D. A. (1974) Methods Enzymol. 38, 290-299) protein kinases I and II can be classified as cAMP-dependent holoenzymes consisting of regulatory and catalytic subunits. Protein kinase III is a cAMP-independent protein kinase.

Ovarian cyclic adenosine monophosphate-dependent protein kinase activity: ontogeny and effect of gonadotropins.

The ontogeny of ovarian cyclic AMP-binding and protein kinase activities during the postnatal development of the rat, as well as the effect of LH and FSH administration on ovarian cyclic AMP-binding and protein kinase activities in 5-day-old and in hypophysectomized rats was examined. Ovaries of 4 to 8-day-old rats possessed little or no measureable cyclic AMP-binding and protein kinase activities. Subsequent postnatal development occurred in three distinct phases. During the first phase, ovarian cyclic AMP-binding and protein kinase activities increased progressively from age 8 days to age 23 days, when adult levels were observed. Protein kinase activity declined markedly during the second postnatal developmental phase from days 24 to 26, lost its cyclic AMP-dependency, and became refractory to stimulation by cyclic AMP. Studies employing a heat-stable protein kinase inhibitor protein isolated from rabbit skeletal muscle suggest that ovarian protein kinase activity during the refractory period was largely of the cyclic AMP-independent variety. During the third postnatal phase, comprising days 30 to 40, ovarian cyclic AMP-binding and protein kinase activities increased to levels seen in sexually mature rats. Protein kinase cyclic AMP-dependency which was lost during the refractory second postnatal period was fully restored during the third phase. Administration of FSH or LH led to a marked increase of ovarian cyclic AMP-binding and protein kinase activities in 5-day-old rats. Hypophysectomy of 20-day-old rats caused a significant reduction of the cyclic AMP-binding and protein kinase activities in a 27,000 X g supernatant fraction, as well as in the mitochondrial, microsomal, and 105,000 X g supernatant fraction. The decreased cyclic AMP-binding and protein kinase activities of these fractions could be partially restored by FSH or LH treatment of the hypophysectomized rats. The results indicate that ovarian cyclic AMP-binding and protein kinase activities, as well as the ability of ovarian protein kinase to respond to cyclic AMP are gradually acquired after the first postnatal week. The postnatal development of ovarian protein kinase and cyclic AMP-binding activities presumably involves the participation of FSH and LH, although the precise mechanism of LH and FSH action remains to be established.

Studies on the binding of 1-anilino-8-naphthalene sulfonate to very low density and high density himan serum lipoproteins.

Very low density and high density lipoproteins have been isolated from human plasma and their interaction with 1-anilin0-8-naphthalene sulfonate has been studied under different conditions of pH and added salt. Intrinsic fluorescence of bound 1-anilino-8-naphthalene sulfonate was higher for high density lipoproteins then for very low density lipoproteins, but was unaffected by salt in both systems. Binding of 1-anilino-8-naphthalene sulfonate by both these lipoproteins was saturable and was higher in the presence of added NaCl or CaCl2, Ca2+ having a greater effect than Na+ in enhancing fluorescence. The binding data were analyzed by Scatchard plots; the number of binding sites and the affinity of 1-anilino-8-naphthalene sulfonate for the site increased with increasing salt concentration. Fluorescence pH curves were similar to those published for phospholipids. From these and previous observations it is suggested that the phospholipids probably represent the major binding sites for 1-anilino-8-naphthalene sulfonate.