Cytochemical identification of the regulatory subunit of the cAMP-dependent protein kinase by use of fluorescently labeled catalytic subunit. Examination of protein kinase dissociation in hepatoma cells responding to 8-Br-cAMP stimulation

Homogeneous catalytic subunit from the cAMP-dependent protein kinase, when derivatized with a fluorophore, was used as a cytochemical probe to locate intracellular sites of the protein kinase regulatory subunit. After conjugation, the fluoresceinated catalytic subunit (F:C), derivatized to a stoichiometry of approximately 1 mol/mol, retained near full activity as judged by specific activity and by titration against either regulatory subunit or Inhibitor Protein of the protein kinase. With this molecular probe the dissociated regulatory subunit was localized by direct cytochemistry in Reuber H-35 hepatoma cells that had been exposed, while intact, for 0-120 min to 10(-4) M 8-Br-cAMP. After stimulation, cultures were fixed and washed and then incubated for 16 h with F:C. Following 8-Br-cAMP stimulation, extensive binding of the probe to both cytoplasmic and nucleolar sites was observed. This binding was diminished but not eliminated when 50 microM cAMP was present during the incubation of the fixed cells with F:C that was eliminated by a 40-fold molar excess of underivatized catalytic subunit but not by heat-denatured catalytic subunit, and was not reduced by a 20-fold molar excess of cGMP-dependent protein kinase, examined plus or minus cGMP. Collectively, the results allow the conclusion that the F:C probe binds free regulatory subunit. The time course of its change with 8-Br-cAMP (measured as the difference between binding in the presence or absence of cAMP during the postfixation treatment) mirrors that previously reported for changes in the catalytic subunit in these cells, also identified cytochemically (Byus, C. V., and Fletcher, W.H. (1982) J. Cell Biol. 93, 727-734). The binding of the F:C probe, detected when cAMP is present during postfixation treatment, may possibly represent binding to free Inhibitor Protein of the cAMP-dependent protein kinase. If so, it was at a level of approximately 20% of the maximal level of detectable regulatory subunit, and it also showed cytosolic and nucleolar localization.     

Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor

A specific and sensitive procedure has been developed that reliably localizes intracellular sites of free catalytic unit (C) dissociated from cAMP-dependent protein kinase. The method is based on a FITC conjugate (F:PKI) of affinity column-purified heat-stable protein inhibitor (PKI) of free C. The fidelity of this cytochemical probe was determined using cultures of Reuber H-35 hepatoma cells that had been stimulated for 2 h with 0.1 mM DBcAMP, or with diluent, then fixed with anhydrous acetone at -30 degrees C. In these preparations the F:PKI probe complexed with free C in cytoplasm, nucleolus, and, to a minor extent, in nucleoplasm. Binding of the F:PKI molecule to free C was competitively diminished by arginine analogues, guanidinium HCI and polyarginine, each used over a 2-log dose range. When the inhibitor's arginine residues were blocked by reaction with cyclohexanedione it no longer inhibited phosphotransferase activity of free C, and when fluorescinated it failed to localize C in stimulated cells. Similarly, when F:PKI was preabsorbed with excess pure C it no longer functioned as a cytochemical stain. Affinity column-purified antibody to free C also reduced significantly the ability of F:PKI to complex with C in cell cultures stimulated with 0.1 mM DBcAMP. 1 microgram of antibody reduced by approximately 10% the binding of F:PKI to all cell compartments while 5 microgram of antibody diminished binding by greater than 50%. Together, these results indicate that the F:PKI binds specifically, perhaps exclusively, to the catalytic units of cAMP- dependent protein kinase. The cytochemical procedure, unlike its biochemical counterparts, is able to locate the dissociation of cAMP- dependent protein kinase in individual cells of functionally or histologically complex cultures. Also, it reveals variations in the time- and dose-dependent activation of the kinase amongst clonal cells stimulated with cyclic nucleotide analogues or hormones.     

Localization of cyclic AMP-dependent protein kinase subunits in rat hepatocyte nuclei by immunogold electron microscopy

We have applied the indirect colloidal immunogold technique to examine the ultrastructural localization of the catalytic subunit C and the regulatory subunits RI and RII of cyclic AMP-dependent protein kinase in rat hepatocyte nuclei before and after glucagon or dibutyryl cyclic AMP administration. The technique allowed the identification and localization of all three subunits in hepatocyte nuclei. Morphometric quantitation of the relative staining density of nuclear subunits indicated an increase of immunogold staining of nuclear catalytic subunit but not of the regulatory subunits after glucagon or dibutyryl cyclic AMP stimulation. The increase of catalytic subunit occurred in a biphasic manner with peak levels 2-30 min and 90-150 min after stimulation. Our experiments represent the first reported use of the immunogold procedure to identify and localize protein kinase subunits in the nucleus.     

Stimulation by glucagon and adenosine-3',5'-cyclic monophosphate of protein degradation in Reuber H35 hepatoma monolayers

Protein degradation in Reuber H35 hepatoma monolayers was measured as release of radioactive trichloroacetic acid-soluble material from intracellular protein labelled with [3H]leucine for 16 hr followed by 3-hr chase period. Proteolysis in this system was stimulated by physiological concentration of glucagon reaching a maximum at 10(-7) M with an increase of 30%. Dibutyryl cyclic AMP also had a stimulatory effect. When both glucagon and dibutyryl cyclic AMP were present at optimal concentrations, their effects were not additive suggesting that glucagon may act via the formation of cyclic AMP. In the presence of protein synthesis inhibitor, cycloheximide or puromycin, proteolysis remained responsive to glucagon. Glucagon counteracted the inhibitory effect of insulin on proteolysis.     

Modulation of nuclear cyclic AMP-dependent protein kinase in dibutyryl cyclic AMP-treated rat H4IIE hepatoma cells.

Biochemical and immunochemical studies were undertaken to quantify the effects of cyclic AMP on cyclic AMP-dependent protein kinase subunit levels in nuclei of H4IIE hepatoma cells. Dibutyryl cyclic AMP (10 microM) caused a significant biphasic (10 and 120 min after stimulation) increase in total nuclear protein kinase activity. The increase observed 10 min after dibutyryl cyclic AMP stimulation was primarily due to an approx. 3-fold increase of catalytic (C) subunit activity, whereas the change observed 120 min after stimulation consisted of an increase in both C subunit and cyclic AMP-independent protein kinase activities. Analysis of nuclear protein extracts by photoaffinity labelling with 8-azido cyclic [32P]AMP identified only the type II regulatory subunit (RII), but not the type I regulatory subunit (RI). Analysis of nuclear RII variants by two-dimensional gel electrophoresis demonstrated that dibutyryl cyclic AMP caused the appearance of two RII variant forms which were not present in the nuclei of unstimulated cells. Using affinity-purified polyclonal antibodies and immunoblotting procedures, we identified an approx. 2-fold increase in the RII and C subunits in nuclear extracts of dibutyryl cyclic AMP-treated hepatoma cells. Finally, the RI, RII and C subunits were quantified by an e.l.i.s.a. which indicated that dibutyryl cyclic AMP increased nuclear RII and C subunits levels biphasically, reaching peak values 10 and 120 min after the initial stimulation. Nuclear RI subunit levels were not affected. These results provide qualitative as well as quantitative evidence for a modulation by cyclic AMP of the nuclear RII and C subunit levels in rat H4IIE hepatoma cells, and indicate a relatively rapid but temporarily limited dibutyryl cyclic AMP-induced translocation of the RII and C subunits to nuclear sites.     

Direct evidence that the protein kinase catalytic subunit mediates the effects of cAMP on tyrosine aminotransferase synthesis

The effect of purified beef heart cAMP-dependent protein kinase catalytic subunit on tyrosine aminotransferase activity in intact cultured rat H35 hepatoma cells was directly tested by micro-injection using human red blood cell ghosts as vehicles. Although the micro-injection procedure itself produced temporary fluctuations in protein synthesis and in tyrosine aminotransferase activity in H35 cells, after a recovery period of 8-12 h, these parameters returned to normal in parallel with restoration of full inducibility of the aminotransferase by both 8-Br-cAMP and dexamethasone. Eight to sixteen hours after fusion of H35 cells with unloaded ghosts, ghosts loaded with bovine serum albumin or mock-loaded with the partially purified protein kinase catalytic subunit, no significant change in the activity of the aminotransferase was detected. In contrast, fusion with ghosts loaded with the catalytic subunit at concentrations between 0.1-2 mg/ml caused reproducible 2-3-fold increases in enzyme activity. Homogeneous preparations of the catalytic subunit exhibited even greater potency as an inducer. The effect was both time- and concentration-dependent and was abolished by inactivation of the catalytic subunit with N-ethylmaleimide prior to loading. The partially purified inhibitor of protein kinase from beef heart, while not affecting basal tyrosine aminotransferase activity, selectively inhibited the ability of 8-Br-cAMP but not that of dexamethasone to stimulate the activity of this enzyme. In addition, micro-injection of the pure regulatory subunit of the kinase blocked the response of the aminotransferase to low concentrations of 8-Br-cAMP. These results provide strong support for the proposition that the catalytic subunit of protein kinase mediates the effects of cAMP on the synthesis of tyrosine aminotransferase.     

Studies of cAMP metabolism in cultured hepatoma cells: presence of functional adenylate cyclase despite low cAMP content and lack of hormonal responsiveness.

The ability of isoproterenol, glucagon, PGE1 and cholera toxin to stimulate the synthesis of cAMP and protein kinase activity in line of liver cells (BRL) and a line of rat hepatoma cells (H35) has been determined. The concentration of cAMP in BRL cells (approximately 10 pmoles/mg protein) is in the range reported for other cultured cell lines but H35 cells contain extraordinarily low amounts of this cyclic nucleotide (approximately 0.05 pmoles/mg protein). Isoproterenol and PGE1 caused an increase in cAMP content, and protein kinase activation in BRL cells, although glucagon was ineffective. H35 cells, in contrast, were completely insensitive to all hormonal agonists. Despite this fact, cholera toxin was able to produce a marked increase in cAMP content, adenylate cyclase activity and protein kinase activation in H35 cells. binding studies with [125 I]-iodohydroxybenzylpindolol, a specific beta-adrenergic receptor antagonist, revealed that each H35 cell possesses fewer than 10 beta-adrenergic receptors whereas BRL cells contain 2-5,000 receptors per cell. The low level of cAMP in H35 cells appears to result from a combination of totally unstimulated adenylate cyclase and apparently elevated phosphodiesterase activities.     

Activation of endogenous phosphorylase kinase in liver glycogen pellet by cAMP-dependent protein kinase

Liver glycogen phosphorylase associated with the glycogen pellet was activated by a MgATP-dependent process. This activation was reduced by 90% by ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid, not affected by the inhibitor of the cAMP-dependent protein kinase, and increased 2.5-fold by the catalytic subunit of cAMP-dependent protein kinase. Low levels of free Ca2+ (8 x 10(-8) M) completely prevented the effects of the chelator. The activation of phosphorylase by MgATP was shown not to be due to formation of AMP. DEAE-cellulose chromatography of the glycogen pellet separated phosphorylase from phosphorylase kinase. The isolated phosphorylase was no longer activated by MgATP in the presence or absence of the catalytic subunit of cAMP-dependent protein kinase. The isolated phosphorylase kinase phosphorylated and activated skeletal muscle phosphorylase b and the activation was increased 2- to 3-fold by the catalytic subunit of cAMP-dependent protein kinase. Mixing the isolated phosphorylase and phosphorylase kinase together restored the effects of MgATP and the catalytic subunit of cAMP-dependent protein kinase on phosphorylase activity. These findings demonstrate that the phosphorylase kinase associated with liver glycogen has regulatory features similar to those of muscle phosphorylase kinase.     

Hormonal regulation of carbamoyl-phosphate synthetase I synthesis in primary cultured hepatocytes and Reuber hepatoma H-35. Defective regulation in hepatoma cells

Regulation of carbamoyl-phosphate synthetase I (CPS) synthesis by various hormones was compared in primary cultured hepatocytes from adult rat and in Reuber hepatoma H-35 by pulse labeling of the cells with [35S]methionine. CPS synthesis in hepatocytes was stimulated 8-fold and 5-fold by dexamethasone and glucagon respectively. CPS synthesis in hepatocytes was synergically (about 50-fold) stimulated by a combination of dexamethasone and glucagon. Less synergic stimulation was observed by combining dexamethasone with N6, O2'-dibutyryladenosine 3',5'-monophosphate (dibutyryl-cAMP) or with isoproterenol. The basal level of CPS synthesis in hepatoma cells was higher than that in hepatocytes. CPS synthesis in hepatoma cells was stimulated by dexamethasone and dibutyryl-cAMP but the extent was only 3-fold and 1.8-fold respectively. The synergic effect of combination of dexamethasone and dibutyryl-cAMP was not observed in hepatoma cells. Neither glucagon nor isoproterenol exhibited an appreciable effect on CPS synthesis in hepatoma cells. Insulin and epinephrine suppressed CPS synthesis both in hepatocytes and hepatoma cells. The effect of epinephrine was indicated to be through alpha-adrenergic receptors. The effects of insulin and epinephrine were additive on CPS synthesis both in hepatocytes and hepatoma cells.     

Regulation of glycogen metabolism in liver by the autonomic nervous system. VI. Possible mechanism of phosphorylase activation by the splanchnic nerve.

The effects of autonomic-nerve stimulation on the activities of phosphorylase (EC 2.4.1.1), dephospho-phosphorylase kinase (EC 2.7.1.38) and phosphorylase phosphatase (EC 3.1.3.17), and on the concentration of adenosine 3', 5'-monophosphate in rabbit liver were investiaged. Results were compared with the effects of epinephrine and glucagon on these enzymes. 1. The acitivity of liver phosphorylase increased rapidly and markedly on electrical stimulation of the splanchnic nerve, or after intraportal administration of epinephrine or glucagon. The activity was not affected by vagal stimulation. 2. The activity of dephospho-phosphorylase kinase increased about 2--3-fold 1 min after injections of epinephrine and glucagon, glucagon causing more activation than epinephrine. The enzyme activity was not altered by splanchnic-nerve, or vagal stimulation. 3. Injections of epinephrine and glucagon caused marked elevation of liver adenosine 3', 5'-monophosphate within a few minutes. With epinephrine, the nucleotide concentration rose to a maximum after 1 min and amounted to about 3-fold increase, while with glucagon the maximum increase of approximately 8-fold increase was observed after 2 min. Stimulation of the splanchnic nerve for 10 min did not affect the adenosine 3', 5'-monophosphate level in the liver. Vagal stimulation also had no effect on the level. 4. The activity of phosphorylase phosphatase decreased promptly (within 30 s) and markedly on splanchnic-nerve stimulation, but did not change significantly on administration of epinephrine of glucagon. A small but insignificant increase in phosphatase activity wasobserved upon vagal stimulation. 5. The effect of Ca-2+ on purified dephospho-phosphorylase kinase was studied. The activity was found to depend partially on free Ca-2+ at low Ca-2+ concentrations (1-10-minus 7--1-10-minus 5 M). 6. These results suggest that the rise in hepatic phosphorylase content upon splanchnic-nerve stimulation, unlike that induced by epinephrine and glucagon, is not mediated by adenosine 3', 5'-monophosphate and subsequent activation of dephospho-phosphorylase kinase, but rather by inactivation of phosphorylase phosphatase. The possible existence of a new factor in this mechanism is discussed.     

The effect of turpentine-induced inflammation on rat liver glycosyltransferases and Golgi complex ultrastructure

The effect of vasopressin on the toad urinary bladder has been shown to be mediated by cyclic AMP. It has been assumed that, as demonstrated for other systems, this involves activation of cyclic AMP-dependent protein kinase. In order to test this hypothesis we investigated the effect of vasopressin on cyclic AMP-dependent protein kinases in epithelial cells of toad bladders. About 80% of protein kinase activity and cyclic AMP-binding capacity was found to be in the cytosol. DEAE-cellulose chromatography showed a pattern of 15--20% type I and 80--85% type II cyclic AMP-dependent protein kinase. Cytosolic kinase was activated 3--4-fold by cyclic AMP with half-maximal activation at 5 . 10(-8) M. Similarly, half-maximal binding of cyclic AMP occurred at 7 . 10(-8) M. Incubation of toad bladders in Ringer's solution containing 0.1 mM 3-isobutyl-1-methylxanthine, prior to homogenization and assay, showed stable cyclic AMP-binding capacity and protein kinase ratio --cyclic AMP/+cyclic AMP. Exposure of bladders to 10 mU/ml of vasopressin for 10 min caused intracellular activation of protein kinase and decrease in cyclic AMP-binding capacity that were maintained for at least 30 min. Incubation of bladders with increasing concentrations of vasopressin (0.5--100 mU/ml) resulted in a discrepancy between a progressive increase in cyclic AMP levels and a levelling off at 10 mU/ml of vasopressin for the changes in protein kinase ratio and cyclic AMP-binding capacity. The increase in kinase ratio was due to higher activity in the absence of exogenous cyclic AMP and was fully inhibitable by a specific protein kinase inhibitor. Using Sephadex G-25-CM50 column chromatography for separation of holoenzyme and free catalytic subunit we demonstrated that the activation of protein kinase in the vasopressin-treated bladders is due to intracellular dissociation of the kinase. These results show that the effect of vasopressin on the toad bladder involves activation of a cytosolic cyclic AMP-dependent protein kinase. The time course and the dose-response curve of the kinase activation closely parallel vasopressin's effect on osmotic water flow.     

An insulin-stimulated (ribosomal S6) protein kinase from soluble extracts of H4 hepatoma cells

Insulin stimulates the phosphorylation of the 40 S ribosomal subunit protein, S6, in intact 32P-labeled H4IIE-C3 cells, a rat hepatoma line. Cell-free cytosolic extracts from H4 cells exhibit a 5- to 10-fold increase in S6 protein kinase activity (measured by transfer of 32P to exogenous 40 S rat liver ribosomal subunits) when prepared from cells exposed to insulin prior to homogenization. Stimulation of S6 phosphorylation in intact cells and activation of S6 protein kinase in cell-free extracts are both detectable within 2 min after insulin, and are maximally stimulated by 10 min. Half-maximal stimulation is observed at 10(-11) M insulin. The stimulated S6 kinase activity requires ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to be present during the kinase assay for full expression. Despite the presence of a 5- to 10-fold increase in S6 protein kinase activity, the extracts from insulin-treated cells exhibit no stimulated kinase activity toward casein, histone, or ATP-citrate lyase assayed under the conditions employed for S6. Thus, insulin mediates the rapid activation of protein kinase specific for ribosomal protein S6 by an as yet unidentified mechanism.     

Variations in some molecular events during the early phases of the Reuber H 35 cell cycle. II.-Chromatin protein phosphorylation and protein kinases

Reuber H 35 hepatoma cells were synchronized by transfer in a serum free medium. Growth was re-initiated by addition of serum. Under these conditions DNA synthesis exhibited a maximum after 24 hours. Chromatin non-histone proteins prepared from cells at various phases of the cell cycle were incubated with [gamma-32P] ATP and the radioactive pattern of protein bound 32P was analysed by electrophoresis on polyacrylamide gels. No radioactive peak was observed in G0. Several peaks appeared 3 hours after the addition of serum. The radioactivity progressively increased until the cells reached the S phase. When most of the cells were in the S phase the radioactivity strongly decreased. Chromatin protein kinase activities were found to increase in late G1 and continued to increase in the S phase. The increase was 65% when phosvitin was the substrate, 100% with casein and histone H1. It is suggested that chromatin phosphorylated proteins could be involved in the mechanism which initiates DNA synthesis in G1 phase cells.     

Differential translational effects of myristic acid and eicosapentaenoic acid on 3-hydroxy-3-methylglutaryl-CoA reductase from Reuber H35 hepatoma cells

The mechanisms by which saturated and polyunsaturated fatty acids may exert their effects on levels of blood cholesterol and human atherosclerosis have not been fully established. In this work, we studied the translational effects of myristic (14:0) and eicosapentaenoic (20:5) acids on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Reuber H35 hepatoma cells. This enzyme is an intrinsic membrane, 96-kDa protein whose proteolysis releases an enzymatically active, 52- to 56-kDa, soluble fragment. We optimized an immunoblot procedure for quantifying small amounts of both the native and the soluble forms of HMG-CoA reductase from Reuber H35 hepatoma cells. We demonstrated that the upregulation of HMG-CoA reductase by a acid is due to an increase of the HMG-CoA reductase protein; therefore, protein synthesis would be required for the increase of HMG-CoA reductase activity caused by this fatty acid. In contrast, the downregulation of HMG-CoA reductase caused by eicosapentaenoic acid is not due to decreased protein synthesis, since similar levels of protein were found in the presence and absence of this fatty acid. Results obtained with cycloheximide as a protein-synthesis inhibitor confirm these findings.     

8-Bromo-cAMP decreases the Ca2+ sensitivity of airway smooth muscle contraction through a mechanism distinct from inhibition of Rho-kinase

To clarify whether cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) activation and Rho-kinase inhibition share a common mechanism to decrease the Ca2+ sensitivity of airway smooth muscle contraction, we examined the effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a stable cAMP analog, and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a Rho-kinase inhibitor, on carbachol (CCh)-, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-, 4beta-phorbol 12,13-dibutyrate (PDBu)-, and leukotriene D4 (LTD4)-induced Ca2+ sensitization in alpha-toxin-permeabilized rabbit tracheal and human bronchial smooth muscle. In rabbit trachea, CCh-induced smooth muscle contraction was inhibited by 8-BrcAMP and Y-27632 to a similar extent. However, GTPgammaS-induced smooth muscle contraction was resistant to 8-BrcAMP. In the presence of a saturating concentration of Y-27632, PDBu-induced smooth muscle contraction was completely reversed by 8-BrcAMP. Conversely, PDBu-induced smooth muscle contraction was resistant to Y-27632. In the presence of a saturating concentration of 8-BrcAMP, GTPgammaS-induced Ca2+ sensitization was also reversed by Y-27632. The 8-BrcAMP had no effect on the ATP-triggered contraction of tracheal smooth muscle that had been treated with calyculin A in rigor solutions. The 8-BrcAMP and Y-27632 additively accelerated the relaxation rate of PDBu- and GTPgammaS-treated smooth muscle under myosin light chain kinase-inhibited conditions. In human bronchus, LTD4-induced smooth muscle contraction was inhibited by both 8-BrcAMP and Y-27632. We conclude that cAMP/PKA-induced Ca2+ desensitization contains at least two mechanisms: 1) inhibition of the muscarinic receptor signaling upstream from Rho activation and 2) cAMP/PKA's preferential reversal of PKC-mediated Ca2+ sensitization in airway smooth muscle.     

Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase

We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE-cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work.     

The role of the cyclic AMP-dependent protein kinase in the glucagon-stimulated phosphorylation of ATP-citrate lyase

We have examined the mechanism whereby glucagon stimulates the phosphorylation of ATP-citrate lyase in intact rat hepatocytes. Purified ATP-citrate lyase is phosphorylated in vitro by the catalytic subunit of the cyclic AMP-dependent protein kinase, in a reaction wherein 2-3 mol phosphate/mol lyase are incorporated, at an initial rate that approaches that observed for mixed histone. This reaction is completely abolished by the protein kinase inhibitor protein. Limited tryptic digestion of ATP-citrate lyase phosphorylated in vitro by the cyclic AMP-dependent protein kinase yields a pattern of 32P-labeled peptides, indistinguishable from those observed in parallel digests of lyase isolated from 32P-labeled, glucagon-stimulated hepatocytes. Phosphorylase b kinase catalyzes the incorporation of 1 mol phosphate/mol lyase, albeit at less than 1/160 the rate observed for phosphorylase b. The phosphorylation of purified ATP-citrate lyase is also catalyzed by homogenates of hepatocytes. This reaction is stimulated by cyclic AMP. At 30 degrees C, in the presence of maximally stimulating concentrations of cyclic AMP, the addition of excess protein kinase inhibitor protein inhibits the phosphorylation of ATP-citrate lyase by 67%. Thus, hepatocytes contain both cyclic AMP-dependent and cyclic AMP-independent ATP-citrate lyase kinase activities. Pretreatment of hepatocytes with glucagon (10(-8) M for 2 min) prior to homogenization results in activation of an endogenous hepatocyte ATP-citrate lyase kinase, as well as histone kinase and phosphorylase b kinase; the glucagon-stimulated increment in lyase kinase (and histone kinase) is observed only when homogenates are assayed in the absence of added cyclic AMP, and is completely abolished by an excess of the protein kinase inhibitor protein. We conclude that the glucagon-stimulated phosphorylation of ATP-citrate lyase in intact hepatocytes is catalyzed directly by the cyclic AMP-dependent protein kinase.     

Fluorescence analysis of denaturation and reassembly of dansylated creatine kinase

Upon exposure of rabbit muscle creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) that has been dansylated at the two reactive lysines to 8 M urea, the maximum emission of the extrinsic fluorophore shifts 4 nm towards the blue, this being accompanied by a small decrease in intensity. The fluorescence emission and excitation spectra of the reassembled and native proteins are the same. Denaturation is accompanied by a rapid decrease in fluorescence which is complete in 10 s. This suggests that denaturation is accompanied by an early disorganization at the catalytic center, where the reactive lysines are located. Reassembly is associated with a rapid increase in dansyl fluorescence followed by a slower decrease that is complete in 6 min. Since reactivation is not complete until 20 min, minor additional structural changes are needed for the reacquisition of catalytic activity. The intrinsic protein fluorescence (eight tryptophans per dimer) of dansylated creatine kinase is approximately 60% less than that of the unlabelled enzyme, which may be attributed to resonance energy transfer, indicating that the reactive lysine is located near one or more of the tryptophans. A more limited quenching of intrinsic fluorescence is observed when dansylated creatine kinase is exposed to 8 M urea. Reassembly, monitored by a decrease in intrinsic fluorescence, reveals that the dansylated protein achieves its final fluorescence after 18 min of renaturation compared with 30 min for unlabelled enzyme. The powerful quenching by the dansyl group may limit the ability to monitor changes in the tryptophan environment. Kinetics of fluorescence polarization changes during denaturation are consistent with a mechanism involving rapid dissociation, followed by a subunit disorganization and possible aggregation. Reassembly would appear to involve first a refolding of the disorganized monomers and subsequent association. These results correspond to our previous observations that subunit renaturation precedes dimerization.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.