Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. II. Temporal and spatial kinetics.

The activation of cyclic AMP-dependent protein kinase has been found to be the predominant mode by which cyclic AMP (cAMP) leads to alterations of a large variety of cellular functions. The activation of the kinase results in the release of the catalytic subunit which as the free enzyme possesses phosphotransferase activity for a variety of specific protein substrates. Using a sensitive and specific cytofluorometric technique we monitored the appearance of free catalytic subunit in Reuber H35 hepatoma cells in culture after incubation with N6-1'-O-dibutyryl-cyclic AMP (DBcAMP), 8-bromoadenosine-3':5'-cyclic monophosphate (8-BrcAMP), and glucagon. The cytochemical method employs the heat-stable inhibitor of the free catalytic subunit which has been conjugated to fluorescein isothiocyanate (F:PKI) and was validated as described in the companion paper (Fletcher and Byus. 1982. J. Cell Biol. 93:719-726). Here we studied the temporal and spatial kinetics of the free catalytic subunit following activation of cAMP-dependent protein kinase by increasing concentrations of DBcAMP,8-BrcAMP, and glucagon. Under similar conditions protein kinase activation was also assessed biochemically in H35 cell supernatants by assaying the protein kinase activity ratio. Incubation of the hepatoma cells with DBcAMP (0.1 mM) led to an increase in the activity ratio from 0.2 in control cultures to a value of nearly 1.0 within a 1- to 2-h period. During this same period using the F:PKI probe, a significant increase in cytoplasmic and nucleolar fluorescence indicative of the release of the free catalytic subunit was coincidentally observed. In contrast to the rapid appearance of catalytic subunit in the cytoplasm and nucleolus of the cell within 5-15 min of the addition of DBcAMP, discernible nucleoplasmic fluorescence did not occur until after 1 h. H35 cell cultures incubated with 8-BrcAMP (0.01-1.0 mM) exhibited a more rapid activation of the protein kinase measured cytochemically compared to the cells treated with DBcAMP. Cultures incubated with 8-BrcAMP had significantly increased cytoplasmic and nucleolar fluorescence compared to unstimulated cells within 1 min of the addition of the analogue and reached a maximal level within 15 min. By employing a microspectrophotometer a distinct dose-dependent increase in cellular fluorescence (i.e., free catalytic subunit) was observed as the concentration of 8-BrcAMP was increased from 0.01 to 1.0 mM at 1, 5, 15, and 60 min following stimulation. The addition of glucagon (10(-6) M) to the culture also led to the activation of cAMP-dependent protein kinase as determined by an increase in the activity ratio. This increase was paralleled throughout the incubation period by a marked elevation in cytoplasmic and nucleolar fluorescence. The results reported herein suggest that both cyclic nucleotide analogues and a polypeptide hormone lead to the activation of cAMP-dependent protein kinase in similar intracellular compartments in Reuber H35 hepatoma cells...

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AMY-1 Interacts with S-AKAP84 and AKAP95 in the Cytoplasm and the Nucleus, Respectively, and Inhibits cAMP-dependent Protein Kinase Activity by Preventing Binding of Its Catalytic Subunit to A-kinase-anchoring Protein (AKAP) Complex

Journal of Biological Chemistry ◽

10.1074/jbc.m206387200 ◽

2002 ◽

Vol 277 (52) ◽

pp. 50885-50892 ◽

Cited By ~ 26

Author(s):

Makoto Furusawa ◽

Takahiro Taira ◽

Sanae M. M. Iguchi-Ariga ◽

Hiroyoshi Ariga

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Catalytic Subunit ◽

Protein Kinase Activity ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Anchoring Protein ◽

Dependent Protein

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Involvement of cAMP-dependent protein kinase in the regulation of heart contractile force. II

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1979.236.1.h84 ◽

1979 ◽

Vol 236 (1) ◽

pp. H84-H91

Author(s):

S. L. Keely ◽

A. Eiring

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Activity Ratio ◽

Contractile Force ◽

Protein Kinase Activity ◽

Phosphorylase Activity ◽

Dependent Protein Kinase ◽

Guinea Pig Heart ◽

Camp Dependent Protein Kinase ◽

Dependent Protein

The effects of histamine on heart cAMP-dependent protein kinase activity, cAMP levels, phosphorylase activity, and contractile force was investigated in the perfused guinea pig heart. To accurately determine the protein kinase activity ratio in guinea pig heart, it was necessary to measure kinase activity in whole homogenates immediately after homogenization of the tissue. Histamine produced a rapid dose-dependent increase in cAMP and the protein kinase activity ratio followed by increased in contractile force and phosphorylase activity. There was a good correlation between the degree of protein kinase activation and the increase in phosphorylase and force. The beta-adrenergic blocking agent propranolol did not reduce the effects of histamine, but metiamide, a potent H2-receptor antagonist, greatly attenuated all the effects of histamine. The data support the hypothesis that increases in heart cAMP-dependent protein kinase activity produce corresponding increases in contractile force and phosphorylase activity.

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

The Journal of Cell Biology ◽

10.1083/jcb.101.3.965 ◽

1985 ◽

Vol 101 (3) ◽

pp. 965-975 ◽

Cited By ~ 36

Author(s):

M R Kuettel ◽

S P Squinto ◽

J Kwast-Welfeld ◽

G Schwoch ◽

J S Schweppe ◽

...

Keyword(s):

Electron Microscopy ◽

Protein Kinase ◽

Hepatoma Cells ◽

Catalytic Subunit ◽

Immunogold Electron Microscopy ◽

Dependent Protein Kinase ◽

Regulatory Subunits ◽

Camp Dependent Protein Kinase ◽

Specific Antisera ◽

Dependent Protein

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.

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Ovarian adenosine 3′:5′-cyclic monophosphate-dependent protein kinase(s). Regulation by choriogonadotropin and lutropin in rat ovarian cells

Biochemical Journal ◽

10.1042/bj1580175 ◽

1976 ◽

Vol 158 (2) ◽

pp. 175-182 ◽

Cited By ~ 17

Author(s):

M R Clark ◽

S Azhar ◽

K M J Menon

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Kinase Activity ◽

Activity Ratio ◽

Gel Filtration ◽

Supernatant Fraction ◽

Protein Kinase Activity ◽

Dependent Protein Kinase ◽

Ovarian Cells ◽

Dependent Protein

Choriogonadotropin and lutropin have been found to activate cyclic AMP-dependent protein kinase in ovarian cells isolated by collagenase dispersion from immature rats. The stimulatory effect of gonadotropins was dependent on both hormone concentration and incubation time. Choriogonadotropin at 1 mug/ml fully stimulated the protein kinase activity within 5 min of incubation, and this effect was specific for choriogonadotropin and lutropin-like activity. In addition, protein kinase activity has been characterized with respect to salt sensitivity, cyclic AMP binding, and its responsiveness to gonadotropins and other peptide hormones. Ovarian protein kinase was susceptible to high salt concentrations. The addition of 0.3-1.0 M-NaCl in incubation medium increased the activity ratio with a concomitant decrease in cycle AMP-dependence. The salt effect on protein kinase was observed both from hormone-treated and untreated cells. The hormone-stimulated and unstimulated protein kinase activity was completely stable in the absence of NaCl. No change in the activity ratio was observed when cellular extracts were assayed for protein kinase activity either immediately or after 2 h in the absence of added salt. Gel filtration in the absence of NaCl of cellular extracts prepared from choriogonadotropin-treated and untreated cells showned only a single peak of protein kinase activity that was sensitive to exogenously added cyclic AMP. By contrast, when 0.5 M-NaCl was included in the column buffer, the chromatography of untreated extract showed two peaks of protein kinase activity. The first peak was sensitive to added cyclic AMP, whereas the second peak was insensitive to it. Under identical experimental conditions, protein kinase from gonadotropin-treated cells showed, on gel filtration, only one peak of activity that was totally insensitive to added cyclic AMP. DEAE-cellulose column chromatography of a 20000 g supernatant fraction resulted in a peak of kinase activity that eluted in approx. 0.15 M-NaCl, similar to the similar to the elution of type II protein kinases as described by Corbin et al. (1975) (J. Biol. Chem. 250, 218-225). Choriogonadotropin stimulation produced a decrease in the capacity of protein kinase to bind exogenous cyclic [3H]AMP, with a concomitant increase in the kinase activity ratio. These results are consistent with the notion that cyclic AMP, GENERATED IN SITU Under hormonal stimulation, binds tot he regulatory subunit of protein kinase with subsequent dissociation of the active catalytic subunit from the holoenzyme.

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The effect of methacholine and histamine on cyclic AMP-dependent protein kinase activity in the guinea-pig isolated trachea

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y92-043 ◽

1992 ◽

Vol 70 (3) ◽

pp. 344-348 ◽

Cited By ~ 4

Author(s):

J. M. Langlands ◽

I. W. Rodger

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Guinea Pig ◽

Kinase Activity ◽

Activity Ratio ◽

Kinase Assay ◽

Protein Kinase Activity ◽

Dependent Protein Kinase ◽

Isolated Trachea ◽

Dependent Protein

The effects of methacholine and histamine were examined on cyclic AMP-dependent protein kinase (A-kinase) activity in guinea-pig isolated trachea, using kemptide as a substrate for phosphorylation during the determination of the enzyme activity. Methacholine (EC90, 10 μM) induced a rapid reduction in the basal A-kinase activity ratio, which was maximal after 30 s. This initial reduction coincided with the early phase of isometric tension development, and returned to control levels 4 min after the addition of methacholine. Pretreatment with atropine inhibited the methacholine response. In contrast, histamine (EQ90, 30 μM) was without effect upon A-kinase activity ratio. The results establish the sensitivity of the A-kinase assay using kemptide and demonstrate that not all contractile agonists have the capacity to inhibit basal activity of A-kinase in airway smooth muscle.Key words: A-kinase, cholinomimetics, guinea-pig trachealis, smooth muscle contraction.

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Adenosine-3′,5′-Monophosphate-Dependent Protein Kinase From Bovine Anterior Pituitary Gland. III. Structural Specificity of the ATP Site of the Catalytic Subunit

Canadian Journal of Biochemistry ◽

10.1139/o74-021 ◽

1974 ◽

Vol 52 (2) ◽

pp. 137-141 ◽

Cited By ~ 19

Author(s):

Simon Lemaire ◽

Fernand Labrie ◽

Marie Gauthier

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Catalytic Subunit ◽

Protein Kinase Activity ◽

Dependent Protein Kinase ◽

Stringent Requirement ◽

Dependent Protein ◽

Atp Analogues ◽

High Concentrations ◽

Derivatives Of

The effect of analogues and derivatives of adenosine 3′,5′-monophosphate (cyclic AMP) and of ATP on the incorporation of 32P from [γ-32P]ATP into histones has been measured using the purified catalytic subunit of adenohypophyseal protein kinase. Gamma-labeled CTP, GTP, and UTP cannot substitute for [γ-32P]ATP but they slightly inhibit the phosphorylation by [γ-32P]-ATP when present as unlabeled compounds. A stringent requirement of the adenine nucleus is observed for the ability to compete at the ATP site, inhibitions of 42, 39, 32, and 63% being observed respectively with adenine, adenosine, 5′AMP, and ADP, while the corresponding purine or pyrimidine derivatives have no effect when present at a 13-fold molar excess relative to [γ-32P]ATP. The N6-benzoyl and N6-butyryl derivatives of cyclic AMP are inactive whereas the 8-substituted derivatives are generally as active as cyclic AMP itself, except for the 8-amino- and 8-hydroxy-derivatives, which exhibit a lower degree of competition. All cyclic AMP and ATP analogues and derivatives that inhibit histone phosphorylation by [γ-32P]ATP act as competitive inhibitors. Such competition at the ATP site of the catalytic subunit of protein kinase probably accounts for the progressive inhibition of cyclic-AMP-dependent protein kinase activity measured at high concentrations (above 10−5 M) of the cyclic nucleotide.

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