In situ localization of cAMP-dependent protein kinases in nuclear and chromosomal substructures: relation to transcriptional activity.

Catalytic (C) and regulatory subunits (RI, RII) of cAMP-dependent protein kinase (PKA) were localized in cell nuclei of various rat tissues by immunogold electron microscopy. A specific labeling within the interchromatin space was found to be associated with perichromatin fibrils, interchromatin granules, and coiled bodies. Condensed chromatin and perichromatin granules were not labeled. In the nucleolus, most gold particles were found in the dense fibrillar component. Quantitation of labeling densities for the C-subunit from cells at different stages of spermatogenesis indicated that the nuclear concentration of PKA varies inversely with the amount of condensed chromatin. No parallel alterations in C-immunoreactivities were observed in the cytoplasm. Immunofluorescence studies on polytene chromosomes of Chironomus thummi with a confocal laser scanning microscope showed a significantly more intense labeling of the C-subunit in transcriptionally active sites (puffs) as compared to inactive bands. Our results indicate that local concentrations of PKA in the nucleus have physiological significance in the control of gene activity. In addition, they suggest that PKA plays a regulatory role also in RNA processing.

Immunoelectron microscopical localization of the catalytic subunit of cAMP-dependent protein kinases in brain microtubules and neurofilaments.

The catalytic subunit of cAMP-dependent protein kinases was localized in microtubules and neurofilaments by immunogold electron microscopy. In microtubules, the label was similarly distributed as an immunolabel for the microtubule associated protein MAP 2. The neurofilaments showed no reaction with the MAP 2-antiserum. Our results support the suggestion of an in vivo role of cAMP-dependent protein kinases in the regulation of microtubules. In addition, this is the first demonstration that cAMP-dependent protein kinase is associated with neurofilaments.

Localization of catalytic and regulatory subunits of cyclic AMP-dependent protein kinases in mitochondria from various rat tissues.

Observation and quantification of the catalytic subunit C of cyclic AMP-dependent protein kinases by immuno-gold electron microscopy suggested a high concentration of cyclic AMP-dependent protein kinases in mitochondria from liver, kidney, heart and skeletal muscle, pancreas, parotid gland and brain cells. The position of gold particles pointed to a localization in the inner membrane/matrix space. A similar distribution was obtained by immunolocalization of the cyclic AMP-dependent protein kinase regulatory subunits RI and RII in liver, pancreas and heart cells. The results indicated the presence of both the type I and the type II cyclic AMP-dependent protein kinases in mitochondria of hepatocytes, and the preferential occurrence of the type I protein kinase in mitochondria from exocrine pancreas and heart muscle. The immunocytochemical results were confirmed by immunochemical determination of cyclic AMP-dependent protein kinase subunits in fractionated tissues. Determinations by e.l.i.s.a. of the C-subunit in parotid gland cell fractions indicated about a 4-fold higher concentration of C-subunit in the mitochondria than in a crude 1200 g supernatant. Immunoblot analysis of subfractions from liver mitochondria supported the localization in situ of cyclic AMP-dependent protein kinases in the inner membrane/matrix space and suggested that the type I enzyme is anchored by its regulatory subunit to the inner membrane. In accordance with the immunoblot data, the specific activity of cyclic AMP-dependent protein kinase measured in the matrix fraction was about twice that measured in whole mitochondria. These findings indicate the importance of cyclic AMP-dependent protein kinases in the regulation of mitochondrial functions.

Localization of cAMP-dependent protein kinase subunits along the secretory pathway in pancreatic and parotid acinar cells and accumulation of the catalytic subunit in parotid secretory granules following beta-adrenergic stimulation.

Catalytic (C) and regulatory (RI and RII) subunits of cAMP-dependent protein kinases were localized by immunoelectron microscopy in cisternae of the rough endoplasmic reticulum (rER) and in the Golgi complex of rat pancreas or parotid cells. Zymogen granules of the exocrine pancreas showed C- and RI-immunoreactivity, secretory granules of parotid acinar cells only RII-immunoreactivity. Injection of rats with isoproterenol (IPR) increased in the parotid gland the number of acinar cells with RII-labeled granules. In addition, it led to the appearance of C-immunoreactivity in the condensing vacuoles and secretory granules with a maximum at 24 h after stimulation. This was confirmed by enzyme-linked immunosorbent assay (ELISA) determinations of C- and RII-subunits in secretory granules isolated from stimulated and control parotid glands. The amount of immunoreactive C-subunits in the secretory granules increased further following repeated injections of the beta-agonist. These findings suggest the existence of secretory forms of cAMP-dependent protein kinase R- and C-subunits and their separate regulation.

Co-ordinated changes in the cyclic AMP signalling system and the phosphorylation of two nuclear proteins of Mr 130,000 and 110,000 during proliferative stimulation of the rat parotid gland by isoprenaline. Possible identity of the two proteins with pp135 and nucleolin.

Parotid glands were stimulated to growth by repeated injection of the beta-agonist isoprenaline into rats. Incubation of intact parotid-gland lobules with [32P]Pi and subsequent analysis of nuclear proteins revealed in the stimulated glands an increased 32P incorporation into two acid-soluble non-histone proteins with apparent Mr values of 110,000 and 130,000 (p110 and p130). After a single injection of isoprenaline, leading to a biphasic increase in DNA synthesis (maximum at 24 h), the same two proteins showed a transiently increased 32P incorporation at 17 h after injection. At this time point at the onset of DNA synthesis the total activity of soluble cyclic AMP-dependent protein kinase decreased. No change in p110/p130 phosphorylation was observed at 0.3 h after stimulation, a time of maximal stimulation of secretion. Administration of the beta-antagonist propranolol 8 h after the injection of isoprenaline suppressed the increase in DNA synthesis, the preceding changes in the concentration of cyclic AMP and in the activity of cyclic AMP-dependent protein kinase, as well as the increased phosphorylation of p110 and p130. Cross-reactivity of p110 and p130 with specific antisera against two nucleolar phosphoproteins of similar molecular mass (nucleolin and pp135), as well as their localization in a nucleolar cell fraction, indicated a possible identity of p110 and p130 with these two proteins. Our results suggest that nucleolin and pp135 are nuclear target proteins of cyclic AMP in the cyclic AMP-influenced regulation of the transition of cells from the G1 to the S phase.

Immunoelectron microscopic localization of catalytic and regulatory subunits of cAMP-dependent protein kinases in the parotid gland.

The subcellular distribution of catalytic (C) and regulatory (RI and RII) subunits of cAMP-dependent protein kinases has been studied by electron microscopy immunocytochemistry. The C-subunit was localized in the inner membrane-matrix space of mitochondria, at the cytoplasmic face of the rough endoplasmic reticulum (rER), in the nucleolus and in peripheral heterochromatin regions. A C-specific immunoreactivity was also found in specific domains of the basal and basolateral plasma membranes of acinar and duct cells, in centrosomes and on keratin filaments which anchor in desmosomes. The RI- and RII-subunits showed a basically similar subcellular distribution. A remarkably high RII-immunoreactivity, in the absence of C-immunoreactivity, was demonstrated in the secretory granules. These results demonstrate the presence of cAMP-dependent protein kinases or their subunits in many subcellular organelles. They also indicate a role for cAMP-dependent protein kinases in the regulation of a number of basal cellular functions as well as their importance in functional and structural cell-cell interactions.

Selective regulation of the amount of catalytic subunit of cyclic AMP-dependent protein kinases during isoprenaline-induced growth of the rat parotid gland.

Stimulation of growth of the rat parotid gland by repeated injection of the beta-agonist isoprenaline led to a significant decrease in the activity of cyclic AMP-dependent protein kinases. Immunochemical quantification of the catalytic (C) and regulatory (RI and RII) subunits of the cyclic AMP-dependent protein kinases type I and type II revealed a loss of 65% of the immunochemically measurable amount of catalytic subunit C. The amount of the regulatory subunits, however, remained constant. The observed decrease in C-subunit was not due to a translocation of the molecule to cellular membranes or to an inhibiting effect of the heat-stable inhibitor of cyclic AMP-dependent protein kinases. A selective decrease in only the C-subunit was also observed after a brief exposure to isoprenaline leading to the stimulation of DNA synthesis. Under these conditions, the decrease was observed at the onset of DNA synthesis (17 h after injection), but not at the the time of an earlier small cyclic AMP peak (13 h after injection) or at the time of maximal DNA synthesis (24 h after injection). The results indicate that the amount of the catalytic subunit of cyclic AMP-dependent protein kinases can be regulated independently from that of the regulatory subunits. The time-limited occurrence of the specific change in the amount of the C-subunit suggests that such a regulation is of physiological significance and that it may participate in cyclic AMP-mediated events involved in the control of cellular proliferation.

Immunocytochemical demonstration of cyclic AMP-dependent protein kinases in duct cells of the rat parotid gland.

The cyclic AMP-dependent protein kinases were immunolocalized in the rat parotid gland using a monospecific antiserum against their catalytic subunit. The kinases were found to be primarily located in the cytoplasm of the parotid duct cells with a preference for the apical cell region. The result questions the traditional view of the control of parotid gland secretion and suggests a role of cyclic AMP not only in the acinar protein secretion but also in ductal functions like fluid and electrolyte transport.

Quantitative changes in the catalytic and regulatory subunits of nuclear cAMP-dependent protein kinases type I and type II during isoproterenol-induced growth of the rat parotid gland.

To quantify the cAMP-dependent protein kinases I and II in parotid gland nuclei independent of the enzyme activity, monospecific antisera against their subunits were applied in a sensitive enzyme immunoassay. About 3% of total catalytic subunit in the homogenate was found in the isolated nuclei. During beta-agonist-induced proliferation of the parotid gland the nuclear concentration of catalytic and regulatory subunits changed. Related to the number of nuclei, the catalytic subunit and the regulatory subunit RI increased about 3-fold whereas the regulatory subunit RII remained unchanged.

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.

Determination of cyclic AMP-dependent protein kinase subunits by an immunoassay reveals a different subcellular distribution of the enzyme in rat parotid than does determination of the enzyme activity.

The distribution of cyclic cAMP-dependent protein kinase in subcellular fractions of rat parotid was determined by two independent biochemical methods, measurement of kinase catalytic activity or by quantitation of the catalytic and regulatory subunits in an enzyme-linked immunosorbent assay using monospecific antibodies. The major amount (85%) of the catalytic activity was found associated with the 100,000 g soluble fraction, whereas only 1/3 of the total catalytic subunit was demonstrated in the soluble fraction by the immunoassay. The immunoassay results furthermore indicated that approximately 50% of the total cellular protein kinase was associated with the extranuclear particulate fraction and that the predominant form of the kinase in the particulate fractions was the type II isoenzyme. The reasons for the differences in the distribution of the protein kinase demonstrated by the two methods were examined. Incomplete extraction of membrane-bound protein kinase and the influence of membrane localized ATPases on activity measurements were, at least in part, responsible for the low percentage of kinase activity measured in the particulate fractions. These results emphasize that the precise quantitation of protein kinase subunits merits investigation by more than one method. For the parotid, the finding that approximately 2/3 of the total catalytic subunit may be particulate associated provides additional evidence that cyclic AMP could be involved in membrane mechanisms of hormone-regulated secretion.

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.

Dibutyryl cAMP treatment of neuroblastoma-glioma hybrid cells results in selective increase in cAMP-receptor protein (R-I) as measured by monospecific antibodies.

The absolute levels of cAMP-dependent protein kinase (cAMP-dPK) subunits (R-I, R-II and C) and cGMP-dependent protein kinase (cGMP-dPK) holoenzyme were studied in neuroblastoma-glioma hybrid cells before and after dibutyryl-cAMP (Bt2cAMP) treatment which results in differentiation of these cells. The levels were determined by two different techniques utilizing antibodies which had been raised against each individual purified protein kinase subunit (or the holoenzyme in the case of the cGMP-dPK). Electrophoretic transfer of samples from SDS-polyacrylamide gels to nitrocellulose paper, followed by immunolabeling of protein kinase subunits with their respective antibodies and [125I]Protein A, demonstrated the monospecific nature of the antibodies, and a selective, several-fold increase in the R-I subunit in Bt2cAMP-treated cells, with no change in the level of R-II or C subunits. A simple enzyme-linked immunosorbent assay (ELISA) capable of measuring nanogram amounts of the various subunits confirmed the selective increase in the R-I subunit. ELISA assay results also indicated that the R-I subunits present before and after Bt2cAMP treatment are antigenically homologous. In conclusion, the specific, sensitive immunological methods described here demonstrate the capacity of neuroblastoma-glioma hybrid cells to regulate separately the levels of the two distinct subunits (R-I and C) of the Type I cAMP-dPK.

Determination and comparative analysis of the catalytic subunit of adenosine 3',5'-cyclic phosphate-dependent protein kinase by an enzyme-linked immunosorbent assay.

A specific antiserum against bovine heart catalytic subunit was used for the determination of the catalytic subunit in an enzyme-linked immunosorbent assay. Under the conditions elaborated the assay has a lower detection limit for catalytic subunit of 0.25 pmol/ml. In crude bovine heart extracts the concentration of catalytic subunit was determined by this method to be 0.18 +/- 0.02 mumol/kg wet wt. The immunochemical comparison of various animal species and cells, including organisms like amoebae and yeast, shows the broad applicability of the assay and provides evidence that the catalytic subunit is a highly conserved molecule.

Modulation of nuclear protein kinase activity and phosphorylation of histone H1 subspecies during the prereplicative phase of rat liver regeneration.

We have measured nuclear protein kinase activity during the prereplicative phase of rat liver regeneration. Total nuclear protein kinase activity increased significantly 15-18 h after partial hepatectomy, with the peak of activity occurring at 16 h. DEAE-Sephacel chromatography resolved nuclear protein kinase activity into two cAMP-independent (Ib and II) and two cAMP-dependent (Ia and III) protein kinases. Sixteen h after partial hepatectomy, there was a marked increase in the activities of the nuclear cAMP-dependent protein kinases and a decrease in the activity of nuclear cAMP-independent protein kinase II. Characterization of the two nuclear cAMP-dependent protein kinases revealed them to be identical with the cytosolic type I and II isozymes. Immunotitration of nuclear catalytic subunit and densitometric analysis of autoradiographs from 8-azido-[32P]cAMP-labeled nuclear RI revealed increases in both subunits 16 h afer partial hepatectomy. Concomitantly with the observed increase in nuclear protein kinase activity, we have observed an increase in the phosphorylation of histone H1 subspecies. Administration of the beta-adrenergic antagonist DL-propranolol, which has been shown to cause delays of equal duration in both the second phase of increased intracellular cAMP levels and the initiation of DNA synthesis (MacManus, J. P., Braceland, B. M., Youdale, T., and Whitfield, J. F. (1973) J. Cell. Physiol. 82, 157-164), results in an equivalent delay of increased nuclear protein kinase activity. Colchicine, which has previously been shown to prevent the onset of DNA synthesis (Walker, P. R., and Whitfield, J. F. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1394-1398), also prevents the increased protein kinase activity normally observed 16 h after partial hepatectomy. We conclude that the onset of DNA synthesis in the regenerating rat liver is preceded by a cAMP-mediated translocation of type I and type II cAMP-dependent protein kinase to the nucleus and phosphorylative modification of histone H1 subspecies. The inhibitory effects of propranolol and colchicine suggest a common cAMP-mediated, colchicine-sensitive link between protein kinase translocation and the initiation of DNA synthesis.