Identification and differential expression of two forms of regulatory subunits (RII) of cAMP-dependent protein kinase II in Friend erythroleukemic cells. Differentiation and 8-bromo-cAMP elicit a large and selective increase in the rate of biosynthesis of only one type of RII

The concentration of regulatory subunits (R) of type II cAMP-dependent protein kinase increased 4- to 5-fold when Friend erythroleukemic cells were either grown in medium containing 0.5 mM 8-bromo-cAMP and 0.2 mM methylisobutylxanthine or stimulated to differentiate. Two species of RII with apparent Mr values of 54,000 (RII-54) and 52,000 (RII-52) are expressed in Friend cells. Both forms of RII were (a) covalently labeled with 8-N3-[32P]cAMP, (b) phosphorylated by the catalytic subunit of protein kinase II, and (c) complexed by polyclonal anti-RII IgGs. RII-52 and RII-54 were not interconverted by phosphorylation or dephosphorylation. A monoclonal antibody that recognizes an internal site in RII resolved the two cAMP-binding proteins by preferentially binding RII-54. The structural diversity suggested by the monoclonal antibody experiment was further examined by comparing two-dimensional maps of tryptic peptides obtained from metabolically labeled [( 35S]met) RII-52 and RII-54. Groups of 35S-labeled peptides that were either uniquely derived from RII-54 or obtained only from RII-52 were readily distinguished, thereby demonstrating that Friend cells produce two separate and distinct forms of type II cAMP-binding subunits. The relative rate of synthesis of RII-52 increased 12- to 14-fold during erythroid differentiation and treatment with 8-bromo-cAMP, while the rate of RII-54 synthesis either declined slowly or was unchanged. Thus, two homologous forms of RII are subject to different modes of physiological (differentiation) and pharmacological (chronic 8-Br-cAMP) regulation, and the accumulation of total RII observed in the present and previous (Schwartz, D. A., and Rubin, C. S. (1983) J. Biol. Chem. 258, 777-784) studies results from a selective increase in the rate of biosynthesis of RII-52.     

Hormonal regulation of nuclear cyclic AMP-dependent protein kinase subunit levels in rat ovaries

Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear cAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity labeling of nuclear extracts with 8-azido-[32P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estrogen-primed hypophysectomized rats. After follicle-stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, Mr = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity. Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density. These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.     

cAMP-dependent protein kinases in the rat testis: regulatory and catalytic subunit associations.

Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RI alpha, RI beta, RII alpha, and RII beta, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14-15-day-old rats. Following separation by DEAE-cellulose, R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14-15 day-old rats, showed the presence of a prominent type I holoenzyme containing RI alpha subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RII beta subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominantly RII alpha and, to a lesser extent RII beta subunits (peak 3). The 53 kDa RI beta protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14-15-day-old, but not adult, rats exhibited high levels of C subunit-free RI alpha, a result not predicted by mRNA studies. This latter result may be attributable to direct RI alpha regulation or to indirect RII beta regulation at a time during testis development prior to germ cell maturation.     

Cyclic AMP-dependent protein kinase in canine pancreatic rough endoplasmic reticulum

A canine pancreas homogenate was subfractionated by several differential centrifugation steps. The distribution of cAMP-dependent protein kinase in the various fractions was monitored by assaying [3H]cAMP binding and photo-cross-linking of the regulatory subunits of the enzyme (RI and RII) with radiolabeled 8-azido-cAMP. The distribution of the kinase was also compared to that of markers for the plasma membrane, the endoplasmic reticulum and the cytosol. While our results confirm previous studies suggesting the presence of cyclic AMP-dependent protein kinase in the cytosol and Golgi, a significant amount of the total [3H] cAMP binding and photolabeled R subunits (both RI and RII) were found in rough microsomes (RM). The association is relatively resistant to extraction with EDTA, low and high ionic strength solutions. These extractions unmasked several new phosphorylation substrates in the "stripped" RM that were inaccessible in the RM, possibly because they were covered by ribosomes or peripheral membrane proteins. RII with a molecular mass of 52 kDa (RII-52 kDa) was the predominant RII found in the cytosolic fraction, whereas RII-52 kDa and RII with a molecular mass of 54 kDa (RII-54 kDa) were approximately equally enriched in the RM fraction. The mobility of the RII-52 kDa-photolabeled band could be shifted to the mobility of the RII-54 kDa band by phosphorylation with purified catalytic subunit and ATP, indicating that they represent "dephospho" and "phospho" forms of RII, respectively. A more precise localization to the rough endoplasmic reticulum was accomplished by isopycnic floatation in sucrose gradients. The enzyme cobanded at the density of rough microsomes and shifted to the lower density of "stripped" microsomes after treatment with puromycin/high salt, which specifically removes ribosomes.     

Protein kinase C activation selectively increases mRNA levels for one of the regulatory subunits (RI alpha) of cAMP-dependent protein kinases in HT-29 cells

We have examined the effect of the protein kinase C activator, TPA, on mRNA levels for subunits of cAMP-dependent protein kinases in the human colonic cancer cell line HT-29, subline m2. Messenger RNA for the regulatory subunit, RI alpha, of cAMP-dependent protein kinases was shown to be present and regulated by TPA. Other mRNAs for subunits of cAMP-dependent protein kinases (RI beta, RII alpha, RII beta, C alpha, C beta) were also present in these cells, but revealed no or only minor changes upon TPA stimulation. When HT-29 cells were cultured in the presence of 10 nM TPA for various time periods, a biphasic response was observed in RI alpha mRNA levels with a maximal increase (approximately 4 fold) after 24 hours. TPA stimulated RI alpha mRNA increased in a concentration-dependent manner and maximal response (4-8 fold) was seen at 3-10 nM. The TPA-induced increase in RI alpha mRNA was not obtained when cells were incubated with TPA together with the protein kinase C inhibitors, staurosporine or H7. The cAMP-analog 8-CPTcAMP alone induced RI alpha mRNA levels 50% more than TPA. Combined treatment with TPA (10 nM) and 8-CPTcAMP (0.1 mM) gave an increase in RI alpha mRNA similar to TPA. These results demonstrate an interaction between the protein kinase C pathway and mRNA levels for the RI alpha subunit of cAMP-dependent protein kinases in HT-29 cells.     

8-Chloro-cAMP inhibits transforming growth factor alpha transformation of mammary epithelial cells by restoration of the normal mRNA patterns for cAMP-dependent protein kinase regulatory subunit isoforms which show disruption upon transformation.

Differential regulation of the regulatory subunits of cAMP-dependent protein kinase isozymes correlates with the growth inhibitory effect of site-selective 8-Cl-cAMP demonstrated in cancer cell lines (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros, D., Ogreid, D., D?skeland, S.O., Jahnsen, T., and Cho-Chung, Y.S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6319-6322). Such selective modulation of protein kinase isozyme regulatory subunits was also found in the 8-Cl-cAMP-induced inhibition of both transformation and transforming growth factor alpha (TGF alpha) production in Ki-ras-transformed rat kidney fibroblasts (Tortora, G., Ciardiello, F., Ally, S., Clair, T., Salomon, D. S., and Cho-Chung, Y. S. (1989) FEBS Lett. 242, 363-367). In this work, we have demonstrated that 8-Cl-cAMP antagonizes the TGF alpha effect in TGF alpha-transformed mouse mammary epithelial cells (NOG-8TFC17) at the level of gene expression for cAMP receptor protein isoforms, RI and RII (the regulatory subunits of protein kinase isozymes). Northern blot analysis demonstrated that in the transformed NOG-8TFC17 cells, compared with the nontransformed counterpart NOG-8 cells, the mRNA levels for the RI alpha cAMP receptor protein markedly increased, whereas the mRNA levels for the RII alpha and RII beta cAMP receptor proteins decreased. 8-Cl-cAMP, which induced growth inhibition and phenotypic reversion in NOG-8TFC17 cells, caused an inverse change in the mRNA patterns of the cAMP receptor proteins; RI alpha cAMP receptor mRNA sharply decreased to levels comparable with that of the nontransformed NOG-8 cells, whereas RII beta mRNA increased to a level even greater than that in the NOG-8 cells. In addition, one mRNA species of RII alpha increased, whereas the other RII alpha mRNA species decreased during the treatment. The mRNA level for the catalytic subunit of protein kinase, however, did not change during 8-Cl-cAMP treatment. In addition, 8-Cl-cAMP brought about a reduction in both TGF alpha mRNA and protein levels. These coordinated changes in the expression of the cAMP receptor proteins and TGF alpha were not observed during cis-hydroxyprolineor TGF beta-induced growth inhibition of the NOG-8TFC17 cells. Thus, the antagonistic effect of 8-Cl-cAMP toward TGF alpha-induced transformation involves modulation of the expression of a specific set of cellular genes.     

Molecular cloning, cDNA structure and deduced amino acid sequence for the hormone-induced regulatory subunit (RII beta) of cAMP-dependent protein kinase from rat ovarian granulosa cells

The regulatory subunit of cAMP-dependent protein kinase designated RII beta (RII51) has previously been shown to be the product of a separate gene. This was accomplished by the molecular cloning of a partial cDNA clone estimated to lack 30-45 nucleotides of the 5' end of the coding region. We hereby report the isolation of a cDNA clone for RII beta from rat granulosa cells, extending 43 nucleotides further 5' compared with the previously published cDNA sequence, and from which the entire amino acid sequence (415 residues) of the rat RII beta protein can be deduced. A cAMP regulated mRNA of 3.2 kilobases (kb) for RII beta was detected by the isolated cDNA in rat Sertoli cells.     

Cyclic AMP-dependent protein kinase type I mediates the inhibitory effects of 3',5'-cyclic adenosine monophosphate on cell replication in human T lymphocytes.

Human T lymphocytes were used as a model system to study the expression and roles of cAMP-dependent protein kinase isozymes (cAKI and cAKII) in cAMP-induced inhibition of cell replication. Human peripheral blood T lymphocytes expressed mRNA for the alpha-subforms (RI alpha and RII alpha) of the regulatory subunits of cAKI and cAKII and for the alpha- and beta-subforms (C alpha and C beta) of the catalytic subunits of cAK. At the protein level, RI alpha represented approximately 75% of the total R subunit activity, whereas RII alpha (phospho and dephospho forms) accounted for the remaining 25%. RII beta was not detected at either the mRNA or the protein level. The RI alpha protein was mainly (greater than 75%) cytosolic, whereas RII alpha was almost exclusively (greater than 90%) particulate associated. Treatment of proliferating T lymphocytes (activated through the CD3 cell surface marker) with 10 different cAMP analogs demonstrated that all inhibited cell replication in a concentration-dependent manner. The potency (as measured by the concentration giving 50% inhibition, IC50) of the cAMP analogs ranged from 30 microM for 8-chlorophenylthio-cAMP to 1100 microM for 8-piperidino-cAMP. A cAMP analog pair directed to activate cAKI (8-aminohexylamino-cAMP and 8-piperidino-cAMP) synergized in the inhibition of T lymphocyte proliferation, whereas a cAKII-directed cAMP analog pair (8-chlorophenylthio-cAMP and N6-benzoyl-cAMP) did not. We conclude that activation of cAKI is sufficient to inhibit T lymphocyte proliferation. The membrane-bound cAKII may mediate cAMP actions not related to cell replication.     

Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.     

Identification and characterization of novel PKA holoenzymes in human T lymphocytes

Cyclic AMP-dependent protein kinase (PKA) is a holoenzyme that consists of a regulatory (R) subunit dimer and two catalytic (C) subunits that are released upon stimulation by cAMP. Immunoblotting and immunoprecipitation of T-cell protein extracts, immunofluorescence of permeabilized T cells and RT/PCR of T-cell RNA using C subunit-specific primers revealed expression of two catalytically active PKA C subunits C alpha1 (40 kDa) and C beta2 (47 kDa) in these cells. Anti-RI alpha and Anti-RII alpha immunoprecipitations demonstrated that both C alpha1 and C beta2 associate with RI alpha and RII alpha to form PKAI and PKAII holoenzymes. Moreover, Anti-C beta2 immunoprecipitation revealed that C alpha1 coimmunoprecipitates with C beta2. Addition of 8-CPT-cAMP which disrupts the PKA holoenzyme, released C alpha1 but not C beta2 from the Anti-C beta2 precipitate, indicating that C beta2 and C alpha1 form part of the same holoenzyme. Our results demonstrate for the first time that various C subunits may colocate on the same PKA holoenzyme to form novel cAMP-responsive enzymes that may mediate specific effects of cAMP.     

Hormonal regulation of the synthesis and mRNA content of the regulatory subunit of cyclic AMP-dependent protein kinase type II in cultured rat ovarian granulosa cells

These studies were undertaken to determine the molecular events by which estradiol and follicle-stimulating hormone (FSH) stimulate in ovarian granulosa cells the increase in the content of one of the regulatory subunits of cAMP-dependent protein kinase type II, RII51 (Mr = 51,000), and its electrophoretic variants RII51.5 (Mr = 51,500) and RII52 (Mr = 52,000). To analyze the de novo synthesis of RII51/52, granulosa cells were cultured (10(6) cells/ml) for 0, 12, 24, or 48 h with estradiol (10 nM) +/- FSH (12.5, 25, and 50 ng/ml), 8-bromo-cAMP (0.25-3 mM), or forskolin (0.5-100 microM) and then pulse-labeled with [35S]methionine (300 microCi/ml; 4 h). Labeled RII51, present either in urea extracts of total cellular protein or after partial purification from a soluble cell extract by cAMP-Sepharose chromatography, was quantitated by autoradiography of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and by excision of the silver-stained spots of the RII51 variants from the gels and counting. Synthesis of RII51 and its electrophoretic variants was low in cells cultured with estradiol alone for 48 h, whereas it was increased 4-5-fold in cells cultured with estradiol and FSH. Changes in the synthesis of actin were minor throughout the culture period regardless of hormone treatment. Pulse-chase experiments using [35S]methionine provided evidence that the isoelectric variants RII51.5 and RII52 may be derived from RII51 by post-translation modification, such as phosphorylation. Labelling with [32P]orthophosphate showed that RII52 contained more radioactivity than RII51.5 and RII51. Northern and filter hybridization assays demonstrated a 6-10-fold dose- and time-dependent increase in the amount of RII51 mRNA in granulosa cells exposed to estradiol and FSH or estradiol and forskolin compared to those cultured with estradiol alone. In vitro translation of poly(A)+ mRNA of granulosa cells from estradiol- and FSH-treated hypophysectomized rats also demonstrated an increase in the content of translatable RII51 mRNA. These studies indicate that in cultured rat granulosa cells the synthesis of RII51 and the content of its mRNA are selectively increased by estradiol and cAMP in a time- and dose-dependent manner. Based on these observations, RII51 appears to be a useful marker to determine the molecular (genomic?) sites of estradiol and FSH action in differentiating rat granulosa cells.