RII Beta Research Articles

Identification of a high affinity binding protein for the regulatory subunit RII beta of cAMP-dependent protein kinase in Golgi enriched membranes of human lymphoblasts.

Immunocytochemical evidence of an association between the regulatory subunit RII of the cAMP-dependent protein kinase (cAMP-PK) and the Golgi apparatus in several cell types has been reported. In order to identify endogenous Golgi proteins binding RII, a fraction enriched in Golgi vesicles was isolated from human lymphoblasts. Only the RII beta isoform was detected in the Golgi-rich fraction, although RII alpha has also been found to be present in these cells. A 85 kDa RII-binding protein was identified in Golgi vesicles using a [32P]RII overlay of Western blots. The existence of an endogenous RII beta-p85 complex in isolated Golgi vesicles was demonstrated by two independent means: (i) co-immunoprecipitation of both proteins under non-denaturing conditions with an antibody against RII beta and (ii) co-purification of RII beta-p85 complexes on a cAMP-analogue affinity column. p85 was phosphorylated by both endogenous and purified catalytic subunits of cAMP-pKII. Extraction experiments and protease protection experiments indicated that p85 is an integral membrane protein although it partitioned atypically during Triton X-114 phase separation. We propose that p85 anchors RII beta to the Golgi apparatus of human lymphoblasts and thereby defines the Golgi substrate targets most accessible to phosphorylation by C subunit. This mechanism may be relevant to the regulation of processes involving the Golgi apparatus itself, such as membrane traffic and secretion, but also relevant to nearby nuclear events dependent on C subunit.

Identification and characterization of the GC-rich and cyclic adenosine 3',5'-monophosphate (cAMP)-inducible promoter of the type II beta cAMP-dependent protein kinase regulatory subunit gene.

A rat genomic clone containing 4.5 kilobases of 5'-flanking DNA and the first exon of the type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase was isolated, restriction mapped, and sequenced. The proximal 400-basepair promoter region was GC rich, lacked TATA/CAAT box motifs, and initiated transcription at multiple sites. Bandshifting and DNase-I footprinting experiments using this region of the RII beta promoter detected several related specific DNA-protein complexes formed using crude and fractionated nuclear extracts from rat ovary, brain, adrenal gland, and liver. All binding in these experiments mapped to a domain within the same region found to confer cAMP inducibility to a chloramphenicol acetyltransferase (CAT) reporter gene when transfected into primary cultures of rat granulosa cells. Although GC boxes (putative SP1-binding sites) and activator protein-2 (AP-2) elements were present in this functional region, and although expression vectors containing AP-2 sites conferred high levels of cAMP regulation of the CAT gene in cultured ovarian cells, neither the GC boxes nor the AP-2 sites were protected by footprint analyses or required for band shift activity of nuclear extract protein. These known regulatory elements, therefore, may be involved in functional activity of the RII beta promoter, but additional cis-acting DNA and trans-acting factors (yet to be characterized) also appear to interact with the functional promoter of the RII beta gene and regulate the hormone-specific expression of the A-kinase subunit in ovarian and neuronal cells.

Protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate modulates messenger ribonucleic acid levels for two of the regulatory subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinases (RII beta and RI alpha) via multiple and distinct mechanisms.

Messenger RNAs (mRNA) for two of the regulatory subunits of cAMP-dependent protein kinases (PKA), RII beta and RI alpha, are transiently (maximal levels at 6 h) stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in cultured rat Sertoli cells in a time- and concentration-dependent manner. Whereas TPA (10(-7) M) stimulated RII beta mRNA 11 +/- 2.8 fold (mean +/- SEM), mRNA levels for RI alpha increased only 2.5 +/- 0.6-fold (mean +/- SEM). No effects of TPA on the other subunits of PKA (RII alpha, C alpha) were observed. TPA-dependent accumulation of mRNAs for RII beta and RI alpha was observed to the same extent in nucleus and cytoplasm. We have previously shown that mRNA levels for all the PKA subunits are increased by cAMP, particularly that of RII beta (greater than 50-fold). TPA modulated the stimulatory effects of cAMP on RII beta and RI alpha mRNAs in opposite directions. Whereas treatment with both 8-CPTcAMP and TPA gave an additive effect on RI alpha mRNA, TPA reduced the cAMP-dependent increase in RII beta mRNA. Although the mRNA for RII beta had returned to basal levels after 24 h of incubation with TPA, the presence of TPA still inhibited cAMP-dependent induction of mRNA for RII beta. In contrast, similar TPA treatment did not influence the subsequent cAMP-dependent stimulation of RI alpha mRNA. Preincubation with 8-CPTcAMP did not influence TPA-dependent stimulation of mRNAs for either RII beta or RI alpha. TPA induction of RII beta mRNA was completely blocked by cycloheximide (an inhibitor of protein synthesis), whereas that of RI alpha was not. The inhibitory effect of TPA on cAMP stimulation of RII beta mRNA was independent of ongoing protein synthesis. These results indicate that TPA induction of mRNAs for RI alpha and RII beta involves multiple and distinct mechanisms. The stimulatory effect of TPA on RI alpha mRNA levels and the inhibitory effect of TPA on cAMP-stimulated RII beta mRNA expression are probably mediated through stable factors, whereas proteins with rapid turnover or factors induced by TPA are involved in the stimulatory effect of TPA on RII beta mRNA.

Identification, purification, and characterization of subunits of cAMP-dependent protein kinase in human testis. Reverse mobilities of human RII alpha and RII beta on sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared with rat and bovine RIIs.

We have previously identified and characterized regulatory (R) subunits of cyclic AMP-dependent protein kinase, particularly the RII subunits in rat tissues (Jahnsen, T., Lohmann, S. M., Walter, U., Hedin, L., and Richards, J. S. (1985) J. Biol. Chem. 260, 15980-15987; Jahnsen, T., Hedin, L., Lohmann, S. M., Walter, U., and Richards, J. S. (1986) J. Biol. Chem. 261, 6637-6639; Jahnsen, T., Hedin, L., Kidd, V. J., Beattie, W. G., Lohmann, S. M., Walter, U., Durica, J., Schulz, T. Z., Schiltz, E., Browner, M., Lawrence, C. B., Goldman, D., Ratoosh, S. L., and Richards, J. S. (1986) J. Biol. Chem. 261, 12352-12361). These studies showed that rat RII alpha and RII beta had apparent molecular masses of 54 and 52 kDa, respectively. The aim of the present study was to purify and characterize cAMP-dependent protein kinase R subunits in human testis and to examine which of the subunits (mRNAs and proteins) are present in this tissue. Our results show that human testis contains mRNAs for five out of the seven known subunits of cAMP-dependent protein kinase. We observed strong expression of mRNAs for RI alpha (1.5 and 3.2 kilobases (kb)), RII alpha (2.2, 2.4, and 7.0 kb), and RII beta (3.3 kb). We also demonstrated mRNAs for two of the three catalytic subunits, C alpha (2.7 kb) and C gamma (1.7 kb). Purification of R subunits by DEAE-cellulose and cAMP affinity chromatography revealed three distinct forms with apparent molecular masses of 49, 51, and 53 kDa, respectively. Characterization of these R subunits by their 8-azido-cAMP photoaffinity labeling and immunoreactivity, as well as by a phosphorylation-dependent mobility shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicated subunit sizes of RII beta (53 kDa) greater than RII alpha dephosphoform (51 kDa) greater than RI alpha (49 kDa). This conclusion was verified by the analysis of RII subunits produced by in vitro transcription/translation of full-length cDNAs for both human RII alpha and RII beta in wheat germ lysates. The in vitro translated products were the same size as the purified human testis subunits, and only the smallest RII subunit (RII alpha) revealed a distinct mobility shift on SDS-PAGE after phosphorylation/dephosphorylation. This study supports the conclusion that the mobilities of human RII subunits (RII alpha, RII beta) on SDS-PAGE are reversed in contrast with those of other species such as rat and bovine.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate modulates messenger ribonucleic acid levels for two of the regulatory subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinases (RII beta and RI alpha) via multiple and distinct mechanisms

Protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate modulates messenger ribonucleic acid levels for two of the regulatory subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinases (RII beta and RI alpha) via multiple and distinct mechanisms

Cloning and expression of an intron-less gene for AKAP 75, an anchor protein for the regulatory subunit of cAMP-dependent protein kinase II beta.

The A-Kinase Anchor Protein AKAP 75 (formerly designated bovine brain P75) is a particulate brain protein that avidly binds the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (Bregman, D. B., Hirsch, A.H. and Rubin, C.S. (1991) J. Biol. Chem. 266, 7207-7213). The formation of stable AKAP 75.RII beta complexes provides a potential mechanism for targeting physiological signals carried by cAMP to specific effector sites within neurons and other brain cells. We have now cloned and characterized the AKAP 75 gene. Its coding sequence is novel and unexpectedly short (1284 base pairs) and contains no introns. When the AKAP 75 gene was transfected into HEK 293 cells, a new RII beta-binding protein with an apparent Mr of 75,000 accumulated. A high proportion (approximately 65%) of the AKAP 75 gene product was excluded from the cytoplasm and was recovered in the 40,000 x g pellet derived from disrupted transfected cells. In contrast, cells transfected with a construct encoding 249 amino acids from the central and C-terminal regions of AKAP 75 produced an RII beta-binding protein (apparent Mr = 45,000) that was exclusively cytosolic. AKAP 75 is a novel protein composed of only 428 amino acid residues (Mr = 47,878). A highly acidic C-terminal region mediates the binding of RII beta (and cAMP-dependent protein kinase II beta), whereas a positively charged N-terminal segment contains structural features that are essential for the association of AKAP 75 with the cytoskeleton and/or intracellular membranes.

5097026 DNA for human regulatory subunits (RI alpha and RII beta) of cAMP-dependent protein kinases: Tor Jahnsen, Ski, Norway

5097026 DNA for human regulatory subunits (RI alpha and RII beta) of cAMP-dependent protein kinases: Tor Jahnsen, Ski, Norway

Hormonal activation of gene transcription in ras-transformed NIH3T3 cells overexpressing RII alpha and RII beta subunits of the cAMP-dependent protein kinase.

ras-Transformed NIH3T3 (R3T3) cells were transfected with expression vectors for the RII alpha and RII beta regulatory subunits of the type II isozyme of cAMP-dependent protein kinase, and the effects on gene activation by corticotropin-releasing factor (CRF) and prostaglandin E1 (PGE1) were analyzed. In RII alpha and RII beta-overexpressing cells, type II isozyme levels were increased, and type I isozyme levels were eliminated, demonstrating that both RII regulatory subunits compete efficiently with RI for catalytic subunit. The type II isozyme separated into three peaks on high performance liquid chromatography, referred to as A, B, and C. Western blot analysis strongly suggests that peak A and peak C correspond to holoenzymes containing RII beta and RII alpha, respectively. Overexpression of RII alpha resulted in the loss of peak A and a dramatic reduction in RII beta protein with no change in RII beta mRNA, indicating that the level of RII beta protein is controlled posttranscriptionally and that RII beta protein may become unstable when displaced from C. The role of type I and II kinases in transcriptional activation was investigated by comparing the response of control and RII expressing clones to site-selective cAMP analogs and the hormones, CRF and PGE1. The site-selective analogs demonstrated that either type I or type II kinase could activate the cAMP-responsive alpha-subunit promoter. The response to various concentrations of CRF or PGE1 was identical in control cells and transfected clones containing very little type I kinase. These experiments suggest that in the CRF and PGE1 response pathways leading to gene induction, the magnitude and sensitivity of the response are not influenced by the presence or absence of type I cAMP-dependent protein kinase.

Adenosine 3',5'-monophosphate-dependent stabilization of messenger ribonucleic acids (mRNAs) for protein kinase-A (PKA) subunits in rat Sertoli cells: rapid degradation of mRNAs for PKA subunits is dependent on ongoing RNA and protein synthesis.

cAMP treatment of primary cultures of Sertoli cells is associated with a transient stimulatory effect on mRNA levels for various protein kinase-A (PKA) subunits. We have previously shown that the induction of mRNA for regulatory subunit II beta (RII beta) is due at least partly to transcriptional activation. In the present study we investigate possible regulatory effects of (Bu)2cAMP on the degradation of mRNAs for various PKA subunits in rat Sertoli cells. We demonstrate subunit specific differences in the decay of mRNAs for the various PKA subunits. When (Bu)2cAMP was removed from Sertoli cell cultures after 6 h of stimulation, there was a rapid decay of mRNAs for both RII beta and RI alpha (half-lives, approximately 3 h). In contrast, mRNA levels for RII alpha continued to increase. Removal of (Bu)2cAMP after a longer period of treatment revealed a similar decay of mRNAs for all of the PKA subunits, with half-lives of approximately 3 h. Incubation of Sertoli cells for 12 h with (Bu)2cAMP, followed by continued incubation in the absence and presence of (Bu)2cAMP as well as in the presence of actinomycin-D (an inhibitor of RNA synthesis), revealed (Bu)2cAMP mediated stabilization of mRNA for the RII beta subunit. Interestingly, actinomycin-D as such stabilized mRNAs for all PKA subunits. Similar treatment with cycloheximide (an inhibitor of protein synthesis) revealed distinct differences between the RI alpha and C alpha subunits vs. the RII subunits; cycloheximide reduced the decay of both RII beta and RII alpha mRNAs, whereas steady state levels of mRNAs for RI alpha and C alpha actually increased after cycloheximide treatment of previously (Bu)2cAMP-stimulated cultures. Cycloheximide treatment also increased basal levels of mRNAs for RI alpha and C alpha, whereas basal levels of RII beta and RII alpha mRNAs were not influenced. These studies indicate that the degradation of mRNAs for the various PKA subunits is subject to different regulation by (Bu)2cAMP, and that ongoing RNA and protein synthesis is required for rapid degradation of all PKA subunits.

Identification, characterization, and hormonal regulation of 3', 5'-cyclic adenosine monophosphate-dependent protein kinases in rat Sertoli cells.

Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA) at the protein and messenger RNA (mRNA) levels. The purpose of the present study was to identify, characterize, and quantify individual R subunits in rat Sertoli cells both at the mRNA and protein levels. Unstimulated Sertoli cells contain high levels of R (approximately 9.2 +/- 0.8 pmol/mg protein) and C (approximately 7.3 +/- 0.7 pmol/mg protein). Stimulation with (Bt)2cAMP (0.1 mM) for 24 and 48 h revealed a time-dependent increase in [3H]cAMP-binding activity. During the same time period the catalytic activity remained relatively constant, resulting in an increase in the R/C ratio from approximately 1.3 to 3.0. Using diethylaminoethyl cellulose chromatography, 8-N3-[32P]cAMP photoaffinity labeling, autophosphorylation by gamma-[32P]ATP, and specific antibodies, we show that unstimulated Sertoli cells contain approximately 75% RI alpha, 25% RII alpha, and very low levels of RII beta. Stimulation of Sertoli cells with (Bt)2cAMP (0.1 mM, 48 h) was associated with a 2.1-fold increase in RI alpha (6.6-14 pmol/mg) and a 10- to 20-fold increase in RII beta (less than 0.1-1.1 pmol/mg), with little or no change in RII alpha (1.9-2.3 pmol/mg). Treatment with cAMP was associated with a slight increase in RI/RII ratio (3.3-4.1). mRNA levels for RII beta increased 30- to 50-fold after (Bt)2cAMP stimulation, whereas only minor changes in mRNA levels for RI alpha, RII alpha, and C alpha were observed (1.5- to 2.0-fold). mRNA levels for RI beta, C beta, and C gamma were not detected in either unstimulated or in cAMP-stimulated Sertoli cells. It is concluded that chronic treatment with cAMP changes the relative proportion of R subunits of PKA in a manner reflecting the changing levels in respective mRNAs. Furthermore, such treatment is associated with the appearance of a new PKA R subunit (RII beta), which is absent in untreated Sertoli cells.

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.

Molecular characterization of bovine brain P75, a high affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II beta.

In mammalian brain, physiological signals carried by cAMP seem to be targeted to intraneuronal sites by the association of cAMP-dependent protein kinase II beta with anchoring proteins that bind the regulatory subunit (RII beta) of the enzyme. Previously, an RII beta-binding domain was characterized in a large (Mr approximately 150,000) candidate anchor protein, rat brain P150 (Bregman, D. B., Bhattacharyya, N., and Rubin, C. S. (1989) J. Biol. Chem. 264, 4648-4656). RII beta-binding proteins with Mr values of 65,000-80,000 were detected in the brains of other species. Since little was known about the structural features of these lower Mr proteins, we undertook the characterization of bovine brain P75 as a prototype. A cDNA encoding 258 amino acid residues at the C terminus of P75 was cloned by probing a lambda gt11 expression library with 32P-RII beta. The cDNA insert was ligated into the pET-3b expression plasmid, and large amounts of the partial P75 polypeptide (designated P47) were produced in Escherichia coli. A purification scheme that yielded 9 mg of soluble P47 from a 1-liter bacterial culture was devised. Antibodies directed against the P47 polypeptide revealed that P75 is expressed almost exclusively in brain. The sequence of 117 amino acid residues at the C terminus of P75 contains the RII beta-binding site and is 80% identical to the corresponding region of P150. In contrast, a lower level of identity (36%) between P75 and P150 at a more N-terminal region indicates that the two RII beta-binding proteins are related, but distinct proteins. P75 is not homologous to microtubule-associated protein 2, an RII alpha-selective binding protein, or any other previously studied proteins. C-terminal truncation analysis disclosed that the final 26 residues in P75 are essential for binding RII beta.

Differentiation of HL-60 leukemia by type I regulatory subunit antisense oligodeoxynucleotide of cAMP-dependent protein kinase.

A marked decrease in the type I cAMP-dependent protein kinase regulatory subunit (RI alpha) and an increase in the type II protein kinase regulatory subunit (RII beta) correlate with growth inhibition and differentiation induced in a variety of types of human cancer cells, in vitro and in vivo, by site-selective cAMP analogs. To directly determine whether RI alpha is a growth-inducing protein essential for neoplastic cell growth, human HL-60 promyelocytic leukemia cells were exposed to 21-mer RI alpha antisense oligodeoxynucleotide, and the effects on cell replication and differentiation were examined. The RI alpha antisense oligomer brought about growth inhibition and monocytic differentiation, bypassing the effects of an exogenous cAMP analog. These effects of RI alpha antisense oligodeoxynucleotide correlated with a decrease in RI alpha receptor and an increase in RII beta receptor level. The growth inhibition and differentiation were abolished, however, when these cells were exposed simultaneously to both RI alpha and RII beta antisense oligodeoxynucleotides. The RII beta antisense oligodeoxynucleotide alone has been previously shown to specifically block the differentiation inducible by cAMP analogs. These results provide direct evidence that RI alpha cAMP receptor plays a critical role in neoplastic cell growth and that cAMP receptor isoforms display specific roles in cAMP regulation of cell growth and differentiation.

Signal transduction mechanisms through Fcγ receptors on the mouse macrophage surface

Mouse macrophages and macrophage cell lines such as P388D1 or J774 carry at least two distinct Fc gamma receptors (Fc gamma R): one specific for the Fc portion of IgG2a (Fc gamma aR, also classified as Fc gamma RI) and another for IgG2b (Fc gamma 2bR, also classified as Fc gamma RII beta). These Fc gamma Rs should transmit, upon binding of an appropriate ligand, a specific signal that leads to the regulation of macrophage functions, as the interaction of immune complex with cell surface receptor has been shown to lead to suppression of the humoral immune response or B cell differentiation, to the destruction of target cells by antibody-dependent cell-mediated cytotoxicity, to activation of arachidonic acid metabolic cascade, to the phagocytosis of opsonized particles, or to the generation of superoxide anion. In this review, we first describe evidence that Fc gamma 2aR and Fc gamma 2bR are associated with casein kinase II and phospholipase A2 activity, respectively. We will then discuss a potential role for these enzymatic activities in signal transduction pathways that leads to the activation of the arachidonic acid metabolic cascade and adenylate cyclase, to the regulation of phagocytosis, and to the suppression of interferon-gamma action to induce Ia antigens.

Fetal Development and Neuronal/Glial Cell Specificity of cAMP-Dependent Protein Kinase Subunit mRNAs in Rat Brain

All known actions of cAMP in the brain require cAMP-dependent protein kinase (cAMPdPK), which consists of regulatory (R) and catalytic (C) subunits (R2C2). Using homologous rat cDNAs for all known cAMPdPK subunit isoforms found in the brain (RI alpha, RI beta, RII alpha, RII beta, C alpha, C beta) we observe that, in the fetal rat brain from 12 days of gestation to birth, while alpha subunit (RI alpha, RII alpha, C alpha) mRNA levels are abundant, beta subunit (RI beta, RII beta, C beta) mRNA levels increase from undetectable or very low levels to abundant levels. Furthermore, while alpha subunit mRNA levels are abundant in both primary neuronal and primary glial cultures, beta subunit mRNA levels are very low (C beta) or undetectable (RI beta, RII beta) in primary glial cultures, but are abundant in primary neuronal cultures. Thus, prior to about 12 days of gestation, cAMP in the brain may act only via the alpha cAMPdPK subunits in neuronal and glial precursor cells. After 12 days of gestation, coincident with the onset of final cell division in neurons, beta cAMPdPK subunits may also mediate the effects of cAMP predominantly in neurons.

Different Mechanisms are Involved in cAMP-Mediated Induction of mRNAs for Subunits of cAMP-Dependent Protein Kinases

The present study addresses possible mechanisms through which cAMP mediates its effects on mRNA levels for the subunits of protein kinase A (PKA) and the cellular protooncogene, c-fos. Messenger RNAs for the PKA subunits (RI alpha, RII alpha, RII beta, and C alpha) were regulated by cAMP with similar kinetics in Sertoli cells. However, effects of cAMP on the PKA mRNAs were slow compared to a well characterized cAMP responsive gene, c-fos. The magnitude of stimulation was dramatically different between the various PKA subunits, in that RII beta mRNA increased more than 50-fold while the mRNAs for the other subunits were induced only two to four times. Separation of nuclear and cytoplasmic RNA demonstrated that mRNAs for PKA subunits were stimulated to the same extent in these two cellular compartments. The more rapid induction of c-fos mRNA by cAMP, compared to the mRNA for RII beta, was also seen at the level of transcription. Maximal transcription rate for c-fos, RI alpha, and C alpha were observed after 30 min, whereas that for RII beta was increasing during the 2-h period examined. Transcriptional activation of the RI alpha gene also appeared faster than that for RII beta. When Sertoli cells were incubated with 8-(4-chlorophenylthio) cAMP and cycloheximide, a potent inhibitor of protein synthesis, we observed a super-induction of the mRNAs for c-fos (10-fold) and RI alpha (2-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the MAP2- and P75-binding domain in the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase. Cloning and expression of the cDNA for bovine brain RII beta.

cDNA clones coding for the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in lambda gt11. The cDNA codes for a protein of 418 amino acids which is 98% homologous to the rat and human RII beta proteins. A series of expression vectors coding for truncated RII beta proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli. Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots. The expressed RII beta-fusion proteins were used in overlay assays to identify the region in RII beta which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C.S. (1984) J. Biol. Chem. 259, 9844-9846) in bovine brain. Fusion protein containing amino acids 1-50 of the RII beta NH2 terminus (RII beta(1-50)] bound to both MAP2 and P75 immobilized on nitrocellulose filters. A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H.M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J. (1989) Neuron 3, 631-638) also bound RII beta-fusion proteins containing RII beta amino acids 1-50. Three fusion proteins, RII beta(1-25), RII beta(25-96), and RII beta(1-265,25-96 deleted) did not bind to MAP2(31) nor P75. The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RII beta. Preincubation of bovine heart protein kinase II alpha and RII beta(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins.

Inhibition of growth and modulation of gene expression in human lung carcinoma in athymic mice by site-selective 8-Cl-cyclic adenosine monophosphate : Ally S, Clair T, Katsaros D et al. Cellular Biochemistry Section, Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. Cancer Res 1989;49:5650-5

Site-selective cyclic AMP (cAMP) analogues inhibit growth and induce changes in morphology in a spectrum of human cancer cell lines (D. Katsaros et al., FEBS Lett., 223:97, 1987). The cellular events underlying such effects of cAMP analogues include differential regulation of type I versus type II cAMP-dependent protein kinase isozymes (S. Ally et al., Proc. Natl. Acad. Sci. USA, 85: 6319, 1988). Infusion (i.p.) of 8-Cl-cAMP, the most potent site-selective cAMP analogue, for 7 days produced regression of LX-1 lung carcinoma in athymic mice in a dose-dependent manner. The tumor regression correlated with the changing levels of cAMP receptor proteins, RI alpha and RII beta, the regulatory subunits of cAMP-dependent protein kinase type I and type II, respectively. By photoaffinity labeling with 8-N3-[32P]cAMP and immunoblotting with a monospecific anti-RII antibody, RI alpha (Mr 49,000) and RII beta (Mr 51,000) were identified in the untreated control tumors. 8-Cl-cAMP treatment induced a rapid increase of both RI alpha and RII beta in tumor cytosols and translocation (within 1 h) of only RII beta from the cytosol to the nucleus. RII beta in both cytosols and nuclei remained elevated during 8-Cl-cAMP treatment, whereas RI alpha in the cytosols gradually decreased with time of treatment after its initial transient increase. Northern blot analyses demonstrated that the RII beta mRNA level increased within 6 h of 8-Cl-cAMP treatment and remained elevated during treatment, whereas the RI alpha mRNA level decreased to below that of the untreated control tumor level after its transient increase during 1-6 h of treatment. 8-Cl-cAMP treatment also caused a sharp decrease in both N-ras and c-myc mRNA levels. These results suggest that the fundamental basis for the antineoplastic activity of 8-Cl-cAMP may reside in the restoration of normal gene regulation in neoplasms in which cAMP receptor proteins play a role.

Type II regulatory subunit of protein kinase restores cAMP-dependent transcription in a cAMP-unresponsive cell line.

cAMP-dependent protein kinase appears to play a role in cAMP-induced gene expression in mammalian cells. There exist two major types of cAMP-dependent protein kinase, type I and type II, which are distinguished by their regulatory subunits, RI and RII, respectively. We investigated the role of type I and type II protein kinase in the cAMP-induced gene expression by either stable or co-transfection of RI alpha, RII alpha, or RII beta gene in an expression vector together with somatostatin-chloramphenicol acetyltransferase (SS-CAT) fusion gene using a cAMP-unresponsive mutant pheochromocytoma cell line (A126-1B2). Introduction of the RII beta gene restored the capability of these cells to induce the SS-CAT gene expression in response to forskolin stimulus and induced a changed morphology which resembled that of wild type. The RII alpha gene also induced SS-CAT gene expression but to a lesser degree than that achieved by the RII beta gene, whereas the RI alpha gene had no effect. The induction of SS-CAT gene expression by the RII beta gene was specifically blocked by the 21-mer RII beta antisense oligodeoxynucleotide. These results show for the first time that type II but not type I regulatory subunit of cAMP-dependent protein kinase is essential for a cAMP-induced gene transcription.

Expression of murine Fc receptors for IgG.

There are two distinct genes that encode murine low affinity Fc gamma RII, murine Fc gamma RII alpha, and murine Fc gamma RII beta, which are transcribed in specific cell lineages. Fc gamma RII alpha transcripts are present in macrophages, NK cells, and mesangial cells; Fc gamma RII beta transcripts are expressed in Fc gamma R-bearing B cells, T cells, and macrophages. We have devised a sandwich ELISA to quantify the expression of Fc gamma RII alpha protein. The ELISA is specific for Fc gamma RII alpha, and does not detect the closely related Fc gamma RII beta protein. Upon stimulation with IFN-gamma the Fc gamma RII beta- macrophage cell line J774a expressed a twelvefold enhanced level of Fc gamma RII alpha protein. Peritoneal macrophages synthesized varying amounts of Fc gamma RII alpha. High levels of Fc gamma RII alpha were observed in resident and thioglycollate-elicited peritoneal macrophages, but no Fc gamma RII alpha was detected in Bacillus Calmette Guérin-elicited macrophages. J774a cells stimulated with rIL-6 bound approximately twice as much anti-Fc gamma RII mAb 2.4G2 IgG as did unstimulated controls. However, the Fc gamma RII alpha-specific ELISA showed no change in the amount of Fc gamma RII alpha expressed. A probe encompassing the extracellular coding sequence of Fc gamma RII beta hybridized to two distinct transcripts that were elevated in rIL-6-stimulated J774a cells. One of these transcripts had the same mobility in electrophoresis as Fc gamma RII alpha mRNA and hybridized to an Fc gamma RII alpha-specific probe, whereas the other transcript was larger and did not hybridize to probes specific for either Fc gamma RII alpha or Fc gamma RII beta. Moreover, we confirmed, with an Fc gamma RII beta-specific probe, that J774a cells do not make Fc gamma RII beta mRNA. Thus, the larger transcript appears to encode a novel Fc gamma RII. We suggest that the increased level of binding of the anti-Fc gamma RII mAb 2.4G2 to rIL-6-induced cells represents translation of a Fc gamma R distinct from Fc gamma RII alpha or Fc gamma RII beta.