Total Protein Kinase A Activity Research Articles

Bone Abnormalities in Mice with Protein Kinase A (PKA) Defects Reveal a Role of Cyclic AMP Signaling in Bone Stromal Cell-Dependent Tumor Development.

Protein kinase A (PKA) is an important enzyme for all eukaryotic cells. PKA phosphorylates other proteins, thus, it is essential for the regulation of many diverse cellular functions, including cytoplasmic trafficking and signaling, organelle structure and mitochondrial oxidation, nuclear gene expression, the cell cycle, and cellular division. The PKA holoenzyme is composed of 2 regulatory and 2 catalytic subunits. Four regulatory (R1α, R1β, R2α, and R2β) and 4 catalytic subunits (Cα, Cβ, Cγ, and Prkx) have been identified, giving rise to mainly PKA-I (when the 2 regulatory subunits are either R1α or R1β), or PKA-II (when the 2 regulatory subunits are either R2α or R2β). Mutations in the PKA subunits can lead to altered total PKA activity or abnormal PKA-I to PKA-II ratio, leading to various abnormalities in both humans and mice. These effects can be tissue-specific. We studied the effect of PKA subunit defects on PKA activity and bone morphology of mice that were single or double heterozygous for null alleles of the various PKA subunit genes. Bone lesions including fibrous dysplasia, myxomas, osteo-sarcomas, -chondromas and -chondrosarcomas were found in these mice. Observational and molecular studies showed that these lesions were derived from bone stromal cells (BSCs). We conclude that haploinsufficiency for different PKA subunit genes affected bone lesion formation, new bone generation, organization, and mineralization in variable ways. This work identified a PKA subunit- and activity-dependent pathway of bone lesion formation from BSCs with important implications for understanding how cyclic AMP affects the skeleton and its tumorigenesis.

Role of cAMP-dependent protein kinase A activity in low-dose endothelial monocyte-activating polypeptide-II-induced opening of blood-tumor barrier.

Our previous studies demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can selectively increase the permeability of blood-tumor barrier (BTB). In addition, low-dose EMAP-II significantly decreases the cyclic adenosine monophosphate (cAMP) concentration and the protein kinase A (PKA) expression level in tumor tissues in the rat C6 glioma model. In this study, an in vitro BTB model was used to investigate the potential role of cAMP/PKA signaling cascade in EMAP-II-induced BTB hyperpermeability. Our data revealed that low-dose EMAP-II (0.05nM) induced a significant decrease in total intracellular cAMP concentration and PKA activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to increase intracellular cAMP nearly completely blocked the EMAP-II-induced decrease in transendothelial electric resistance and increase in horseradish peroxidase flux across the BTB. Similar pretreatment completely prevented the EMAP-II-induced changes in RhoA/Rho kinase activity, expression and distribution of tight junction-associated protein ZO-1, and myosin light chain phosphorylation, as well as actin cytoskeleton arrangement in RBMECs. Pretreatment with 6Bnz-cAMP to activate PKA significantly attenuated these EMAP-II-induced alterations in RBMECs. In summary, our present study demonstrates that the cAMP/PKA signaling cascade works as a crucial signaling pathway in EMAP-II-induced BTB hyperpermeability.

Threat bias in mice with inactivating mutations of Prkar1a

Anxiety disorders are associated with abnormalities in the neural processing of threat-related stimuli. However, the neurobiological mechanisms underlying threat bias in anxiety are not well understood. We recently reported that a Prkar1a heterozygote (Prkar1a+/−) mouse with haploinsufficiency for the main regulatory subunit (R1α) of protein kinase A (PKA) exhibits an anxiety-like phenotype associated with increased cAMP signaling in the amygdala. Prkar1a+/− mice provide a novel model to test the direct effect of altered PKA expression and subsequent anxiety-like behavioral phenotype on the response to threat. We hypothesized that Prkar1a+/−mice would exhibit a bias in threat detection since increased amygdala activity during emotional stimuli is associated with a maladaptive response. We measured behavior and PKA activity in brain areas after exposure to predator or control odor exposure in male Prkar1a+/− and wild-type (WT) littermates. Indeed, there were significant differences in the behavioral response to threat detection; WT mice showed the expected response of decrease in exploratory behavior during predator vs. control odor exposure, while Prkar1a+/− mice did not alter their behavior between conditions. Basal and total PKA activity was independently associated with genotype, with an interaction between genotype and threat condition. Prkar1a+/− mice had higher PKA activity in amygdala and ventromedial hypothalamus in response to predator odor. In contrast, WT mice had higher PKA activity in amygdala and orbitofrontal cortex after exposure to control odor.Dysregulated PKA activity in the amygdala–prefrontal cortex circuitry in Prkar1a+/− mice is associated with behavioral phenotype of anxiety and a bias for threat. This is likely related to a failure to inhibit the amydgala response, which is an effect of the genotype. These results suggest that the alteration in PKA signaling in Prkar1a+/− mice is not ubiquitous in the brain; tissue-specific effects of the cAMP/PKA pathway are related to threat detection and fear sensitization.

Insulin Regulates Adipocyte Lipolysis via an Akt-Independent Signaling Pathway

After a meal, insulin suppresses lipolysis through the activation of its downstream kinase, Akt, resulting in the inhibition of protein kinase A (PKA), the main positive effector of lipolysis. During insulin resistance, this process is ineffective, leading to a characteristic dyslipidemia and the worsening of impaired insulin action and obesity. Here, we describe a noncanonical Akt-independent, phosphoinositide-3 kinase (PI3K)-dependent pathway that regulates adipocyte lipolysis using restricted subcellular signaling. This pathway selectively alters the PKA phosphorylation of its major lipid droplet-associated substrate, perilipin. In contrast, the phosphorylation of another PKA substrate, hormone-sensitive lipase (HSL), remains Akt dependent. Furthermore, insulin regulates total PKA activity in an Akt-dependent manner. These findings indicate that localized changes in insulin action are responsible for the differential phosphorylation of PKA substrates. Thus, we identify a pathway by which insulin regulates lipolysis through the spatially compartmentalized modulation of PKA.

Evidence that a wortmannin-sensitive signal transduction pathway regulates aflatoxin biosynthesis

A signal transduction pathway involving cAMP and protein kinase A (PKA) regulates aflatoxin accumulation and nor-1 and ver-1 (aflatoxin structural genes) promoter function in Aspergillus parasiticus by modulating expression of a key transcriptional activator, AflR. To understand the function of this pathway in greater detail we treated A. parasiticus in culture with wortmannin, a frequently used probe of phosphatidyl inositol (PI)-3 kinase activity. A. parasiticus D8D3 (nor-1::GUS reporter) and I4 (ver-1::GUS reporter) were grown on a defined solid growth medium (GMS agar) under aflatoxin-inducing conditions. GMS containing wortmannin (1 microM) reduced aflatoxin B1 accumulation up to 15-fold accompanied by a similarly large decrease in ver-1 and nor-1 promoter activity. Wortmannin inhibited growth (colony diameter) and asexual sporulation but to a much smaller extent. Wortmannin treatment increased intracellular cAMP levels up to 25-fold; total PKA activity also increased within 10 min of wortmannin exposure. These data support a regulatory model in which PI-3 kinase activity modulates intracellular cAMP accumulation and PKA activity. This in turn regulates AflR expression and activity, aflatoxin gene expression and aflatoxin accumulation.

Evidence that a wortmannin-sensitive signal transduction pathway regulates aflatoxin biosynthesis

A signal transduction pathway involving cAMP and protein kinase A (PKA) regulates aflatoxin accumulation and nor-1 and ver-1 (aflatoxin structural genes) promoter function in Aspergillus parasiticus by modulating expression of a key transcriptional activator, AflR. To understand the function of this pathway in greater detail we treated A. parasiticus in culture with wortmannin, a frequently used probe of phosphatidyl inositol (PI)-3 kinase activity. A. parasiticus D8D3 (nor-1::GUS reporter) and I4 (ver- ::GUS reporter) were grown on a defined solid growth medium (GMS agar) under aflatoxin-inducing conditions. GMS containing wortmannin (1 μM) reduced aflatoxin B1 accumulation up to 15-fold accompanied by a similarly large decrease in ver-1 and nor-1 promoter activity. Wortmannin inhibited growth (colony diameter) and asexual sporulation but to a much smaller extent. Wortmannin treatment increased intracellular cAMP levels up to 25-fold; total PKA activity also increased within 10 min of wortmannin exposure. These data support a regulatory model in which PI-3 kinase activity modulates intracellular cAMP accumulation and PKA activity. This in turn regulates AflR expression and activity, aflatoxin gene expression and aflatoxin accumulation.

17q22–24 Chromosomal Losses and Alterations of Protein Kinase A Subunit Expression and Activity in Adrenocorticotropin-Independent Macronodular Adrenal Hyperplasia

Primary adrenocortical hyperplasias leading to Cushing syndrome include primary pigmented nodular adrenocortical disease and ACTH-independent macronodular adrenal hyperplasia (AIMAH). Inactivating mutations of the 17q22-24-located PRKAR1A gene, coding for the type 1A regulatory subunit of protein kinase A (PKA), cause primary pigmented nodular adrenocortical disease and the multiple endocrine neoplasia syndrome Carney complex. PRKAR1A mutations and 17q22-24 chromosomal losses have been found in sporadic adrenal tumors and are associated with aberrant PKA signaling. The objective of the study was to examine whether somatic 17q22-24 changes, PRKAR1A mutations, and/or PKA abnormalities are present in AIMAH. We studied fourteen patients with Cushing syndrome due to AIMAH. Fluorescent in situ hybridization with a PRKAR1A-specific probe was used for investigating chromosome 17 allelic losses. The PRKAR1A gene was sequenced in all samples, and tissue was studied for PKA activity, cAMP responsiveness, and PKA subunit expression. We found 17q22-24 allelic losses in 73% of the samples. There were no PRKAR1A-coding sequence mutations. The RIIbeta PKA subunit was overexpressed by mRNA, whereas the RIalpha, RIbeta, RIIalpha, and Calpha PKA subunits were underexpressed. These findings were confirmed by immunohistochemistry. Total PKA activity and free PKA activity were higher in AIMAH than normal adrenal glands, consistent with the up-regulation of the RIIbeta PKA subunit. PRKAR1A mutations are not found in AIMAH. Somatic losses of the 17q22-24 region and PKA subunit and enzymatic activity changes show that PKA signaling is altered in AIMAH in a way that is similar to that of other adrenal tumors with 17q losses or PRKAR1A mutations.

Glucose-dependent activation of protein kinase A activity in Saccharomyces cerevisiae and phosphorylation of its TPK1 catalytic subunit

Protein kinase A (PKA), in yeast, plays a major role in controlling metabolism and gene expression in connection with the available nutrient conditions. We here measure, for the first time, a transient change in the in vivo PKA activity, along a cAMP peak produced by 100 mM glucose addition to glycerol-growing cells as well as a change in the phosphorylation state of its catalytic subunit (Tpk1p) following PKA activation. PKA activity was measured in situ in permeabilized cells, preserving its intracellular localization. Comparison of total PKA activity, measured in situ in permeabilized cells with data obtained from in vitro assays in crude extracts, underscores the inhibitory potency of the regulatory subunit within the cell. Tpk1p phosphorylation was detected through non-denaturing gel electrophoresis. Phosphorylation of Tpk1p increases its specificity constant toward kemptide substrate. The use of mutants of the cAMP pathway showed that phosphorylation depends on the activation of PKA via the G-protein coupled receptor pathway triggered by glucose. The phosphorylation state of Tpk1p was followed during the diauxic shift. Tpk1p phosphorylation is dynamic and reversible: its up-regulation correlates with a fully fermentative metabolism, while its down-regulation with stationary phase or respiratory metabolism. Reversible phosphorylation can thus be considered a new control mechanism possibly pointing to a fine-tuning of PKA activity in response to environmental conditions.

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.

Effects of long-term high-altitude hypoxia on myocardial protein kinase A activity and troponin I isoforms in fetal and nonpregnant sheep

In fetal sheep, we found that the augmentation of cardiac contractility by beta-adrenergic receptor (beta-AR) stimulation was reduced after exposure to long-term hypoxia. However, cyclic adenosine monophosphate (cAMP) production after beta-AR stimulation was higher in long-term hypoxic fetal sheep than in normoxic ones. Therefore, we studied the potential role of changes in myocardial protein kinase A (PKA) activity and troponin I (TnI) isoforms in fetal and nonpregnant sheep exposed to approximately 112 days of hypoxia at high altitude (3820 m). Resting and maximally stimulated (by cAMP) PKA activity was measured by phosphorylation of the artificial peptide, Kemptide. Specificity was confirmed by inhibition with PKI, a specific PKA inhibitor. For TnI isoforms, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to resolve the proteins. We used monoclonal anti-cardiac TnI antibody (clone C5), which also cross-reacted with slow skeletal muscle TnI, to identify TnI isoforms. For the fetal hearts, resting PKA activity was significantly higher in the high-altitude group than the control group, but total PKA activity was not different between the normoxic and hypoxic groups. In the adult hearts, no significant difference was observed in either resting or total PKA activity between normoxic and hypoxic groups. For both the fetal and adult sheep, the predominant TnI was the cardiac isoform, and hypoxic exposure produced no change in the TnI isoform composition. Neither a reduction in PKA activity nor a change in TnI isoforms could explain the reduction in beta-receptor augmentation of cardiac contractility in fetal sheep exposed to long-term hypoxia.

Role of protein kinase A in GABAA receptor dysfunction in CA1 pyramidal cells following chronic benzodiazepine treatment.

One-week treatment with the benzodiazepine (BZ) flurazepam (FZP), results in anticonvulsant tolerance, associated with reduced GABAA receptor (GABAR) subunit protein and miniature inhibitory post-synaptic current (mIPSC) amplitude in CA1 neurons of rat hippocampus. Because protein kinase A (PKA) has been shown to modulate GABAR function in CA1 pyramidal cells, the present study assessed whether GABAR dysfunction is associated with changes in PKA activity. Two days after 1-week FZP treatment, there were significant decreases in basal (- 30%) and total (- 25%) PKA activity, and a 40% reduction in PKA RIIbeta protein in the insoluble fraction of CA1 hippocampus. The soluble component of CA1 showed a significant increase in basal (100%) but not total PKA activity. Whole-cell recording in vitro showed a 50% reduction in mIPSC amplitude in CA1 pyramidal cells, with altered sensitivity to PKA modulators. Neurons from FZP-treated rats responded to 8-bromo-cAMP with a significant increase (31%) in mIPSC amplitude. Likewise, vasoactive intestinal polypeptide (VIP), an endogenous PKA activator, caused a significant 36% increase in mIPSC amplitude in FZP-treated cells. Neither agent had a significant effect on mIPSC amplitude in control cells. This study supports a role for PKA in GABAR dysfunction after chronic FZP treatment.

Regulatory subunit type I-alpha of protein kinase A (PRKAR1A): a tumor-suppressor gene for sporadic thyroid cancer.

The tumor-suppressor gene encoding the cyclic AMP-dependent protein kinase A type I-alpha regulatory subunit PRKAR1A has been mapped to chromosome 17 (17q22-24) and is mutated in Carney complex, a familial neoplasia syndrome that is associated with thyroid tumors. Other genes implicated in cyclic nucleotide-dependent signaling have been investigated in thyroid tumorigenesis. We studied protein kinase A (PKA) activity in noninherited follicular thyroid adenomas and follicular, papillary, and undifferentiated (anaplastic) thyroid carcinomas. We then examined these and additional thyroid tumors for losses of the 17q22-24 PRKAR1A region, mutations of the PRKAR1A gene, and expression of its peptide product. Total PKA activity was markedly increased in carcinomas over that in adenomas, whereas the ratio of free vs. total PKA activity was decreased in cancer. Consistent with these findings, the 17q22-24 region was frequently lost in cancer but not in benign adenomas. A novel inactivating mutation of the PRKAR1A gene (leading to premature termination of the predicted protein) was found in an aggressive thyroid cancer. The tumor with PRKAR1A gene mutation, as well as the tumors with 17q allelic losses, showed decreased PRKAR1A expression by immunostaining. We conclude that PRKAR1A, the most abundant regulatory subunit of protein kinase A and a principal cyclic AMP-signaling modulator, acts as a tumor-suppressor gene in sporadic thyroid cancer. Published 2002 Wiley-Liss, Inc.

Protein kinase A: regulation and receptor-mediated delivery of antisense oligonucleotides and cytotoxic drugs.

Protein kinases help regulate eukaryotic cell division. We investigated the regulation of cAMP-dependent protein kinase A (PKA) and casein kinase (CK) type I activity in normal cells and in cancer. To assess this activity in biopsies we suggest a new parameter--the ratio of CK activity and total PKA activity divided by cAMP concentration: CK/PKA/cAMP. In 98 samples of colon mucosa in normal, inflamed, polyp, and adenocarcinoma cells, we found this parameter to be fairly constant in normal conditions and increased 10-fold in colon cancer; the ratio does not depend on the place of biopsy or the patient's age or sex. Experiments with model systems of concanavalin A-stimulated lymphocytes and regenerating rat liver showed that in normal cell proliferation the parameter increases 2-3-fold, as compared to a 30-fold increase in cancer. Unlike normal cells, malignant cells show CK activation and decrease of cAMP; therefore, PKA activity decreases. This suggests a correlation of CK and PKA activity and significant damage to their regulation at pathological changes of tissue proliferation. To further study concerted CK and PKA regulation we used monoclonal antibodies (mAbs) against cAMP-dependent protein kinase regulatory subunit RKII beta. We produced 11 antibodies in three groups: inhibiting, which block cAMP binding with RII beta and inhibit holoenzyme formation (RS6); activating, which enhance cAMP binding and do not affect holoenzyme formation (RS28); and neutral (RS17). To investigate mAb influence on protein kinase regulation in live cells we permeabilized pheochromocytoma PC12 by digitonin. When used at 5-microM concentration for 5 min, digitonin allowed us to deliver mAb into PC12 cells at 30-34-nM concentration, leaving 68-75% viable cells. Protein kinase activity was measured within 0.5 and 4 h after incorporation of mAbs into cells. After 30 min incorporation, mAb RS6 blocked PKA activation in PC12 cells under the influence of cAMP; other mAbs showed no effect. mAb RS6 caused a 4-fold increase of free C subunit activity 4 h after incorporation. mAb RS38 decreased R2C2 activity and did not influence C subunit activity. The change of free C subunit activity caused by mAb incorporation was followed by a synchronized, well-balanced change of CK type I activity, which suggests a correlation between the two phosphorylation systems of cell proteins.

Association of deficient type II protein kinase A activity with aberrant nuclear translocation of the RII beta subunit in systemic lupus erythematosus T lymphocytes.

Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by abnormal T cell signal transduction and altered T cell effector functions. We have previously observed a profound deficiency of total protein kinase A (PKA) phosphotransferase activity in SLE T cells. Here we examined whether reduced total PKA activity in SLE T cells is in part the result of deficient type II PKA (PKA-II) isozyme activity. The mean PKA-II activity in SLE T cells was 61% of normal control T cells. The prevalence of deficient PKA-II activity in 35 SLE subjects was 37%. Deficient isozyme activity was persistent over time and was unrelated to SLE disease activity. Reduced PKA-II activity was associated with spontaneous dissociation of the cytosolic RIIbeta2C2 holoenzyme and translocation of the regulatory (RIIbeta) subunit from the cytosol to the nucleus. Confocal immunofluorescence microscopy revealed that the RIIbeta subunit was present in approximately 60% of SLE T cell nuclei compared with only 2-3% of normal and disease controls. Quantification of nuclear RIIbeta subunit protein content by immunoprecipitation and immunoblotting demonstrated a 54% increase over normal T cell nuclei. Moreover, the RIIbeta subunit was retained in SLE T cell nuclei, failed to relocate to the cytosol, and was associated with a persistent deficiency of PKA-II activity. In conclusion, we describe a novel mechanism of deficient PKA-II isozyme activity due to aberrant nuclear translocation of the RIIbeta subunit and its retention in the nucleus in SLE T cells. Deficient PKA-II activity may contribute to impaired signaling in SLE T cells.

Transcriptional induction of cyclooxygenase-2 gene by okadaic acid inhibition of phosphatase activity in human chondrocytes: Co-stimulation of AP-1 and CRE nuclear binding proteins

The involvement of serine/threonine protein phosphatases in signaling pathways that control the expression of the cyclooxygenase-2 (COX-2) gene in human chondrocytes was examined. Okadaic acid (OKA), an inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A), induced a delayed, time-dependent increase in the rate of COX-2 gene transcription (runoff assay) resulting in increased steady-state mRNA levels and enzyme synthesis. The latter response was dose dependent over a narrow range of 1-30 nmol/L with declining expression and synthesis of COX-2 at higher concentrations due to cell toxicity. The delayed increase in COX-2 mRNA expression was accompanied by the induction of the proto-oncogenes c-jun, junB, junD, and c-fos (but not FosB or Fra-1). Increased phosphorylation of CREB-1/ATF-1 transcription factors was observed beginning at 4 h and reached a zenith at 8 h. Gel-shift analysis confirmed the up-regulation of AP-1 and CRE nuclear binding proteins, though there was little or no OKA-induced nuclear protein binding to SP-1, AP-2, NF-kappaB or NF-IL-6 regulatory elements. OKA-induced nuclear protein binding to 32P-CRE oligonucleotides was abrogated by a pharmacological inhibitor of protein kinase A (PKA), KT-5720; the latter compound also inhibited OKA-induced COX-2 enzyme synthesis. Calphostin C (CalC), an inhibitor of PKC isoenzymes, had little effect in this regard. Inhibition of 12P-CRE binding was also observed in the presence of an antibody to CREB-binding protein (265-kDa CBP), an integrator and coactivator of cAMP-responsive genes. The binding to 32P-CRE was unaffected in the presence of excess radioinert AP-1 and COX-2 NF-IL-6 oligonucleotides, although a COX-2 CRE-oligo competed very efficiently. 32P-AP-1 consensus sequence binding was unaffected by incubation of chondrocytes with KT-5720 or CalC, but was dramatically diminished by excess radioinert AP-1 and CRE-COX-2 oligos. Supershift analysis in the presence of antibodies to c-Jun, c-Fos, JunD, and JunB suggested that AP-1 complexes were composed of c-Fos, JunB, and possibly c-Jun. OKA has no effect on total cellular PKC activity but caused a delayed time-dependent increase in total PKA activity and synthesis. OKA suppressed the activity of the MAP kinases, ERK1/2 in a time-dependent fashion, suggesting that the Raf-1/MEKK1/MEK1/ERK1,2 cascade was compromised by OKA treatment. By contrast, OKA caused a dramatic increase in SAPK/JNK expression and activity, indicative of an activation of MEKK1/JNKK/SAPK/JNK pathway. OKA stimulated a dose-dependent activation of CAT activity using transfected promoter-CAT constructs harboring the regulatory elements AP-1 (c-jun promoter) and CRE (CRE-tkCAT). We conclude that in primary phenotypically stable human chondrocytes, COX-2 gene expression may be controlled by critical phosphatases that interact with phosphorylation dependent (e.g., MAP kinases:AP-1, PKA:CREB/ATF) signaling pathways. AP-1 and CREB/ATF families of transcription factors may be important substrates for PP-1/PP-2A in human chondrocytes.

Dissociation of negative inotropic effect of carbachol from changes in cAMP and PKA in perfused rat hearts.

The purpose of this investigation was to determine whether muscarinic receptors are selectively coupled to the pool of adenylyl cyclase that is responsible for the elevation of particulate (membrane-bound) adenosine 3',5'-cyclic monophosphate (cAMP), because this appears to lead to translocation of particulate cAMP-dependent protein kinase A (PKA) to the soluble fraction and the positive inotropic response. Perfusion of hearts with carbachol and isoproterenol concurrently for 1.5 min prevented the increase in left ventricular pressure (LVP) found with isoproterenol alone, although isoproterenol-induced changes in total and particulate cAMP levels and soluble and particulate PKA activity were unaffected. However, perfusion of hearts with carbachol for 1 min then with isoproterenol and carbachol for 1.5 min abolished the isoproterenol-induced increase in LVP and in total and particulate cAMP levels, although changes in total and particulate PKA activity were only partially attenuated. Perfusion of hearts with carbachol for 1 min then carbachol plus forskolin for 2 or 5 min also completely prevented the forskolin-induced increase in LVP without affecting the changes in either total or particulate cAMP levels or soluble or particulate PKA activity produced by this agent. Therefore, muscarinic receptors do not appear to selectively couple to the pool of adenylyl cyclase responsible for elevation of particulate cAMP levels. These data provide further evidence that antagonism of adenylyl cyclase activity is not required for the inhibition by carbachol of positive inotropic responses of ventricular muscle to cAMP generating agents.

The Effect of Circulating Serum Factors from Patients with Systemic Lupus Erythematosus on Protein Kinase A (PKA) Activity and PKA-Dependent Protein Phosphorylation in T Lymphocytes

T lymphocytes from subjects with active systemic lupus erythematosus (SLE) exhibit reduced cAMP-inducible, protein kinase A (PKA)-dependent phosphorylation of several intracellular substrates compared with healthy and disease controls. To ascertain whether the persistent T cell activation observed during active SLE can result in impaired PKA-dependent protein phosphorylation, normal T cells were activated in vitro by monoclonal anti-CD3-ϵ antibody and recombinant IL1-α (rIL1-α) for 24 hr. T cell activation, verified by IL2 mRNA, IL2 receptor-α (IL2R-α) mRNA, and IL2R-β mRNA expression, did not diminish cAMP-inducible, PKA-dependent protein phosphorylation. We also tested the hypothesis that circulating factors present in active SLE serum can decrease cAMP-inducible total PKA phosphotransferase activity and PKA-dependent protein phosphorylation in normal T lymphocytes. T cells cultured for 24 hr in medium supplemented with 10% active SLE sera (from subjects who exhibited the defect of PKA-dependent protein phosphorylation) exhibited similar total PKA phosphotransferase activity and substrate phosphorylation as cells cultured in normal AB serum. Moreover, the addition of interferon-α (IFN-α) and/or immune complexes (IC) did not diminish either total PKA activity or PKA-dependent substrate phosphorylation. Lastly, we found that the defect of PKA-dependent protein phosphorylation in active SLE T cells could not be reversed by culturing the cells in culture medium supplemented with 10% AB serum for 24 hr. In conclusion, (a) deficient cAMP-inducible, PKA-dependent phosphorylation in SLE T cells is not reversible by culturing cells in vitro; (b) there is no evidence to support the concept that serum factors, including IC and IFN-α, can induce a defect of PKA-dependent protein phosphorylation in normal T cells.