Isozymes of cyclic AMP-dependent protein kinases (PKA) in human lymphoid cell lines: Levels of endogenous cAMP influence levels of PKA subunits and growth in lymphoid cell lines

Activation of the cAMP signaling pathway in lymphoid cells is known to inhibit cell proliferation of T and B cells as well as cytotoxicity of natural killer (NK) cells. In order to find suitable model systems to study cAMP-mediated processes, we have examined the expression of cAMP-dependent protein kinase (PKA), endogenous levels of cAMP, and cell proliferation in eight cell lines of B lineage origin, four cell lines of T lineage origin, and normal human B and T cells. We demonstrated that the expression of mRNA and protein for one of the regulatory (R) subunits of PKA (RIα) was present in all the cells investigated, in contrast to the other R subunits (RIβ, RIIα, and RIIβ). Furthermore, three T cell lines and one B cell line expressed only RIα and C, implying these cells to contain solely PKA type I. Moreover, for the RI subunit, we observed an apparent reciprocal relationship between levels of mRNA and protein. Generally, RIα protein was low in cell lines where mRNA was elevated and vice versa. This was not the case for the RII subunits, where high levels of mRNA were associated with elevated levels of protein. Interestingly, we demonstrated an inverse correlation between levels of endogenous cAMP and cell growth as determined by [³H]-thymidine incorporation and cell-doubling rate (P < 0.05). Taken together, our results demonstrate great differences in PKA isozyme composition, which should be taken into consideration when using lymphoid cell lines as model system for cAMP/PKA effects in normal lymphocytes. J. Cell. Physiol. 177:85–93, 1998. © 1998 Wiley-Liss, Inc.     

Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester.

Protein phosphorylation was studied in primary cultures of thyroid epithelial cells after the addition of different mitogens: thyrotropin (TSH) acting through cyclic AMP, epidermal growth factor (EGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA). EGF or TPA increased the phosphorylation of five common polypeptides. Among these, two 42-kilodalton proteins contained phosphotyrosine and phosphoserine with or without phosphothreonine. Their characteristics suggested that they are similar to the two 42-kilodalton target proteins for tyrosine protein phosphorylation demonstrated in fibroblasts in response to mitogens. No common phosphorylated proteins were detected in TSH-treated cells and in EGF- or TPA-treated cells. The differences in the protein phosphorylation patterns in response to TSH, EGF, and TPA suggested that the newly emerging cyclic AMP-mediated mitogenic pathway is distinct from the better known growth factor- and tumor promoter-induced pathways.     

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.     

佛波酯PMA促进碱性成纤维细胞生长因子（bFGF）释放及机理研究

Basic fibroblast growth factor (bFGF) is a strong mitogenic factor and inducer of angiogenesis. It may play an important role in the growth of solid tumors. Whereas bFGF is known to act extracellularly, the protein lacks a transient signal peptide. No defined mechanism for bFGF secretion has been characterized besides release from dead or injured cells. To explore molecular mechanism that modulates bFGF release, we treated CNE-2 cells with PMA for two days and found that the treatment increased bFGF gene expression in the cytoplasm and bFGF release significantly after 48 hours. This result suggests that protein kinase C is very likely to be involved in the bFGF release regulation. Our results have also shown that PKC-alpha activated by PMA in CNE-2 cells can phosphorylate bFGF (18 KDa) in CNE-2 cells. The result suggests that PKC-alpha translocation and activation can phosphorylate bFGF in CNE-2 cells and increase bFGF release.     

Epidermal growth factor activates extracellular signal-regulated protein kinases (ERK) in freshly isolated porcine granulosa cells.

We investigated the ability of EGF to stimulate the phosphorylation (i.e. activation) of extracellular signal-regulated kinases (ERKs) in freshly isolated porcine granulosa cells (pGC) held in suspension. pGCs were isolated from the ovaries of prepubertal pigs at slaughter, and equilibrated for 24 h at 37 degrees C in 12 x 75 mm culture tubes. The cells were then treated with 0-10 ng/ml EGF for 1-240 min. Treatments were terminated, and the total cell lysates were subjected to SDS-PAGE and Western analysis. The Westerns were blotted with anti-panERK and with anti-phosphoERK, antibodies that recognize all forms of ERKs and the phosphorylated (i.e. activated) forms of ERKs, respectively. Western blot analysis with the panERK antibody revealed a gel shift of ERKs, suggesting hyperphosphorylation after treatment with as little as 0.1 ng/ml of EGF. Phosphorylation of the ERKs was confirmed by using the phosphoERK antibody, which indicated increased phosphorylation of ERKs above control with 0.1 ng/ml EGF and maximal phosphorylation of ERK with 5-10 ng/ml EGF. Activation of ERK by EGF, as measured by both gel shift analysis and active ERK blotting, in the freshly isolated pGC was rapid, increasing above controls after 1 min of treatment, maintaining high levels through 40 min, and declining from 60 to 240 min. These data indicate that EGF stimulates active ERK in a time- and concentration-dependent manner in freshly isolated pGCs and that this experimental approach represents an effective manner with which to evaluate the role of EGF and the ERK signal transduction pathway in freshly harvested pGC.     

Epidermal growth factor (EGF) inhibits stimulated thyroid hormone secretion in the mouse

It is known that epidermal growth factor (EGF) inhibits iodide uptake in the thyroid follicular cells and lowers plasma levels of thyroid hormones upon infusion into sheep and ewes. In this study, the effects of EGF on basal and stimulated thyroid hormone secretion were investigated in the mouse. Mice were pretreated with 125I and thyroxine; the subsequent release of 125I is an estimation of thyroid hormone secretion. It was found that basal radioiodine secretion was not altered by intravenous injection of EGF (5 micrograms/animal). However, the radioiodine secretion stimulated by both TSH (120 microU/animal) and vasoactive intestinal peptide (VIP; 5 micrograms/animal) were inhibited by EGF (5 micrograms/animal). At a lower dose level (0.5 microgram/animal), EGF had no influence on stimulated radioiodine secretion. In conclusion, EGF inhibits stimulated thyroid hormone secretion in the mouse.     

Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1)

Dog thyroid epithelial cells in primary culture constitute a physiologically relevant model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (thyroid-stimulating hormone [TSH]). As previously shown in this system, the cAMP-dependent mitogenic pathway differs from growth factor cascades as it stimulates the accumulation of p27(kip1) but not cyclins D. Nevertheless, TSH induces the nuclear translocations and assembly of cyclin D3 and cdk4, which are essential in cAMP-dependent mitogenesis. Here we demonstrate that transforming growth factor beta(1) (TGFbeta(1)) selectively inhibits the cAMP-dependent cell cycle in mid-G1 and various cell cycle regulatory events, but it weakly affects the stimulation of DNA synthesis by epidermal growth factor (EGF), hepatocyte growth factor, serum, and phorbol esters. EGF+serum and TSH did not interfere importantly with TGFbeta receptor signaling, because they did not affect the TGFbeta-induced nuclear translocation of Smad 2 and 3. TGFbeta inhibited the phosphorylation of Rb, p107, and p130 induced by TSH, but it weakly affected the phosphorylation state of Rb-related proteins in EGF+serum-treated cells. TGFbeta did not inhibit c-myc expression. In TSH-stimulated cells, TGFbeta did not affect the expression of cyclin D3, cdk4, and p27(kip1), nor the induced formation of cyclin D3-cdk4 complexes, but it prevented the TSH-induced relocalization of p27(kip1) from cdk2 to cyclin D3-cdk4. It prevented the nuclear translocations of cdk4 and cyclin D3 without altering the assembly of cyclin D3-cdk4 complexes probably formed in the cytoplasm, where they were prevented from sequestering nuclear p27(kip1) away from cdk2. This study dissociates the assembly of cyclin D3-cdk4 complexes from their nuclear localization and association with p27(kip1). It provides a new mechanism of regulation of proliferation by TGFbeta, which points out the subcellular location of cyclin D-cdk4 complexes as a crucial factor integrating mitogenic and antimitogenic regulations in an epithelial cell in primary culture.     

Modulation of T cell activation by differential regulation of the phosphorylation of two cytosolic proteins. Implication of both Ca2+ and cyclic AMP-dependent protein kinases

Interleukin 2 production by activated Jurkat T cells is markedly decreased by prostaglandin E2 (PGE2). The target of PGE2 action has been investigated in the present study. Among the biochemical events occurring after CD3.TCR triggering by anti-CD3 monoclonal antibody, phosphorylation of two cytosolic proteins, pp21 and pp23, was strongly inhibited by PGE2, forskolin, and 8-bromo-cAMP, whereas anti-CD3 monoclonal antibody-induced CD3.TCR modulation and Ca2+ influx were not affected. The inhibition of both pp21 and pp23 phosphorylation and interleukin 2 synthesis by PGE2 can be largely reversed by the cAMP-dependent protein kinase inhibitor, N-[2-(methylamino)-ethyl-1]-5-isoquinoline sulfonamide. Together with the demonstration of a cAMP-dependent protein kinase activity in Jurkat T cells, these results are consistent with the participation of the cAMP-dependent protein kinase mediating the inhibitory action of PGE2, probably through the inhibition of pp21 and pp23 phosphorylation. Thus, it appears that the modulation of the phosphorylation of these cytosolic proteins represents an essential step in the regulation of T lymphocyte activation.     

Isoform-specific regulation of immune cell reactivity by the catalytic subunit of protein kinase A (PKA)

There are two major genes encoding the catalytic subunits of protein kinase A, Cα and Cβ. The functional significance of these isoforms is enigmatic. Lymphoid cells of the immune system express both Cα and Cβ. In this study we tested the role of Cα and Cβ in regulating immune cell reactivity to antigens using mice carrying a targeted disruption of the Cα and Cβ gene respectively. Cα and Cβ ablation both resulted in a 50% reduction in PKA-specific kinase activity and the level of PKA type I but not PKA type II. Moreover, despite that C subunit ablation did not affect immune cell development and homeostasis, Cα but not Cβ ablation augmented expression of the activation marker CD69 on lymphocytes. CD69 induction coincided with immune cell hyperresponsiveness and was associated with reduced sensitivity to cAMP-mediated inhibition of anti-CD3 induced T cell proliferation. Our results imply that Cα is required for normal immune cell reactivity and demonstrates isoform-specific effects and non-redundant functions of C subunit isoforms expressed in the same cell.     

Phosphorylation of P-Rex1 by the cyclic AMP-dependent protein kinase inhibits the phosphatidylinositiol (3,4,5)-trisphosphate and Gbetagamma-mediated regulation of its activity

Rac activation is a key step in chemotaxis of hematopoietic cells, which is both positively and negatively regulated by receptors coupled to heterotrimeric G proteins. P-Rex1, a Rac-specific guanine nucleotide exchange factor, is dually activated by phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)) and the Gbetagamma subunits of heterotrimeric G proteins. This study explored the regulation of P-Rex1 by phosphorylation with the cAMP-dependent protein kinase (protein kinase A) in vitro and by G(i)- and G(s)-coupled receptors in HEK293T cells. P-Rex1 isolated from Sf9 and HEK293T cells migrates as two distinct bands that are partially phosphorylated. Phosphorylation of P-Rex1 with protein kinase A (PKA) inhibits the PIP(3)- and Gbetagamma-stimulated P-Rex1 guanine nucleotide exchange activity on Rac. The guanine nucleotide exchange factor activity of three different forms of P-Rex1 (native Sf9, de-phosphorylated, and phosphorylated) was examined in the presence of PIP(3) and varying concentrations of Gbeta(1)gamma(2). Gbeta(1)gamma(2) was 47-fold less potent in activating the phosphorylated form of P-Rex1 compared with the de-phosphorylated form. HEK293T cells expressing P-Rex1 were labeled with (32)P and stimulated with lysophosphatidic acid (LPA) to release Gbetagamma or isoproterenol to activate PKA. Treatment with isoproterenol or S(p)-cAMPS, a potent activator of PKA, increased the incorporation of (32)P into P-Rex1. LPA increased the amount of GTP-bound Rac in the cells and isoproterenol reduced basal levels of GTP-bound Rac and blunted the effect of LPA. Treatment of the cells with S(p)-cAMPS also reduced the levels of GTP-bound Rac. These results outline a novel mechanism for G(s)-linked receptors to regulate the function of P-Rex1 and inhibit its function in cells.     

Activation of cyclic AMP-dependent protein kinase(s) by growth hormone in the liver and adrenal gland of the rat.

A single dose of growth hormone (10 mg/kg, i.p.) was injected into male weanling rats (50--60 g), and the temporal changes in cyclic AMP concentration, protein kinase activation, and ornithine decarboxylase activation were measured in the liver and adrenal gland. The level of cyclic AMP did not change significantly from control values in either liver or adrenal following growth hormone administration. Cyclic AMP-dependent protein kinase(s); however, was markedly activated in liver and adrenal within 30 min. Protein kinase remained activated for more than 4 hr in the liver, while activation of protein kinase in the adrenal returned to control value within 2 hr. Ornithine decarboxylase activity was elevated 20-fold in liver within 4 hr of injection and was increased 7- to 8-fold in be adrenal within l hr. These observations are discussed with regard to the generality of the role of cyclic AMP as the second messenger for target-specifici trophic hormone action and the significance of protein kinase activiation as an index of the cyclic nucleotide involvement in the growth response.     

Activation and glucagon regulation of mitogen-activated protein kinases (MAPK) by insulin and epidermal growth factor in cultured rat and human hepatocytes

Many hepatocellular activities may be proximally regulated by intracellular signalling proteins including mitogen-activated protein kinases (MAPK). In this study, signalling events from epidermal growth factor (EGF) and insulin were examined in primary cultured human and rat hepatocytes. Using Western immunoblots, rat and human hepatocytes were found to produce a rapid tyrosine phosphorylation of the EGF receptor and MAPK following 0·5–1 min exposure to EGF. Phosphorylation of p42 and p44 MAPK was observed following 2·5 min exposure to EGF. Insulin treatment produced phosphorylation of the insulin receptor β subunit; shc phosphorylation was not observed. MAPK phosphorylation corresponded with a shift in molecular weight and an increase in kinase activity. Insulin-dependent activation of MAPK was unequivocally observed only in human hepatocytes, though a slight activation was detected in rat. Co-treatment with insulin and EGF produced phosphorylation and complete electrophoretic shift in molecular weight of MAPK, with an additive or synergistic increase in enzyme activity in rat but not human hepatocytes; human hepatocyte MAPK was maximally stimulated by EGF alone. Glucagon pretreatment blocked phosphorylation, gel mobility shift and kinase activity of MAPK induced by insulin but only partially blocked EGF-induced MAPK activation in human hepatocytes. Glucagon also reduced the activation of MAPK by EGF in rat hepatocytes. Pre-treatments with forskolin or cyclic AMP analogues diminished in the insulin-, EGF- and insulin plus EGF-dependent activation of MAPK in rat hepatocytes without effecting phosphorylation of receptors or MAPK. These results indicate that although EGF and insulin may both signal through the MAPK/ras/raf/MAPK pathway, the response for MAPK differs between these ligands and between species. Further, in both rat and human, glucagon exerts its effects through a cyclic AMP-dependent mechanism at a level in the insulin and EGF signal transduction pathways downstream of MAPK but promixal to MAPK. The partial inhibition of EGF-induced MAPK phosphorylation by glucagon in human hepatocytes provides further evidence for a raf-1-independent pathway for activation of MAPK. © 1998 John Wiley & Sons, Ltd.     

Na+-dependent elevation of the acidic cell surface pH (microclimate pH) of rat jejunal villus cells induced by cyclic nucleotides and phorbol ester: possible mediators of the regulation of the Na+/H+ antiporter

The effects of cyclic nucleotides and phorbol ester on the acidic cell surface pH of rat jejunal villi were studied by using single-barrelled pH-sensitive microelectrodes. Addition of dibutyryl cAMP (1 mM) to the mucosal bathing solution caused an elevation of the cell surface pH from 6.19 +/- 0.04 (n = 12 measurements from three animals) to 6.53 +/- 0.03 (12) in the presence of Na+ in the medium. However, dibutyryl cAMP had no significant effect in the absence of Na+ and presence of 1 mM amiloride. Dibutyryl cGMP (1 mM) also had an Na+-dependent inhibitory effect on the cell surface pH. A phorbol ester, phorbol 12-myristate 13-acetate, caused an elevation of the cell surface pH only in the presence of Na+ from 6.14 +/- 0.07 (12) to 6.46 +/- 0.08 (12). Phorbol and phorbol 13-acetate, which do not stimulate protein kinase C, were without significant effects. These results suggest that increased levels of the intracellular cyclic nucleotides and activation of protein kinase C raise the acidic cell surface pH by inhibiting the activity of the brush-border Na+/H+ antiporter in the rat jejunal villus cells.     

Epidermal growth factor increases P incorporation into phosphatidylcholine and protein kinase C activity in colon carcinoma cell line (HT29)

There are conflicting data about the effect of the epidermal growth factor (EGF) on protein kinase C (PKC) enzyme activity. The aim of our study was to find out which type of phospholipids [phosphatidylinositol 4,5-bisphosphate P14,5P₂ or the other phospholipids-phosphatidylcholine (PC) or phosphatidic acid (PA)] could be the source of 1,2-diacylglycerol (1,2-DAG) in PKC activation. In colon carcinoma cells (HT29) we observed a more than 2-fold increase in the PC pool and at the same time decreased tyrosine kinase activity (50%). With increasing incubation time EGF affects the pools of both phosphatidylinositols and other phospholipids parallel with the activation of the tyrosine kinase activity. EGF increases the activity of PKC in the HT29 cell line and PC could be the source of 1,2-DAG which may stimulate PKC activity.     

Mechanism of CD150 (SLAM) down regulation from the host cell surface by measles virus hemagglutinin protein

Measles virus has been reported to enter host cells via either of two cellular receptors, CD46 and CD150 (SLAM). CD46 is found on most cells of higher primates, while SLAM is expressed on activated B, T, and dendritic cells and is an important regulatory molecule of the immune system. Previous reports have shown that measles virus can down regulate expression of its two cellular receptors on the host cell surface during infection. In this study, the process of down regulation of SLAM by measles virus was investigated. We demonstrated that expression of the hemagglutinin (H) protein of measles virus was sufficient for down regulation. Our studies provided evidence that interactions between H and SLAM in the endoplasmic reticulum (ER) can promote the down regulation of SLAM but not CD46. In addition, we demonstrated that interactions between H and SLAM at the host cell surface can also contribute to SLAM down regulation. These results indicate that two mechanisms involving either intracellular interactions between H and SLAM in the ER or receptor-mediated binding to H at the surfaces of host cells can lead to the down regulation of SLAM during measles virus infection.     

Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD

We studied 11 new kindreds with primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC) and found that 82% of the kindreds had PRKAR1A gene defects (including seven novel inactivating mutations), most of which led to nonsense mRNA and, thus, were not expressed in patients' cells. However, a previously undescribed base substitution in intron 6 (exon 6 IVS +1G-->T) led to exon 6 skipping and an expressed shorter PRKAR1A protein. The mutant protein was present in patients' leukocytes and tumors, and in vitro studies indicated that the mutant PRKAR1A activated cAMP-dependent protein kinase A (PKA) signaling at the nuclear level. This is the first demonstration of an inactivating PRKAR1A mutation being expressed at the protein level and leading to stimulation of the PKA pathway in CNC patients. Along with the lack of allelic loss at the PRKAR1A locus in most of the tumors from this kindred, these data suggest that alteration of PRKAR1A function (not only its complete loss) is sufficient for augmenting PKA activity leading to tumorigenesis in tissues affected by CNC.     

Sodium and potassium uptake in primary cultures of rat astroglial cells induced by long-term exposure to the basic astroglial growth factor (AGF2)

Astroglial cell cultures were derived from newborn rat forebrain and cultured for 5 days in serum containing-, and for an additional 4 days in a serum-free, defined medium. At the end of this 9-day-long period, basic astroglial growth factor (AGF2) was administered to the culture medium (10 ng per ml). Cells were subsequently cultured in AGF2 containing serum-free, defined medium for further two weeks. At definite intervals of culturing, unidirectional influx of both Na⁺ and K⁺ (I_Na and I_K, respectively) was determined by applying²²Na and⁴²K. The AGF2-treated cultures showed highly increased, amiloride-sensitive I_Na at the early exposure period (2–8 hours), similar to that we have reported about cultured astroglia exposed to AGF2 for minutes. They also exhibited significant furosemide-sensitive-, while relatively poor ouabain-sensitive component of I_Na. However, at later periods of exposure to AGF2, I_Na was significantly reduced, particularly due to the decrease of its amiloride-sensitive component, while its furosemide-sensitive component further increased with the time of AGF2 treatment. In contrast to I_Na, the I_K in the cultures exposed to AGF2 increased significantly in the course of the long-term exposure period, particularly the ouabain-, and furosemide-sensitive-components, while its amiloride-sensitive component, similarly to that of I_Na, decreased. Our findings show that the initial activation of the Na⁺/H⁺ (or K⁺/H⁺) exchange, what characterized the cation transport changes by short-term exposure of astroglial cells to AGF2 in our previous study, comes relatively soon to a cessation but activation of the Na⁺, K⁺-pump and the furosemide-sensitive Na⁺ and K⁺ influxes further increases. Thus, they suggest the possibility that furosemide-sensitive cation movements play a role, besides the Na⁺, K⁺-pump, in the control of glial cell differentiation.Cente de Neurochimie du CNRS.Special issue dedicated to Dr. Paola S. Timiras.