Isolation of CHO-K1 clones defective in cAMP-dependent proteolysis, as determined by the stability of exogenously expressed GATA-6

Degradation of the GATA-6(Delta50) protein expressed in a CHO-K1 clone (tc1-17a) is stimulated in the presence of dbcAMP through proteasome without new protein synthesis [FEBS Lett. 408 (1997) 301], whereas the intrinsic GC-box-binding protein was stable. To examine the cellular mechanism responsible for this specific degradation of GATA-6(Delta50), we initially introduced the blasticidin-S deaminase gene carrying a promoter with GATA motifs that are recognized by GATA-6. The resulting cell line (tc2G2) grew in the presence of blasticidin S. However, the presence of both blasticidin S and dbcAMP was lethal due to degradation of GATA-6. Cells resistant to such lethality were isolated by chemical mutagenesis. The GATA-6(Delta50) in these resistant cells was stable in the presence of dbcAMP in contrast to that in the parent tc2G2 cells, as determined by gel-mobility shift analysis and Western blotting. These clones could be beneficial for identification and characterization of the components participating in the signaling pathway for both protein degradation and cAMP-dependent biological processes.     

cAMP-dependent proteolysis of GATA-6 is linked to JNK-signaling pathway

A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 by proteasomes around its IC50. We further examined the effects of SP600125 on the degradation of GATA-6 in detail, since an activator of JNK (anisomycin) is available. Interestingly, anisomycin immediately stimulated the export of nuclear GATA-6 into the cytoplasm, and then the cytoplasmic content of GATA-6 decreased slowly through degradation by proteasomes. Such an effect of anisomycin was inhibited by SP600125, indicating that the observed phenomenon might be linked to the JNK signaling pathway. The inhibitory effect of SP600125 could not be ascribed to the inhibition of PKA, since phosphorylation of CREB occurred in the presence of dbcAMP and SP600125. The nuclear export of GATA-6 was inhibited by leptomycin B, suggesting that CRM1-mediated export could be activated by anisomycin. Furthermore, it seems likely that the JNK activated by anisomycin may stimulate not only the nuclear export of GATA-6 through CRM1 but also the degradation of GATA-6 by cytoplasmic proteasomes. In contrast, A-kinase might activate only the latter process through JNK.     

Sphingosine inhibits angiotensin-stimulated aldosterone synthesis.

Sphingosine and other protein kinase C inhibitors were tested for their ability to inhibit aldosterone synthesis by bovine adrenal glomerulosa cells. Sphingosine inhibited angiotensin (AII)-stimulated aldosterone synthesis (IC50 of 5 microM). At doses that totally blocked steroidogenesis, sphingosine did not affect protein synthesis or [125I]AII binding to cells. Sphingosine also inhibited dibutyryl cyclic AMP (dbcAMP)-stimulated aldosterone synthesis. Sphingosine inhibited pregnenolone synthesis from cholesterol, but not the conversion of progesterone or 20 alpha-hydroxycholesterol to aldosterone. These results suggest that sphingosine inhibits steroidogenesis at a locus close to that where stimulation occurs by AII and dbcAMP. Other protein kinase C inhibitors were tested. Retinal, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and staurosporine inhibited aldosterone synthesis stimulated by AII and dbcAMP. Retinal and H-7 also inhibited progesterone conversion to aldosterone, and retinal blocked [125I]AII binding. Staurosporine was more specific, inhibiting AII-stimulated aldosteronogenesis at concentrations which had little effect on conversion of progesterone to aldosterone. Because they inhibited dbcAMP stimulation, none of the inhibitors was sufficiently specific to use as a probe of the role of protein kinase C. The IC50 of sphingosine suggests that this or related products of lipid hydrolysis could act as endogenous regulators of adrenal cell function.     

Interleukin-10 and cAMP-elevating agents cooperate to induce suppressor of cytokine signaling-3 via a protein kinase A-independent signal

We have recently shown that IL-10 represents an efficient stimulus for suppressor of cytokine signalling (SOCS)-3 mRNA expression in human neutrophils and PBMC. Herein, we identify cAMP-elevating agents such as prostaglandin E2 (PGE2), PGE1, forskolin, dibutyryl cAMP (dbcAMP) and cholera toxin as a novel class of agonists able to induce SOCS-3 mRNA and protein expression in human leukocytes, cooperating with interleukin-10 (IL-10) in such activities. While PGE2 or dbcAMP prolonged the stability of SOCS-3 mRNA isolated from IL-10-treated leukocytes, inhibitors of cAMP-dependent protein kinase A (H89, KT5720, and St-Ht31 peptide) did not influence the action of PGE2/dbcAMP and/or IL-10 on SOCS-3 mRNA and protein expression, implying that their effect are mediated through a PKA-independent pathway. Taken together, our data identify cAMP-elevating substances as a novel class of agonists able to trigger SOCS-3 expression, and suggest that SOCS-3 might be involved in the regulatory effects of cAMP-elevating substances.     

A Nerve Growth Factor-Dependent Protein Kinase That Phosphorylates Microtubule-Associated Proteins In Vitro: Possible Involvement of Its Activity in the Outgrowth of Neurites from PC12 Cells

We have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF-pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three- to eightfold in terms of phosphorylation of microtubule-associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP(dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP-dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF; it required Mg²⁺ for activity but not Mn²⁺ or Ca²⁺. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF-dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K-252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF-dependent MAP kinase was selectively abolished. We isolated several mutant clones of PC12D cells that were deficient in the ability to induce neurites in response to either of the two stimulators. In these variant cells, the activity of the relevant protein kinase was decreased, in parallel with the deficiency in the neurite response to NGF or dbcAMP. These observations suggest that the NGF-dependent MAP kinase may play an important role in the outgrowth of neurites from PC12 cells in response to NGF.     

N-Acetyl-d-Glucosamine Induces Germination in Candida albicans through a Mechanism Sensitive to Inhibitors of cAMP-Dependent Protein Kinase

The present study examines the involvement of cAMP-dependent protein kinase (PKA) in the dimorphic transition of Candida albicans by assessing the in vivo effect of two permeable PKA inhibitors on N-acetyl-d-glucosamine (GlcNAc)- and serum-induced differentiation. The permeable myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI), which inhibited C. albicans PKA in vitro, caused a concentration-dependent inhibition of germ-tube formation in cultures induced to germinate by GlcNAc; germination halted irrespective of the time of addition of the inhibitor. MyrPKI also blocked dibutyryl-cAMP (dbcAMP)- and glucagon-stimulated germination but did not affect serum-induced germination. H-89, another highly specific PKA inhibitor, displayed the same effect on germination. Neither MyrPKI nor H-89 had any effect on budding of yeast cells. In conclusion, our results indicate that cAMP-mediated activation of PKA plays a pivotal role in the biochemical mechanism underlying morphogenesis.     

N-Acetyl-d-Glucosamine Induces Germination in Candida albicans through a Mechanism Sensitive to Inhibitors of cAMP-Dependent Protein Kinase

The present study examines the involvement of cAMP-dependent protein kinase (PKA) in the dimorphic transition of Candida albicans by assessing the in vivo effect of two permeable PKA inhibitors on N-acetyl-d-glucosamine (GlcNAc)- and serum-induced differentiation. The permeable myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI), which inhibited C. albicans PKA in vitro, caused a concentration-dependent inhibition of germ-tube formation in cultures induced to germinate by GlcNAc; germination halted irrespective of the time of addition of the inhibitor. MyrPKI also blocked dibutyryl-cAMP (dbcAMP)- and glucagon-stimulated germination but did not affect serum-induced germination. H-89, another highly specific PKA inhibitor, displayed the same effect on germination. Neither MyrPKI nor H-89 had any effect on budding of yeast cells. In conclusion, our results indicate that cAMP-mediated activation of PKA plays a pivotal role in the biochemical mechanism underlying morphogenesis.     

Role of cAMP-dependent pathway in eosinophil apoptosis and survival

The survival and apoptosis of eosinophils is of pivotal importance for controlling allergic diseases such as asthma and rhinitis. In this study we have investigated the role for cAMP in regulating eosinophil survival and apoptosis in the absence of eosinophil-active cytokines. The treatment with dibutyryl cyclic AMP (dbcAMP) increased eosinophil survival with a concomitant decrease of apoptosis in a dose-dependent manner. The pretreatment with a protein kinase A (PKA) inhibitor blocked the effects of dbcAMP on survival and apoptosis of eosinophils. The catalytic subunit of PKA was translocated to nucleus in parallel with a robust increase of intracellular cAMP levels upon exposure to dbcAMP but not IL-5, suggesting the separation of PKA activation from the IL-5-induced suppression of eosinophil apoptosis. When eosinophils were treated with pharmacological inhibitors of protein kinases prior to exposure to dbcAMP or IL-5, only the mitogen-activating protein kinase (MAPK) inhibitor, PD098059, was partly able to block dbcAMP-induced augmentation of eosinophil viability, whereas both Janus kinase 2 and MAPK inhibitors effectively interrupted the IL-5-induced prolongation of eosinophil survival. The effects of dbcAMP and these protein kinase inhibitors on eosinophil apoptosis were confirmed by morphologic analysis. We propose that a cAMP-dependent pathway may constitute an important component for regulating eosinophil survival/apoptosisand that cAMP may inhibit eosinophil apoptosis through the activation of PKA and of subsequent MAPK in part.     

Picrosides I and II,selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells

Picrosides I and II caused a concentration-dependent (> 0.1 microM) enhancement of basic fibroblast growth factor (bFGF, 2 ng/ml)-, staurosporine (10 nM)- and dibutyryl cyclic AMP (dbcAMP, 0.3 mM)-induced neurite outgrowth from PC12D cells. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, blocked the enhancement of bFGF (2 ng/ml)-, staurosporine (10 nM)- or dbcAMP (0.3 mM)-induced neurite outgrowth by picrosides, suggesting that picrosides activate MAP kinase-dependent signaling pathway. However, PD98059 did not affect the bFGF (2 ng/ml)-, staurosporine (10 nM)- and dbcAMP (0.3 mM)-induced neurite outgrowth in PC12D cells, indicating the existence of two components in neurite outgrowth induced by bFGF, staurosporine and dbcAMP, namely the MAP kinase-independent and the masked MAP kinase-dependent one. Furthermore, picrosides-induced enhancements of the bFGF-action were markedly inhibited by GF109203X (0.1 microM), a protein kinase C inhibitor. The expression of phosphorylated MAP kinase was markedly increased by bFGF (2 ng/ml) and dbcAMP (0.3 mM), whereas that was not enhanced by staurosporine (10 nM). Picrosides had no effect on the phosphorylation of MAP kinase induced by bFGF or dbcAMP and also unaffected it in the presence of staurosporine. These results suggest that picrosides I and II enhance bFGF-, staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the intracellular MAP kinase-dependent signaling pathway. Picrosides I and II may become selective pharmacological tools for studying the MAP kinase-dependent signaling pathway in outgrowth of neurites induced by many kinds of neuritogenic substances including bFGF.     

Interplay between acid phosphatase and cysteine proteases in mediating vitellin degradation during early embryogenesis of Periplaneta americana

In this work, we characterized the activities of two classes of proteases and AcP during early embryogenesis of Periplaneta americana. AcP activity was first detected at day 6 and reached a maximum level at day 10 of development. Using phosphoamino acids, phosphatase activity was shown to be directed only against phosphotyrosine at day 6 while at day 10 it was also active against phosphoserine. In parallel, two classes of proteases were detected and located within yolk granules: a clan CA-cysteine protease, which was inhibited by E-64, insensitive to CA 074 and activated by acidic pH at day 3; and a neutral serine protease, which was inhibited by aprotinin at day 6. Assays of vitellin (Vt) degradation evidenced that incubations at neutral pH induced slight proteolysis, while the incubations at acidic pH did not result in Vt degradation. However, pre-incubations of Vt with AcP increased the levels of Vt acidic proteolysis and this could be inhibited by the addition of phosphatase inhibitors. On the other hand, the same pre-incubations showed no effects on the profile of degradation at neutral pH. We propose that AcP and cysteine protease cooperate to assure Vt breakdown during early embryogenesis of P. americana.     

Cyclic AMP-dependent proteolysis of GATA-6 expressed on the intracellular membrane

Cyclic AMP-dependent proteolysis of GATA-6 was characterized by fusing GATA-6 with the carboxyl-terminal membrane domain of SREBP-2. When the fusion protein was stably expressed in CHO-K1 cells, it was recovered in the ER membrane. This protein was processed in a similar manner to SREBP-2 upon cholesterol starvation, and the GATA-6 moiety moved into the nucleus. The GATA-6 moiety on the membrane became undetectable in the presence of dbcAMP or cholera toxin. However, H-89, K-252a, MG115 and lactacystin inhibited this decrease, suggesting that the cytoplasmic GATA-6 moiety of the fusion protein was degraded by proteasomes though A-kinase upon elevation of the cellular cAMP concentration.     

Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain.

The effects of cAMP-dependent protein kinase (cAMP-PK) phosphorylation on the degradation of the microtubule-associated protein tau by calpain were studied. Purified bovine brain tau that had been phosphorylated by cAMP-PK had a slower migration pattern on sodium dodecyl sulfate-polyacrylamide gels and a more acidic, less heterogeneous pattern on two-dimensional, nonequilibrium pH gradient electrophoresis (NEPHGE) gels compared with untreated tau. Phosphorylation of tau by cAMP-PK significantly inhibited its proteolysis by calpain compared with untreated tau. To our knowledge this is the first demonstration that phosphorylation of tau by a specific kinase results in increased resistance to hydrolysis by calpain. Tau dephosphorylated by alkaline phosphatase migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gels and also showed an altered two-dimensional NEPHGE pattern. Dephosphorylation of tau had no effect on its susceptibility to calpain proteolysis, indicating that regulation of the susceptibility to calpain hydrolysis is due to the phosphorylation of a specific site(s). These results suggest a role for phosphorylation in regulating the degradation of tau. Abnormal phosphorylation could result in a protease-resistant tau population which may contribute to the formation of paired helical filaments in Alzheimer's disease.     

Modulation of the human sperm acrosome reaction by effectors of the adenylate cyclase/cyclic AMP second-messenger pathway.

The acrosome reaction of spermatozoa appears to be analogous to various somatic cell exocytotic events which involve cascade reactions, i.e., transmission of an external signal across the cell membrane resulting in activation of an "amplifier" enzyme and the generation of a second messenger. Using a synchronous acrosome reaction system (De Jonge et al., J. Androl., 10:232-239, '89a), it was found that analogues of the second-messenger cAMP, dibutyryl cAMP (dbcAMP) and 8-bromo cAMP, stimulated the acrosome reaction of capacitated spermatozoa. Additionally, treatment of spermatozoa with either xanthine or non-xanthine phosphodiesterase inhibitors induced a significant (P less than 0.05) increase in the percent acrosome reaction after a period of capacitation in comparison to untreated controls. These results indicate that analogues of cAMP or inhibitors which prevent cAMP hydrolysis can induce the human sperm acrosome reaction. Subsequent experiments were conducted to test whether the amplifier enzyme in the cascade reaction, adenylate cyclase, has a role in the acrosome reaction. Forskolin, an adenylate cyclase stimulator, caused a significant (P less than 0.01) increase in the percent acrosome reaction in comparison to controls. Modulators of adenylate cyclase--adenosine, 2'-0-methyladenosine, and 2',3'-dideoxyadenosine--significantly (P less than 0.01) inhibited the forskolin-induced acrosome reaction. dbcAMP was able to overcome the inhibition by adenosine. Two inhibitors of protein kinase A, the Walsh inhibitor and H-8, caused a significant (P less than 0.01) inhibition of the dbcAMP-induced acrosome reaction. Finally, in the absence of extracellular calcium, dbcAMP induced a significant (P less than 0.01) increase in the acrosome reaction in contrast to A23187. These results suggest that: 1) a molecular mechanism for the human sperm acrosome reaction involves the cAMP second-messenger system; i.e., activation of adenylate cyclase, the amplifier enzyme that produces cAMP, production of cAMP as a second messenger, and activation of cAMP-dependent kinase A; and that 2) activation of adenylate cyclase occurs after calcium influx.     

Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.     

Heat-resistant inhibitors of protein kinase C from bovine brain

Bovine brain cytosol is shown to contain two heat-resistant inhibitors of protein kinase C, with the following characteristics: 1. One protein kinase C inhibitor can be easily purified to homogeneity. Evidence is presented that this polypeptide of Mr 19,000 is calmodulin. It inhibits protein kinase C with an EC50 of about 2.5 microM and the inhibition is Ca2+-independent. It inhibits only intact protein kinase C. Removal of the regulatory domain of protein kinase C, by limited proteolysis with trypsin, abolishes the inhibition. 2. Another protein kinase C inhibitory activity has been partially purified. Its Mr is low (Mr 600-700, as estimated by gel chromatography). It is not digested by proteases, is hydrophilic, acid- and alkali-resistant, acts Ca2+-independently, and, in contrast to calmodulin, inhibits even the catalytic fragment of protein kinase C after removal of the regulatory domain by limited proteolysis. This inhibition is, at least partially, due to a competition with ATP. Besides protein kinase C, calcium/calmodulin-dependent protein kinase II is inhibited to a similar extent. cAMP-dependent protein kinase is not affected.     

Synergistic effects of cAMP-dependent signalling pathways and IL-1 on IL-6 production by H19-7/IGF-IR neuronal cells

cAMP-dependent signalling cascades regulate a number of CNS functions including brain inflammation processes. In this study, we characterized IL-1-induced IL-6 production in hippocampal cells using H19-7/IGF-IR cells and investigated the effect of changes in intracellular cAMP levels on IL-1 activity. IL-1 potently induced IL-6 mRNA expression with a corresponding increase in IL-6 release, in a time- and dose-dependent manner with a maximal at 24 h and with an EC50 value of 0.11 ng/ml. Cell pre-treatment with the IL-1sR antagonist produced a rightward shift of IL-1 dose-response effect with a corresponding decrease in IL-1 potency. IL-1-induced IL 6 release was attenuated in the presence of the p38 MAPK inhibitor SB203580 but was not significantly affected by the PKA inhibitor KT 5720. Western blotting analysis of phospho-CREB cell content showed a marked increase in CREB activation. Similar results were obtained by pharmacologically increasing cAMP using dibutyryl cyclic adenosine monophosphate (dbcAMP) or the cAMP-specific type-4 phosphodiesterase inhibitor rolipram. Both dbcAMP and rolipram increased IL-6 production to about 50% of IL-1 effect. However, in the presence of IL-1, IL-6 production was further potentiated by either dbcAMP and rolipram, reaching 300% and 500% IL-1-induced levels. These data implicate the role of cAMP-dependent pathways on IL-6 production in neuronal cells and suggest novel synergistic mechanisms of regulation of cytokine production in brain.     

Modulation of red cell vesiculation by protease inhibitors

Release of vesicles from human red cell membranes was induced either by ATP-depletion or by incubation of the cells in presence of sonicated dimyristoylphosphatidylcholine (DMPC) vesicles. Vesicles released from ATP-depleted red cells but not the DMPC-induced vesicles contained degradation products of band 3 protein. Furthermore, in ATP-depleted erythrocytes proteolytic breakdown products could be demonstrated that were not detected in cells incubated with DMPC. Proteolysis was neither significantly affected by the protease inhibitor N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK) nor by other protease inhibitors tested in this study (diisopropylfluorophosphate, N-ethylmaleimide and phenylmethylsulfonyl fluoride). Both vesiculation processes were inhibited in a concentration dependent way by TLCK while other protease inhibitors did not significantly influence membrane vesiculation. Phase contrast microscopy showed that TLCK diminished the DMPC-induced formation of echinocytes which is known to precede vesicle release. These results suggest that the influence of TLCK on membrane vesiculation is not primarily due to inhibition of proteolysis but to a direct interaction of the inhibitor with the intrinsic domain of the erythrocyte membrane.