Protein phosphorylation in human peripheral lymphocytes - stimulation by phytohemagglutinin and N6 monobutyryl cyclic AMP.

Protein phosphorylation was studied in human peripheral lymphocytes. The cells were preincubated with

, exposed to phytohemagglutinin (PHA), N⁶ monobutyryl cAMP (MBcAMP) or 8 bromo cGMP (BcGMP), homogenized and analyzed by SDS-polyacrylamide gel electrophoresis. Both PHA and MBcAMP produced early increases in the [³²P]content of multiple proteins in the 30,000–100,000 molecular weight range. After further incubation with PHA there was a shift in the phosphorylation response to smaller molecular weight proteins. BcGMP (1 mM-10 pM) had no effect on protein phosphorylation. These results suggest a role for cAMP in the early action of PHA on human peripheral lymphocytes.     

Protein kinase A and/or C inhibitors potentiate isoproterenol-induced cyclic AMP accumulation in intact human lymphocytes

The objective of the present study was to investigate the roles of protein kinase A and/or C in agonist-induced beta adrenoceptor activation in intact human lymphocytes. LYmphocytes from healthy subjects were incubated with isoproterenol and phosphodiesterase inhibitor (IBMX, 1.0 mM) after 20 minutes of preincubation with (or without) various compounds possessing protein kinase A and/or C inhibitory activities. These compounds included the relatively selective protein kinase C (PK-C) inhibitors (W-7, calmidazolium, polymyxin B, neomycin, tamoxifen and clomiphene), purified protein inhibitors of protein kinase A (PK-A) (obtained synthetically, or purified from bovine hearts and porcine hearts) and the two compounds (H-7, H-9), which have been found to inhibit both PK-A and PK-C. The results showed that all PK-C inhibitors alone decreased cellular basal cAMP levels while inhibitors of PK-A as well as both H-7 and H-9 increased basal cAMP levels in a dose dependent manner at certain concentrations. All inhibitors studied potentiated isoproterenol-induced cAMP accumulation. The protein kinase A and C inhibitor, H-7, also potentiated PGE1 (but not forskolin)-induced cAMP accumulation. In contrast, the protein kinase C activator, PMA, inhibited isoproterenol- and PGE1- (but not forskolin) induced cAMP accumulation. These data suggest that the potentiating effects of PK-A and/or C inhibitors may be related to the inhibition of PK-A and/or PK-C, both of which have been shown to be involved in beta 2 adrenoceptor desensitization and phosphorylation.     

Activation and phosphorylation of the ''dense-vesicle'' high-affinity cyclic AMP phosphodiesterase by cyclic AMP-dependent protein kinase.

Incubation of a hepatocyte particulate fraction with ATP and the isolated catalytic unit of cyclic AMP-dependent protein kinase (A-kinase) selectively activated the high-affinity 'dense-vesicle' cycle AMP phosphodiesterase. Such activation only occurred if the membranes had been pre-treated with Mg2+. Mg2+ pre-treatment appeared to function by stimulating endogenous phosphatases and did not affect phosphodiesterase activity. Using the antiserum DV4, which specifically immunoprecipitated the 51 and 57 kDa components of the 'dense-vesicle' phosphodiesterase from a detergent-solubilized membrane extract, we isolated a 32P-labelled phosphoprotein from 32P-labelled hepatocytes. MgCl2 treatment of such labelled membranes removed 32P from the immunoprecipitated protein. Incubation of the Mg2+-pre-treated membranes with [32P]ATP and A-kinase led to the time-dependent incorporation of label into the 'dense-vesicle' phosphodiesterase, as detected by specific immunoprecipitation with the antiserum DV4. The time-dependences of phosphodiesterase activation and incorporation of label were similar. It is suggested (i) that phosphorylation of the 'dense-vesicle' phosphodiesterase by A-kinase leads to its activation, and that such a process accounts for the ability of glucagon and other hormones, which increase intracellular cyclic AMP concentrations, to activate this enzyme, and (ii) that an as yet unidentified kinase can phosphorylate this enzyme without causing any significant change in enzyme activity but which prevents activation and phosphorylation of the phosphodiesterase by A-kinase.     

The role of cyclic AMP in modulating cytotoxic T lymphocytes. II. Sequential changes during culture in responsiveness of cytotoxic lymphocytes to cyclic AMP-active agents

Agents that increase cyclic AMP (cAMP) levels inhibited the activity of cytotoxic T lymphocytes (CTL) obtained from spleens of mice immunized with allogeneic cells. Cultured CTL, however, were desensitized to cAMP-active agents, in that the capacity of these agents to inhibit the activity of cultured CTL was markedly reduced. The capacity to inhibit CTL activity was reduced more rapidly for some agents than for others; the percent inhibition by histamine and PGE2 was reduced after 4 hr and was reduced more than 20% after 24 hr, whereas the percent inhibition by dibutyryl cAMP, theophylline, and cholera enterotoxin was reduced less than 6% after 24 hr, and was reduced significantly only after 48 hr. Culture in the presence of antigen accelerated desensitization to the latter three agents. CTL populations were also tested for their capacity to increase cAMP levels in response to agonists. The capacity of histamine to increase cAMP levels of the CTL was lost within 4 hr (i.e., as rapidly as its capacity to inhibit CTL activity) and was never restored, whereas the capacity of PGE2 to increase cAMP levels persisted throughout culture. These results suggest that culture induces multiple alterations in cAMP metabolism of CTL. These alterations, which result in dissociation of CTL activity from cAMP-mediated regulatory steps, may include loss of histamine receptors and/or histamine receptor-adenylate cyclase coupling, and also loss of one or more biochemical reactions that link elevated cAMP levels to inhibition of lysis.     

Effects of cyclic AMP on in vivo cytotoxic T lymphocytes generation

Chronic schistosomiasis mansoni is associated with a modulation of schistosome egg-associated cell-mediated granuloma formation. In this study the injection of a cell-free sonicate from either spleen or thymus cells from chronic Schistosoma mansoni-infected mice (> 15 weeks) passively modulated granuloma size in acutely infected (6–8 weeks) syngeneic mice. Cell-free sonicates from age-matched uninfected mice did not suppress granuloma formation. The chronic extracts also suppressed the soluble schistosome egg antigenic preparation-elicited delayed skin test reactivity in 7–8 week infected animals, but failed to suppress the unrelated antigen-elicited reactivity to bovine serum albumin (BSA) of BSA-sensitized animals.     

Protein phosphorylation and compartments of cyclic AMP in the control of cardiac contraction

The roles of cyclic AMP, cyclic AMP-dependent protein kinase and the phosphorylation of specific proteins in the regulation of cardiac contractility are briefly reviewed. Criteria for determining whether changes in cyclic AMP and protein phosphorylation are involved in a physiological response are discussed. Although cyclic AMP-dependent phosphorylation of the voltage-operated Ca^2– channel, phospholamban, troponin-I and C-protein have all been implicated in the response of the heart to inotropic agents which elevate cyclic AMP, none of these phosphorylations satisfy all of the criteria completely. Evidence is presented that there are compartments of cyclic AMP in heart which are coupled to different functional responses.Abbreviations cAMP 3,5 cyclic adenosine monophosphate - PDE cyclic nucleotide phosphodiesterase - cA-PrK cAMP-dependent protein kinase - SR sarcoplasmic reticulum - PGE₁ prostaglandin E₁ - Tn-I troponin I     

Hypoxanthine effects on cyclic AMP levels in human lymphocytes

We have measured hypoxanthine effect on cAMP levels in PBL in basal conditions (no agonist), and with the addition of 2-(p-[2-carboxyethyl] phenylethylamino)-5'-N-ethylcarboxamidoadenosine (CGS-21680, a specific A2 receptor agonist). We have found that hypoxanthine, at 25 microM and 50 microM concentrations, increases cAMP levels in PBL in basal and A2 agonist stimulated conditions.     

Protein kinase A mediates cAMP-induced tyrosine phosphorylation of the epidermal growth factor receptor

An increase in the intracellular cAMP concentration induces tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) followed by activation of extracellular signal-regulated kinases 1/2 (ERK1/2). In this report we demonstrate that these effects of cAMP are mediated via activation of protein kinase A (PKA). Chemical inhibition of PKA suppressed forskolin-induced EGFR tyrosine phosphorylation and ERK1/2 activation in PC12 cells. Furthermore, forskolin failed to induce significant tyrosine phosphorylation of the EGFR and ERK1/2 activation in PKA-defective PC12 cells. Forskolin-induced EGFR tyrosine phosphorylation was also observed in A431 cells and in membranes isolated from these cells. Phosphoamino acid analysis indicated that the recombinant catalytic subunit of PKA elicited phosphorylation of the EGFR on both tyrosine and serine but not threonine residues in A431 membranes. Together, our data indicate that activation of PKA mediates the effects of cAMP on the EGFR and ERK1/2. While PKA may directly phosphorylate the EGFR on serine residues, PKA-induced tyrosine phosphorylation of the EGFR occurs by an indirect mechanism.     

Protein kinase C-epsilon mediates phorbol ester-induced phosphorylation of connexin-43

We have used adenoviral vectors to express dominant negative variants of protein kinase C epsilon (PKCepsilon) or mitogen kinase kinase 1 (MKK1) to investigate their involvement in phorbol ester-induced connexin-43 (Cx43) phosphorylation in cardiomyocytes. Stimulation of cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) increased the fraction of the slower migrating (> or = 45 kDa) and more extensively phosphorylated Cx43 species. Expression of dominant negative MKK1 did not prevent the effect of PMA on Cx43 phosphorylation. Selective inhibition of PKCE significantly decreased baseline levels of Cx43 phosphorylation and the PMA-induced accumulation of > or = 45 kDa Cx43. Thus, production of the more extensively phosphorylated species of Cx43 in cardiomyocytes by PMA requires activation of PKCepsilon.     

Trypsin-induced increase in intracellular cyclic AMP of lymphocytes.

Trypsin increases intracellular levels of cylic AMP (cAMP) in lymphocytes. The trypsin-induced increase in cAMP is blocked by specific trypsin inhibitors and by high concentrations of different proteins. Several proteolytic enzymes from various sources, including other pancreatic proteases, do not cause an increase in cAMP under the same experimental conditions. Immobilized trypsin induced the same increase in cAMP as does free trypsin. The trypsin-induced rise in cAMP is not due to inhibition of cAMP phosphodiesterase, but consistent activation of adenylate cyclase by trypsin could not be demonstrated. The extent of the trypsin-induced increase in intracellular cAMP correlates with the type of the lymphocyte and with the state of maturity attained by the cells. Transformed lymphocytes and nonlymphoid cells do not react at all.     

Protein kinase C potentiates corticotropin releasing factor stimulated cyclic AMP in pituitary

The hypophysiotrophic hormone corticotropin releasing factor (CRF) stimulates the anterior pituitary corticotroph to export stress hormones such as adrenocorticotrophic hormone (ACTH). In rat anterior pituitary cells, CRF-induced elevation of cyclic AMP was profoundly potentiated (by an order of magnitude) by stimulators of protein kinase C. This effect occurred within minutes, was concentration dependent, and exhibited the appropriate pharmacological specificity to attribute the effects to protein kinase C. Phorbol myristate acetate (PMA), phorbol dibutyrate (PDB) and teleocidin were active with appropriate EC50's, while 4-alpha-PMA was inactive. PMA and PDB were also ACTH secretagogues in their own right. We suggest that protein kinase C can modulate CRF receptor coupling to the adenylate cyclase holoenzyme in anterior pituitary cells.     

Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation.

The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA.     

Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells.

Stimulation of human endothelial cells (EC) by thrombin elicits a rapid increase of intracellular free Ca2+ [(Ca2+]i), platelet-activating factor (PAF) production and 1-O-alkyl-2-lyso-sn-glycero-3- phosphocholine (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) activity. The treatment of EC with thrombin leads to a 90% decrease in the cytosolic protein kinase C (PKC) activity; this dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. The role of PKC in thrombin-mediated PAF synthesis has been assessed: (1) by the blockade of PKC activity with partially selective inhibitors (palmitoyl-carnitine, sphingosine and H-7); (2) by chronic exposure of EC to phorbol 12-myristate 13-acetate (PMA), which results in down-regulation of PKC. In both cases, a strong inhibition of thrombin-induced PAF production is observed, suggesting obligatory requirement of PKC activity for PAF synthesis. It is suggested that PKC regulates EC phospholipase A2 (PLA2) activity as thrombin-induced arachidonic acid (AA) release is 90% inhibited in PKC-depleted cells. Brief exposure of EC to PMA strongly inhibits thrombin-induced [Ca2+]i rise, acetyltransferase activation and PAF production, suggesting that, in addition to the positive forward action, PKC provides a negative feedback control over membrane signalling pathways involved in the thrombin effect on EC. Forskolin and iloprost, two agents that increase the level of cellular cAMP in EC, are very effective in inhibiting thrombin-evoked cytosolic Ca2+ rise, acetyltransferase activation and PAF production; this suggests that endogenously generated prostacyclin (PGI2) may modulate the synthesis of PAF in human endothelial cells.     

Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways

Agonist-induced phosphorylation of beta-adrenergic receptors (beta ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of beta ARs, particularly the beta 1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type beta 1AR (WT beta 1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA-beta 1AR), the putative GRK phosphorylation sites (GRK-beta 1AR), and both sets of phosphorylation sites (PKA-/GRK-beta 1AR). Following agonist stimulation, both PKA-beta 1AR and GRK-beta 1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WT beta 1AR and PKA-beta 1AR, sequestration of the GRK-beta 1AR and PKA-/GRK-beta 1AR was independent of beta-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WT beta 1AR and PKA-beta 1AR, whereas caveolae inhibitors prevented internalization only of the GRK-beta 1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the beta 1AR and 2) the pathway selected for beta 1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits.     

A cyclic AMP dependent protein kinase in Dictyostelium discoideum

A cyclic AMP-dependent protein kinase was found to appear during the time course of development of $\text{Dictyostelium}\text{discoideum}$. No cyclic AMP dependency was observed at any stage of development in crude 110,000 X G soluble extracts. After partial purification, however, extracts from post-aggregation stages contained enzyme that was activated up to 6-fold by cyclic AMP, whereas protein kinase from earlier stages was not affected by cyclic AMP. Likewise, cyclic AMP binding activity increased from the aggregation to the slug stage of development. Approximately one-half of the total cyclic AMP binding activity co-purified with the cyclic AMP dependent protein kinase. The enzyme from $\text{Dictyostelium}$ showed similarities to mammalian protein kinases with respect to its kinetic properties but differed in its behavior on ion-exchange chromatography.     

Protein phosphorylation in the larval epidermis of Locusta migratoria: Role of cyclic AMP and 20-hydroxyecdysone

Cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinase (PK) activities were quantified in the wing-pad epidermis of Locusta migratoria during the fifth larval instar. A low level of cyclic nucleotide-independent PK activity was detected in this tissue, whereas no PK-C (Ca²⁺- and phospholipid-dependent enzyme) activity was found. The main cyclic nucleotide-PK activity was cAMP-PK and large changes were observed during the intermoult. Concomitant increases in cAMP-PK activity and cAMP-dependent phosphorylation of several endogenous polypeptide substrates occurred during the last part of the intermoult cycle. The most marked ³²P-incorporation was observed in a 43 kDa polypeptide. These changes could be correlated with the peak of 20-OH-ecdysone in both hemolymph and integument.In vitro incubation of wing-pads with 20-OH-ecdysone (48 h) did not change cAMP-PK activity, but there were major changes in the pattern of endogenous phosphoproteins in particular the appearance of 43 and 45 kDa phosphoproteins. Two-dimensional analysis revealed several groups of phosphorylated spots. The results indicate a close relationship between certain cAMP-dependent phosphorylated polypeptides and the action of 20-OH-ecdysone.     

Protein kinase C mediates platelet-derived growth factor-induced tyrosine phosphorylation of p42

One of the early events after stimulation of Swiss 3T3 cells with either platelet-derived growth factor (PDGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), diacylglycerol, or several other mitogens is the near stoichiometric phosphorylation at tyrosine and serine of a scarce cytoplasmic protein (p42). TPA and diacylglycerol are known to directly stimulate the activity of a protein-serine/threonine kinase, protein kinase C (PKC). PDGF and several other mitogens stimulate tyrosine kinases directly and PKC indirectly. We have therefore examined the involvement of PKC in p42 tyrosine phosphorylation in Swiss 3T3 cells. Firstly, six agents which stimulated phosphorylation of p42 also stimulated phosphorylation of a known PKC substrate, an 80,000-Mr protein (p80). Secondly, in PKC-deficient cells (cells in which PKC activity was reduced to undetectable levels by prolonged exposure to TPA), PDGF-induced p42 phosphorylation was reduced three- to fourfold. Phosphoamino acid analysis of phosphorylated p42 from PDGF-stimulated PKC-deficient cells revealed primarily phosphoserine and only a trace of phosphotyrosine, suggesting that the reduction in PDGF-stimulated tyrosine phosphorylation of p42 resulting from PKC deficiency is greater than three- to fourfold. Finally, comparison of antiphosphotyrosine immunoprecipitates of PKC-deficient versus naive cells revealed that most other PDGF-induced tyrosine phosphorylation events were quite similar. These data suggest that mitogens such as PDGF, which directly stimulate phosphorylation of some proteins at tyrosine, induce p42 tyrosine phosphorylation via a cascade of events involving PKC.     

Protein kinase and cyclic AMP levels in differentiating myoblasts are altered by extracellular calcium

Protein kinase specific activities and cyclic AMP levels show a similar pattern of response, when the Ca²⁺ concentration is altered in the culture medium of differentiating chick skeletal muscle cells; an increase at intermediate Ca²⁺ concentrations (0.05–0.2mM), followed by a decrease at higher concentrations (2mM). Effects of Ca²⁺ on protein kinase appear to be on cyclic AMP-independent enzymes in both nucleus and cytoplasm, and are quite the reverse of Ca²⁺ effects on the muscle-specific enzyme, creatine kinase.     

Protein phosphorylation in chick kidney. Response to parathyroid hormone, cyclic AMP, calcium, and phosphatidylserine

The regulation of endogenous protein phosphorylation by parathyroid hormone (PTH) was investigated using confluent monolayer cultures of chick kidney cells. Homogenates and subcellular fractions of PTH (bovine 1-34)-treated cells were subjected to an endogenous protein phosphorylation assay using ((gamma- 32P]ATP in the presence or absence of 2.0 microM cAMP or 0.5 mM Ca2+ with 25 micrograms/ml of phosphatidylserine and reactions terminated with sodium dodecyl sulfate. In other experiments, cultures were incubated in a phosphate-free 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline containing 50 muCi/ml of [32P]PO4 and incubations were terminated with sodium dodecyl sulfate. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cyclic AMP stimulated 32P incorporation into proteins having molecular weights of 17,000, 22,000, 35,000, 42,000, 54,000, 75,000, 80,000, 120,000, and 143,000. Calcium-phosphatidylserine stimulated the phosphorylation of proteins of 20,000, 52,000, 58,000, 60,000, and 143,000. The protein phosphorylation patterns in cultured kidney cells and freshly isolated kidney tissue were quite similar. Treatment of cultured cells with 5-50 ng/ml of PTH resulted in stimulated phosphorylation of the 35,000 and 42,000 dalton proteins as assessed by endogenous phosphorylation in homogenates. In intact cells incubated with [32P]PO4, PTH stimulated most noticeably the phosphorylation of the 35,000-dalton protein. Based on studies with cultured and fresh kidney cells, the majority of the substrate proteins for cAMP and calcium-dependent protein kinases were located in the cytoplasm with the exception of the 42,000-dalton protein which was located in the brush-border-plasma membrane fraction. The cytoplasmic cAMP-dependent protein kinase activity was responsible for the majority of PTH-stimulated protein phosphorylation.