Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.     

Metabolism of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) by cultured rat Kupffer cells.

In platelets, and in several other cell systems, pre-treatment with protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA) results in the inhibition of receptor-mediated responses, suggesting that protein kinase C may play an important role in the termination of signal transduction. In the present study, we have attempted to locate the site of action of phorbol ester by comparing thrombin-induced (i.e. receptor-mediated) platelet activation with that induced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and NaF, two agents which by-pass the receptor and initiate platelet responses by directly modulating G-protein function. After a 10 s pre-treatment with PMA (16 nM), dense-granule secretion induced by thrombin (0.2 unit/ml), GTP[S] (40 microM) and NaF (30 mM) was potentiated, resulting in a greater than additive response to agent plus PMA. However, after a 5 min pre-treatment, thrombin-induced secretion alone was inhibited, whereas PMA plus GTP[S]/NaF-induced release remained greater than additive. [32P]Phosphatidate formation in response to all three agents, in contrast, was inhibited by 50-70% in PMA (5 min)-treated platelets. That secretion induced by these agents is a protein kinase C-dependent event was demonstrable by using staurosporine, a protein kinase C inhibitor which at concentrations of 1-10 nM inhibited (70-90%) PMA-induced as well as thrombin- and NaF-induced secretion and protein phosphorylation. In membranes from PMA-treated platelets, thrombin-stimulated GTPase activity was significantly enhanced compared with that in untreated membranes (59% versus 82% increase over basal activity). The results suggest that inhibition of receptor-mediated responses by PMA may be directed towards two sites relating to G-protein activation: (i) receptor-stimulated GTPase activity and (ii) G-protein-phospholipase C coupling. Furthermore, the lack of inhibition of NaF- and GTP[S]-induced secretion by PMA suggests that different mechanisms may be involved in thrombin-induced and G-protein-activator-induced secretion.     

Different requirements for protein kinase C activation and Ca2+-independent insulin secretion in response to guanine nucleotides. Endogenously generated diacylglycerol requires elevated Ca2+ for kinase C insertion into membranes

Electrically permeabilized RINm5F cells were used to assess the factors required for activation of protein kinase C (PKC) and insulin secretion. PKC was activated either by phorbol 12-myristate 13-acetate (PMA) or by the generation of endogenous diacylglycerol in response to the nonhydrolyzable guanine nucleotide analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S). As shown previously, both PMA and GTP gamma S elicit Ca2+-independent insulin secretion. This effect was mimicked by guanyl-5'-yl imidodiphosphate (Gpp(NH)p) but not by guanosine 5'-O-(3-fluorotriphosphate) and guanosine 5'-O-(3-phenyltriphosphate) possessing only one negative charge in the gamma-phosphate group. The action of PMA was mediated by PKC, since the agent caused both phosphorylation of specific protein substrates and association of the enzyme with cellular membranes. This translocation was independent of the Ca2+ concentration employed. In contrast, GTP gamma S only promoted association of PKC with membranes at 10(-6) and 10(-5) M Ca2+ and failed to alter significantly protein phosphorylation in the absence of Ca2+. Neither Gpp(NH)p, which stimulates insulin release, nor the other two GTP analogs, increased the proportion of PKC associated with membranes. To verify that the Ca2+-dependent effect of GTP gamma S on PKC is due to activation of phospholipase C, we measured the generation of diacylglycerol. GTP gamma S indeed stimulated diacylglycerol production in the leaky cells by about 50% at Ca2+ concentrations between 10(-7) and 10(-5) M, an effect which was almost abolished in the absence of Ca2+. Thus, at 10(-7) M Ca2+, the concentration found in resting intact cells, the generated diacylglycerol was not sufficient to cause PKC insertion into the membrane, demonstrating that both elevated Ca2+ and diacylglycerol are necessary for translocation to occur. It is concluded that while PKC activation by PMA elicits Ca2+-independent insulin secretion, the kinase seems not to mediate the stimulatory action of GTP analogs in the absence of Ca2+.     

Phorbol ester treatment inhibits thrombin but not stable GTP analogue-induced platelet granule secretion despite inhibition of phosphatidate formation with both agonists

Previous work has demonstrated that pre-treatment of platelets with phorbol esters that activate protein kinase C eg phorbol 12-myristate 13-acetate (PMA) results in an inhibition of inositol phospholipid breakdown and granule secretion induced by physiological agonists such as thrombin and collagen. In the present study, the effect of pre-treatment with PMA on granule secretion and [32P]-phosphatidate (PA) formation induced by the stable GTP analogue, guanosine 5'-[gamma thio] triphosphate (GTP gamma S) was examined in saponin-permeabilized platelets. A low concentration of PMA ie 1.6nM, that did not induce significant 5-hydroxytryptamine (5HT) secretion on its own, but inhibited low-dose thrombin-induced 5HT secretion totally and PA formation by 30-40% in intact as well as permeabilised platelets was chosen. Our results demonstrate a lack of inhibition of GTP gamma S (40 microM)-induced 5HT secretion by PMA in permeabilised platelets, despite significant inhibition (70%) of PA formation, suggesting that apart from the diacylglycerol pathway of secretion which may be common to thrombin and GTP analogues, secretion induced by physiological agonists such as thrombin may involve another mechanism that is inhibitable by phorbol esters.     

Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A2 in response to thrombin in rabbit platelets

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inositol trisphosphate (IP3) in [3H]inositol-labeled platelets, increases of [3H]arachidonic acid [( 3H]AA) release, and [3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A2 or C inhibitor suggested that not only phospholipase C but also phospholipase A2 activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A2 activity. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanyl-5'-(beta,gamma-iminio)triphosphate), or AlF4- caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5'-(2-O-thio)diphosphate) and was also inhibited by decreased Mg2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IP3 formation, was diminished by 100 ng/ml of pertussis toxin, associated with ADP-ribosylation of membrane 41-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTP gamma S-stimulated release from permeabilized platelets were both markedly dependent on Ca2+. However, Ca2+ addition could not enhance AA release without GTP gamma S even when Ca2+ was increased up to 10(-4) M in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase A2 activity, not by phospholipase C activity, and that Ca2+ is an important factor to the activation of phospholipase A2 but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase A2.     

Characterization of an actin-myosin head interface in the 40-113 region of actin using specific antibodies as probes.

A method of membrane permeabilization of T lymphocytes with the bacterial cytotoxin streptolysin O has allowed the effect of guanine nucleotide analogues on phosphatidylinositol metabolism and protein kinase C (PKC) activation to be investigated. The data demonstrate that, in permeabilized cells, phosphorylation of the gamma subunit of the CD3 antigen can be induced in response to the PKC activator phorbol 12,13-dibutyrate, the polyclonal mitogen phytohaemagglutinin (PHA) and the stimulatory guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]). Application of a pseudo-substrate inhibitor of PKC indicated that CD3gamma-chain phosphorylation induced in response to all three agonists was mediated by PKC. PHA and GTP[S] also stimulated inositol phospholipid turnover and inositol phosphate accumulation. The kinetics and concentration-dependence of PHA-induced inositol phospholipid hydrolysis correlated with PHA-induced CD3gamma phosphorylation, suggesting that PHA may regulate CD3gamma phosphorylation via diacylglycerol produced as a consequence of inositol phospholipid hydrolysis. However, there was an inconsistency in that PHA induced greater (greater than 200%) levels of inositol phospholipid turnover than did GTP[S], but much weaker (less than 50%) levels of CD3-antigen phosphorylation. There was also a discrepancy between GTP[S] effects on phosphatidylinositol turnover and PKC activation, in that the half-maximal GTP[S] concentration for inositol phosphate production and CD3gamma-chain phosphorylation was 0.75 microM and 75 microM respectively. Moreover, 10 microM-GTP[S] induced maximal inositol phosphate production, but only 10% of maximal CD3gamma-chain phosphorylation. The data are consistent with the idea that other signal-transduction pathways, in addition to those involving inositol phosphate production, exist for the regulation of PKC in T lymphocytes.     

Ca2(+)-induced secretion by electropermeabilized human neutrophils. The roles of Ca2+, nucleotides and protein kinase C

Studies of stimulus-response coupling have benefitted from the availability of permeabilization techniques, whereby putative second messengers and intracellular modulators can be introduced into the cell interior. Electropermeabilization, which uses high-intensity electric fields to breach the plasma membrane, creates small pores, permitting access of solutes with molecular masses below 700 KDa. Neutrophils permeabilized by this technique, but not intact cells, discharged lysosomal constituents when exposed to micromolar levels of Ca2+. Secretion by electroporated neutrophils was significantly enhanced by the presence of Mg-ATP (0.3-1.0 mM). Contrary to expectations, it was determined that ATP was not the only nucleotide which enhanced Ca2(+)-induced secretion in the presence of Mg2+. Not only could GTP, XTP, ITP, UTP or ADP partially or completely replace ATP, but even non-hydrolyzable nucleotides such as ADP beta S ATP gamma S, and App[NH]p were effective. GTP gamma S and GDP beta S were inhibitory, while Gpp[NH]p was inactive. None of these nucleotides induced secretion on its own. In contrast, neutrophils which were permeabilized and then washed, were only slightly activated by Mg-ATP and other nucleotides; even the response to Ca2+ alone was less. This hyporesponsiveness of washed cells proved to be due to a time-dependent deactivation of the permeabilized neutrophils taking place at 4 degrees C. In an effort to assess the role for protein kinase C (PKC) in secretion in this system, we examined the effects of phorbol myristate acetate (PMA), a PKC agonist. PMA enhanced degranulation induced by Ca2+ by lowering the requirement for this divalent cation; enhancement by PMA was not dependent upon exogenous ATP. Three inhibitors of PKC with varying specificity, namely H-7, K-252a, and staurosporine, all abrogated PMA-enhanced secretion. These agents also inhibited secretion stimulated by Ca2+ plus ATP in parallel with that induced by Ca2+ plus PMA, strongly suggesting a role for PKC in modulation of degranulation by ATP. Our results show that electropermeabilized neutrophils provide a convenient, useful model for stimulus-secretion coupling. These data also suggest that the 'requirement' for Mg-ATP, which has been observed in other permeabilized cell systems, is not simply for metabolic energy or as a substrate for kinases. It is possible that these nucleotides all interact with a recently described neutrophil receptor for adenine nucleotides or with a recently postulated exocytosis-linked G-protein.     

Pertussis Toxin-Insensitive G Protein Mediates Carbachol Activation of Phospholipase D in Rat Pheochromocytoma PC12 Cells

In the present study, an activation mechanism for phospholipase D (PLD) in [3H]palmitic acid-labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [3H]phosphatidylethanol ([3H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [3H]-PEt, which reached a plateau within 1 min, in a concentration-dependent manner. The [3H]PEt formation by CCh was completely antagonized by atropine, demonstrating that the CCh effect was mediated by the muscarinic acetylcholine receptor (mAChR). A tumor promoter, phorbol 12-myristate 13-acetate (PMA), also caused an increase in [3H]-PEt content, which reached a plateau at 30-60 min after exposure, but an inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 microM), blocked PMA-induced [3H]PEt formation by 77%, it had no effect on the CCh-induced formation. These results suggest that mAChR-induced PLD activation is independent of PKC, whereas PLD activation by PMA is mediated by PKC. NaF, a common GTP-binding protein (G protein) activator, and a stable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), also stimulated [3H]PEt formation in intact and digitonin-permeabilized cells, respectively. GTP, UTP, and CTP were without effect. Furthermore, guanosine 5'-O-(2-thiodiphosphate) significantly inhibited CCh- and GTP gamma S-induced [3H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 microM) had no effect on [3H]PEt formation by GTP gamma S.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel mechanism for the Ca(2+)-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase

Mechanisms of Ca2+ sensitization of both myosin light chain (MLC) phosphorylation and force development by protein kinase C (PKC) were studied in permeabilized tonic smooth muscle obtained from the rabbit femoral artery. For comparison, the Ca2+ sensitizing effect of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) was examined, which had been previously shown to inhibit MLC phosphatase in phasic vascular smooth muscle. We now report that PKC activators (phorbol esters, short chain synthetic diacylglycerols and a diacylglycerol kinase inhibitor) and GTP gamma S significantly increase both MLC phosphorylation and force development at constant [Ca2+]. Major phosphorylation site occurring in the presence of phorbol-12,13- dibutyrate (PDBu) or GTP gamma S at constant [Ca2+] is the same serine residue (Ser-19) as that phosphorylated by MLC kinase in response to increased Ca2+ concentrations. In an ATP- and Ca(2+)-free solution containing 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4- diazepine (ML-9), to avoid the kinase activity, both PDBu and GTP gamma S significantly decreased the rate of MLC dephosphorylation to half its control value. However, PDBu inhibited the relaxation rate more than did GTP gamma S. In the presence of microcystin-LR to inhibit the phosphatase activity, neither PDBu nor GTP gamma S affected MLC phosphorylation and force development. These results indicate that PKC, like activation of GTP binding protein, increases Ca2+ sensitivity of both MLC phosphorylation and force production through inhibition of MLC phosphatase.     

Receptor-Induced Diacylglycerol Formation in Permeabilized Platelets; Possible Role for a Gtp-Binding Protein

Abstract

Exposure of human platelets to 10 discharges from a 4.5 μF capacitor charged at 3 kV permitted isolation of a stable preparation of permeabilized platelets that, after equilibration with Ca²⁺ buffers (pCa < 6) for 15 min at 0°C, secreted 5-hydroxytryptamine (5-HT) at 25°C. Thrombin enhanced the sensitivity to Ca²⁺ of the secretion of 5-HT by about 10-fold, whereas Arg -vasopressin and the prostaglandin endoperoxide analogue, U-46619, increased sensitivity to Ca²⁺ by 3 to 4-fold. This action of thrombin was associated with stimulation of diacylglycerol formation, a marked increase in phosphorylation of protein P47 and a smaller increase in phosphorylation of the P-light chain of myosin. Thrombin exerted these effects at a [Ca²⁺ _free] of 0.1 μM, suggesting that the receptor-activated breakdown of platelet phosphoinositides to diacylglycerol may not require prior Ca²⁺ mobilization in intact platelets. In both the presence and absence of thrombin, a higher [Ca²⁺ _free] was required for optimal secretion than for maximal phosphorylation of P47 and myosin light-chain, indicating that Ca²⁺ and possibly diacylglycerol have roles in the secretory mechanism additional to activation of the enzymes that phosphorylate these proteins. Stable GTP analogues such as guanosine-5′-0-(3-thiotriphosphate) (GTPγS), and to a lesser extent GTP itself, enhanced the Ca²⁺ sensitivity of the secretion of 5-HT from permeabilized platelets. Moreover, GTP potentiated the stimulatory action of thrombin. These effects of GTPγS and GTP were associated with increased diacylglycerol formation and were inhibited by guanosine-5′-0-(2-thiodiphosphate) (GDPβS) suggesting that a GTP-binding protein may play a role in the receptor-activated breakdown of phosphoinositides. However, as GDPβS did not inhibit the potentiation of secretion caused by thrombin alone, a GTP-independent pathway of platelet activation may also exist.     

Regulation of bombesin-stimulated inositol 1,4,5-trisphosphate generation in Swiss 3T3 fibroblasts by a guanine-nucleotide-binding protein.

The stimulation of inositol phosphate generation by bombesin and GTP analogues was studied in Swiss 3T3 cells permeabilized by electroporation. Bombesin-stimulated inositol phosphate generation is potentiated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and inhibited by guanosine 5'-[beta-thio]diphosphate at all peptide concentrations tested, with no change in the EC50 value (concn. giving half-maximal response) for the agonist. Kinetic analysis showed that, although bombesin-stimulated [3H]InsP3 generation in [3H]inositol-labelled cells was rapid (maximal by 5-10 s), the response to GTP[S] alone displayed a distinct lag time of 20-30 s. This lag time was significantly decreased by the addition of bombesin, suggesting that in this system agonist-stimulated GTP/GDP exchange occurs. In addition, bombesin-stimulated generation of Ins(1,4,5)P3 mass at 10 s was enhanced by GTP[S] in the absence of a nucleotide response alone, a result consistent with this proposal. Pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent inhibition of bombesin-, but not GTP[S]-, stimulated inositol phosphate generation. Furthermore, although PMA pretreatment did not affect the lag time for InsP3 formation in response to GTP[S] alone, the degree of synergy between bombesin and the nucleotide was severely decreased at early time points. The results therefore demonstrate that the high-affinity bombesin receptor is coupled via a G-protein to phospholipase C in a manner consistent with a general model for receptor-G-protein interactions and that this coupling is sensitive to phosphorylation by protein kinase C.     

Phorbol ester treatment of intact rabbit platelets greatly enhances both the basal and guanosine 5''-[gamma-thio]triphosphate-stimulated phospholipase D activities of isolated platelet membranes. Physiological activation of phospholipase D may be secondary to activation of phospholipase C.

Rabbit platelets were labelled with [3H]glycerol and incubated with or without phorbol 12-myristate 13-acetate (PMA). Membranes were then isolated and assayed for phospholipase D (PLD) activity by monitoring [3H]phosphatidylethanol formation in the presence of 300 mM-ethanol. At a [Ca2+free] of 1 microM, PLD activity was detected in control membranes, but was 5.4 +/- 0.8-fold (mean +/- S.E.M.) greater in membranes from PMA-treated platelets. Under the same conditions, 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated PLD by 18 +/- 3-fold in control membranes, whereas PMA treatment and GTP[S] interacted synergistically to increase PLD activity by 62 +/- 12-fold. GTP[S]-stimulated PLD activity was observed in the absence of Ca2+, but was increased by 1 microM-Ca2+ (3.5 +/- 0.2-fold and 1.8 +/- 0.1-fold in membranes from control and PMA-treated platelets respectively). GTP exerted effects almost as great as those of GTP[S], but 20-30-fold higher concentrations were required. Guanosine 5'-[beta-thio]diphosphate inhibited the effects of GTP[S] or GTP, suggesting a role for a GTP-binding protein in activation of PLD. Thrombin (2 units/ml) stimulated the PLD activity of platelet membranes only very weakly and in a GTP-independent manner. The actions of PMA and analogues on PLD activity correlated with their ability to stimulate protein kinase C in intact platelets. Staurosporine, a potent protein kinase inhibitor, had both inhibitory and, at higher concentrations, stimulatory effects on the activation of PLD by PMA. The results suggest that PMA not only stimulates PLD via activation of protein kinase C but can also activate the enzyme by a phosphorylation-independent mechanism in the presence of staurosporine. However, under physiological conditions, full activation of platelet PLD may require the interplay of protein kinase C, increased Ca2+ and a GTP-binding protein, and may occur as a secondary effect of the activation of phospholipase C.     

Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: Further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDa protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells. we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 μM), on pepsinogen secretion and phosphorylation of the 72-kDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 μM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 μM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with ³²P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 μM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS “phosphorylation/calmodulin binding domain peptide” indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion. J. Cell. Biochem. 64:514–523. © 1997 Wiley-Liss, Inc.     

Activated release of membrane-anchored TGF-alpha in the absence of cytosol

The ectodomain of proTGF-alpha, a membrane-anchored growth factor, is converted into soluble TGF-alpha by a regulated cellular proteolytic system that recognizes proTGF-alpha via the C-terminal valine of its cytoplasmic tail. In order to define the biochemical components involved in proTGF-alpha cleavage, we have used cells permeabilized with streptolysin O (SLO) that have been extensively washed to remove cytosol. PMA, acting through a Ca(2+)-independent protein kinase C, activates cleavage as efficiently in permeabilized cells as it does in intact cells. ProTGF-alpha cleavage is also stimulated by GTP gamma S through a mechanism whose pharmacological properties suggest the involvement of a heterotrimeric G protein acting upstream of the PMA- sensitive Ca(2+)-independent protein kinase C. Activated proTGF-alpha cleavage is dependent on ATP hydrolysis, appears not to require vesicular traffic, and acts specifically on proTGF-alpha that has reached the cell surface. These results indicate that proTGF-alpha is cleaved from the cell surface by a regulated system whose signaling, recognition, and proteolytic components are retained in cells devoid of cytosol.     

Mutations within the uncE gene affecting assembly of the F1F0-ATPase of Escherichia coli.

Activation of protein kinase C (PKC) in human T lymphocytes is an immediate consequence of mitogenic signalling via the antigen-receptor complex and CD2 antigen. In order to investigate further the signal-transduction pathways which result in PKC activation, we have established a novel PKC assay system using streptolysin-O-permeabilized T cells. Known peptide substrates of PKC were introduced into permeabilized cells in the presence of [gamma-32P]ATP, 3 mM-Mg2+ and 150 nM free Ca2+. The peptide found to have the lowest background phosphorylation had the sequence Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys (peptide GS), and the phosphorylation of the peptide was increased up to 6-fold by direct activation of PKC with phorbol 12,13-dibutyrate. Induction of PKC activation with the UCHT1 antibody against the CD3 antigen, or with phytohaemagglutinin (PHA) or guanosine 5'-[gamma-thio]triphosphate (GTP[S]), increased peptide-GS phosphorylation by 2-3 fold. The specificity of PKC action on peptide GS was demonstrated by blocking increases in phosphorylation with a pseudosubstrate peptide PKC inhibitor. PKC activation by this technique could be detected within 1 min of adding external ligand. Dose-response curves revealed that PHA-induced production of inositol phosphates correlated closely with PKC activities, whereas only a partial correlation between these parameters was observed with GTP[S]. Our data are consistent with the presence of more than one G-protein-mediated pathway of PKC regulation in T cells. The quantitative PKC assay system described is both simple and reproducible, and its potential application to a wide range of cell types should prove useful in further investigations of PKC activation mechanisms.     

Evidence for GTP-binding protein involvement in the tyrosine phosphorylation of the T cell receptor zeta chain.

The zeta subunit of the T cell receptor (TCR) is a prominent substrate for a TCR-activated tyrosine kinase. Tyrosine phosphorylation of the zeta subunit in response to antibody-mediated receptor cross-linking was synergized in permeabilized T cells by either of two non-hydrolyzable GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) or guanosine 5'-[beta, gamma-imido]triphosphate Gpp(NH)p. ATP analogues did not significantly affect antibody-induced tyrosine phosphorylation. Unlike the GTP analogues, the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP beta S) did not enhance phosphorylation of zeta. The effect induced by the GTP analogues required TCR occupancy and was independent of protein kinase C. Taken together these observations implicate a GTP-binding protein in the modulation of TCR-induced tyrosine phosphorylation.     

Constitutive GDP/GTP exchange and secretion-dependent GTP hydrolysis activity for Rab27 in platelets

We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTP-Rab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca(2+) concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (beta-gamma-imidoguanosine 5'-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca(2+)-induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.     

Calcium- and guanine-nucleotide-dependent exocytosis in permeabilized rat mast cells. Modulation by protein kinase C.

We have used a digitonin-permeabilized cell system to study the signal transduction pathways responsible for stimulus-secretion coupling in the rat peritoneal mast cell. Conditions were established for permeabilizing the mast cell plasma membrane without disrupting secretory vesicles. Exocytotic release of histamine from digitonin-permeabilized cells required a combination of micromolar concentrations of Ca2+ and the stable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but was independent of exogenous ATP. In the presence of 40 microM-GTP[S], exocytosis was half-maximal at 1.3 microM-Ca2+ and maximal at 10 microM-Ca2+; GTP[S] alone (100 microM) had no effect on histamine release in the absence of added Ca2+. In the presence of 10 microM free Ca2+, 5 microM-GTP[S] was required for half-maximal exocytosis. To examine the possible role of protein kinase C (PKC) in exocytosis, we utilized 12-O-tetradecanoylphorbol 13-acetate (TPA) to activate PKC and studied its effect on histamine release from permeabilized mast cells. Cells that had been incubated with TPA (25 nM for 5 min) exhibited increased sensitivity to both GTP[S] and Ca2+. The PKC inhibitor staurosporine blocked the effect of TPA without inhibiting normal exocytosis in response to the combination of GTP[S] and Ca2+. In addition, down-regulation of mast-cell PKC by long-term TPA treatment (25 nM for 20 h) blocked the ability of the cells to respond to TPA and inhibited exocytosis in response to Ca2+ and GTP[S] by 40-50%. These results suggest that the sensitivity of the exocytotic machinery of the mast cell can be altered by PKC-catalysed phosphorylation events, but that activation of PKC is not required for exocytosis to occur.     

Tyrosine phosphorylation of an SH2-containing protein tyrosine phosphatase is coupled to platelet thrombin receptor via a pertussis toxin-sensitive heterotrimeric G-protein.

SH-PTP1 is a protein tyrosine phosphatase (PTP) predominantly expressed in haematopoietic cells and containing two src homology-2 (SH2) domains. Here we report that SH-PTP1 is phosphorylated on both serine and tyrosine residues in response to thrombin or phorbol myristate acetate (PMA), which increased by 60 and 40%, respectively, SH-PTP1 activity. Thrombin-induced phosphorylation of SH-PTP1 is an early signalling event (maximal within 10 s) involving neither integrin signalling, nor calcium, nor release of ADP or thromboxane A2. Moreover, in contrast with PMA, the effect of thrombin on the tyrosine phosphorylation of SH-PTP1 was hardly affected by GF109203X, a specific protein kinase C (PKC) inhibitor. Finally, phosphorylation of SH-PTP1 could be provoked in permeabilized platelets by thrombin or GTP gamma S. This was abolished by pertussis toxin, the specificity of this effect being verified with the megakaryocytic cell line Dami cell. Our data thus identify SH-PTP1 as an in vivo substrate of a putative protein tyrosine kinase linked to the thrombin receptor by a Gi protein. This might offer some clue to unravel the mechanism of thrombin not only in platelets but also in nucleated cells, where its mitogenic effect is known to involve pertussis toxin-sensitive G-proteins, tyrosine phosphorylation and the ras pathway.     

Guanosine 5'-O-(thiotriphosphate)-dependent inositol trisphosphate formation in membranes is inhibited by phorbol ester and protein kinase C

Phosphoinositide hydrolysis was studied in a washed membrane preparation of 1321N1 astrocytoma cells prelabeled with [3H]inositol. GTP gamma S stimulated the formation of [3H]inositol mono-, bis-, and trisphosphate ([3H]InsP, [3H]InsP2, and [3H]InsP3) with a half-maximal effect on [3H]InsP formation at 5 microM. Carbachol increased the accumulation of [3H]inositol phosphates only in the presence of added guanine nucleotide. Calcium increased [3H]InsP3 accumulation over a range of concentrations (10 nM-3 mM free calcium). When 1321N1 cells were treated with phorbol ester (100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA)) prior to preparation of the membranes, the maximal [3H]InsP formation induced by GTP gamma S or GTP gamma S plus carbachol was decreased by 50-75%. In contrast, the response to a maximal calcium concentration presumed to activate phospholipase C directly was minimally inhibited (approximately 15%). PMA treatment did not affect muscarinic receptor affinity for carbachol or the effect of GTP on agonist binding. PMA treatment was also without effect on the breakdown of exogenous [3H]InsP3 in homogenates, permeabilized cells, and membranes, indicating that the InsP3-phosphatase was not the site of phorbol ester action. PMA treatment inhibited [3H] InsP3 formation only in membranes and not in cytosol prepared from the same cells, suggesting a membrane site of PMA action. Membranes were also required to demonstrate GTP gamma S-stimulated [3H]InsP3 formation although calcium-stimulated [3H]InsP3 formation was demonstrable in both membranes and cytosol. The addition of purified protein kinase C to the membranes mimicked the effect of PMA treatment to decrease GTP gamma S-stimulated [3H]InsP3 production. These data indicate that the effect of PMA on phosphoinositide metabolism is demonstrable in a cell-free system and that it can be mimicked by protein kinase C. We suggest that the ability of PMA to block GTP gamma S-stimulated formation of [3H]InsP3 results from inhibition of the G protein interaction with phospholipase C.