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PubMed1994

Mechanically induced calcium mobilization in cultured endothelial cells is dependent on actin and phospholipase.

We sought to evaluate the mechanisms by which mechanical perturbation elevates intracellular calcium in endothelial cells. We report that the transient elevation in intracellular calcium in cultured bovine aortic endothelial cells (BAEC) in response to gentle perturbation with the side of a micropipette was not blocked by depolarization (external K+, 130 mmol/L), nifedipine (10 mumol/L), or Bay K 8644 R(+) (10 mumol/L). Thus, voltage-dependent calcium channels were not involved in the response. Also, amiloride (10 mumol/L) and tetraethylammonium (1 mmol/L) had no effect on calcium mobilization, indicating that Na+ and K+ transporters were not involved. Pretreatment of the cells with the phospholipase C and phospholipase A2 inhibitor manoalide (10 mumol/L) for 10 minutes at 37 degrees C completely abolished the calcium response, as did a 10-minute pretreatment with the inhibitor of actin polymerization, cytochalasin B (1 mumol/L). We observed an inhibitory effect of the phospholipase A2 and phospholipase C inhibitor 4-bromophenacyl bromide (10 mumol/L) on the mechanical response of BAEC that was not as potent as that observed with manoalide. To examine the role of arachidonic acid (AA) and subsequent metabolites that may be released after a putatively mechanical activation of phospholipase A2, we exposed BAEC to exogenous AA. We found that continued exposure of BAEC for 5 minutes to 10 nmol/L to 10 mumol/L AA caused no elevation of intracellular calcium. If mechanical stimulation activates phospholipase A2, the liberated AA and subsequent metabolites do not appear to have much effect on BAEC intracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1987

Renal phospholipase C and diglyceride lipase activity in spontaneously hypertensive rats.

Phospholipase C activity and diglyceride lipase activity were studied in the renal cortex and medulla of 10- and 40-week-old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched normotensive Wistar-Kyoto rats (WKY). Enhanced phospholipase C activity was found in the cortical and medullary cytosol of kidney from SHRSP, and microsomal diglyceride lipase in SHRSP also increased. In SHRSP, phospholipase C and diglyceride lipase activities increased with age, but this increase was not evident in WKY. Phospholipase C had high substrate specificity for phosphatidylinositol in renal cytosol of both WKY and SHRSP. The increased activities were accompanied by prostaglandin E2 synthesis in renal medullary microsomes of 10-week-old SHRSP and were also present in the kidney of 40-week-old SHRSP. Total phospholipid and arachidonic acid contents in kidney were markedly high in the medulla of 10-week-old SHRSP, but these lipids were decreased in 40-week-old SHRSP. These results suggest that phospholipids and arachidonic acid in SHRSP may be genetically high and that the activated phospholipase C and diglyceride lipase hydrolyze phospholipids, providing arachidonic acid for prostaglandin synthesis, which results in a decrease of phospholipids and arachidonic acid in the kidney of 40-week-old SHRSP. These studies demonstrate that a phosphatidylinositol-specific phospholipase C-prostaglandin synthetic system may play an important role in the course of hypertension in SHRSP.

Animals↗

PubMed1983

Effects of cations, phospholipases, and neuraminidase on calcium binding to "gas-dissected" membranes from cultured cardiac cells.

Sarcolemmal membranes prepared by "gas dissection" from monolayers of cultured neonatal rat heart cells were studied with respect to their ability to bind calcium. Lanthanum displacement of calcium was 168 +/- 7 nmol/mg sarcolammel protein. This represents 3.21 mmol Ca/kg dry weight original cells on the basis of the measured membrane protein: dry cell weight ratio of 19.1 g/kg. Lanthanum-displaceable calcium from whole cells was essentially equal (3.32 mmol/kg dry weight), which indicates that all calcium displaceable from whole cells by lanthanum is localized to sarcolemmal sites. The potency of a series of divalent cations for calcium displacement from the sarcolemma was according to similarity of their crystal radii to that of calcium (cadmium greater than manganese greater than magnesium). This order was the same for the cations' ability to displace calcium from whole cells and for their ability to uncouple excitation from contraction in neonatal papillary muscle. The membranes were treated with four enzymes: phospholipase A2, phospholipase C, phopholipase D, and neuraminidase. Phospholipase A2 and phospholipase D produced significantly increased calcium-binding. The increased binding secondary to phospholipase A2 treatment was eliminated by an albumin wash which was indicative of binding to the fatty acid product of hydrolysis. The increase after phospholipase D treatment can be attributed to an increase in phosphatidate, with attendant increase in net anionic charge on the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

5-Hydroxytryptamine induces phospholipase C-mediated hydrolysis of phosphoinositides through 5-hydroxytryptamine-2 receptors in cultured fetal mouse ventricular myocytes.

5-Hydroxytryptamine (5-HT) stimulates the rate and force of cardiac contraction. However, the molecular mechanisms of 5-HT actions on the heart are unknown. We examined effects of 5-HT on phospholipase C-mediated hydrolysis of phosphoinositides and its regulation in cultured fetal mouse ventricular myocytes labeled with [3H]inositol. Accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate was assessed after stimulation with 5-HT, catecholamines, and AlF4-. Inositol bisphosphate and trisphosphate reached a peak at 15 minutes by 5-HT stimulation and at 30 minutes by AlF4- stimulation. Inositol monophosphate accumulated linearly for at least 30 minutes in the presence of LiCl. The 5-HT effect was dose dependent, and the threshold concentration was 0.1 microM with the half-maximum effective concentration of 1 microM. Ketanserin in nanomolar concentrations inhibited the phospholipase C reaction by 100 microM 5-HT with the half-maximum inhibitory concentration of 0.5 nM. Pertussis toxin (100-1,000 ng/ml) did not influence the phospholipase C reaction by 5-HT, but it partially inhibited the reaction by AlF4-. Protein kinase C-activating phorbol esters like 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate, but not 4 alpha-phorbol 12,13-didecanoate, which is inactive for protein kinase C, completely inhibited the reaction by 5-HT; TPA showed 30% inhibition on the reaction by AlF4-. The magnitude of accumulated inositol phosphates by AlF4- was at least several times greater than that by 5-HT. Norepinephrine- and epinephrine-stimulated phospholipase C reactions were completely abolished by prazosin. These results suggest that 5-HT directly stimulates phospholipase C-mediated hydrolysis of phosphoinositides through 5-hydroxytryptamine-2 (5-HT2) receptors in the ventricular myocytes and that this reaction is negatively regulated by protein kinase C. 5-HT2 receptors may be coupled to phospholipase C via a pertussis toxin-insensitive GTP-binding protein in the myocytes.

Aluminum↗

PubMed1976

The effect of phospholipase C on human blood platelets.

The effect of phospholipase C (EC 3.1.4.3) on human blood platelets has been studied. Phospholipase C from Bacillus cereus was purified to homogeneity as judged by analytical and sodium dodecyl sulphate disc gel electrophoresis and by immunoelectrophoresis. Human platelets isolated from platelet-rich plasma by gel filtration or by centrifugation and washing were incubated with phospholipase C. A loss of 20-45% of the total platelet phospholipid was observed, whereas 88% was hydrolyzed when platelet homogenates were submitted to identical enzyme treatment. Intact platelets lost 50-75% phosphatidylethanolamine, 20-50% phosphatidylcholine, and 20-25% phosphatidylserine. Sphingomyelin was not a substrate for the enzyme under the conditions used. The platelets contained no detectable endogenous phospholipase C activity. The loss of phospholipid was not accompanied by aggregation of the platelets, nor did the platelets lose their ability to aggregate with ADP or thrombin. Total platelet factor 3 releasable by freezing and thawing was reduced. Measurements of releasable platelet factor 4 and the efflux of serotonin showed that no release reaction was triggered even when up to 45% of the total phospholipid in the platelets was hydrolyzed. When sphingomyelinase was added together with, before, or after phospholipase C, aggregation occurred. Sphingomyelinase alone gave no aggregation. The gel-filtered platelets also aggregated upon addition of purified phospholipase C from Clostridium perfringens. The distribution of phospholipids in the platelet membrane is discussed.

Bacillus cereus↗

PubMed1986

Phospholipase C and melittin enhancement of glucose-induced electrical activity.

The influence of exogenous phospholipase C and melittin on electrical activity in islet B cells was determined to assess the extent to which polyphosphatidylinositol turnover serves to generate components that influence the electrical events in the plasma membrane. Phospholipase C hydrolyzes polyphosphatidylinositol to diacylglycerol and polyphosphoinositol, whereas melittin increases the susceptibility of phospholipids to phospholipases and increases the permeability of the membrane to ions. Application of both 20 mU/ml phospholipase C or 0.5 mg/ml melittin to 11.1 mM glucose elicited a time-dependent enhancement of glucose-induced electrical activity that stabilized after 10 min. Phospholipase C increased both the active phase fraction and the burst frequency, whereas melittin only increased the burst frequency. These results indicate that both compounds, which disrupt the phospholipid environment of the plasma membrane, play a role in modulating the oscillatory pattern of electrical activity in the B cell, although melittin is obviously not influencing the factors controlling the ionic events in the same manner as phospholipase C.

Animals↗

PubMed1995

Oxytocin stimulates myometrial guanosine triphosphatase and phospholipase-C activities via coupling to G alpha q/11.

Oxytocin stimulates phosphoinositide turnover in myometrium. To elucidate whether the coupling mechanism involves the interaction of oxytocin receptor with GTP-binding proteins, we examined oxytocin stimulation of guanosine triphosphatase (GTPase) activity and phospholipase-C activity in rat and human myometrial membranes. Oxytocin consistently stimulated both GTPase and phospholipase-C activities, and both stimulations were attenuated by an antibody directed against the carboxyl-terminals of the GTP-binding proteins, G alpha q and G alpha 11. Neutralization of the antibody by preincubation with antigenic peptide reversed this inhibition. [Thr4,Gly7]oxytocin, a specific oxytocin receptor agonist, stimulated both GTPase and phospholipase-C activities, and the stimulations were also inhibited by anti-G alpha q/11 IgG. Immunoreactive GTP-binding proteins, G alpha q and G alpha 11, and phospholipase-C beta 3 isoforms were present in myometrial membranes. These results indicate that stimulation of phospholipase-C activity by oxytocin in myometrium is mediated via G alpha q, G alpha 11, or a closely related GTP-binding protein, probably coupling to phospholipase-C beta.

Animals↗

PubMed1999

[Development from actions of bacterial phospholipases C on eucaryotic plasma membranes to molecular biology of GPI-anchored proteins].

Bacterial phospholipases C are known to act on biomembranes, since they can cleave the phosphodiester linkage between the polar head and the hydrophobic moiety of each phospholipid in these membranes. These enzymes have been classified into three groups; phosphatidylcholine (PC)-, sphingomyelin (SM)- and phosphatidylinositol (PI)-degrading phospholipases C. Enzymatic properties and toxicities of these phospholipases C are reviewed, in relation to author's research. Studies on the hemolytic phospholipases of Clostridium sp., Bacillus cereus etc., revealed that hydrolysis of choline-containing phospholipids such as PC and SM was responsible for the hemolysis of mammalian erythrocytes by these enzymes in the presence of Ca2+ and/or Mg2+. Also, the studies on a structure-activity correlation of SM-hydrolyzing phospholipase C from B. cereus disclosed the similarity of active sites between this enzyme and bovine pancreatic DNase I. By action of PI-degrading phospholipases C, several membrane proteins such as alkaline phosphatase, 5'-nucleotidase, VSG (protozoal surface glycoprotein) etc., were shown to be released from the plasma membranes of eucaryotic cells. From structural analysis, these proteins have been revealed to be glycosylphosphatidylinositol (GPI)-anchored proteins bound to the plasma membranes with carboxyl terminal-attached glycolipid. Biochemistry and molecular biology of GPI-anchored proteins, including the structures and biosynthetic routes of GPI glycolipids as well as the process of GPI attachment to proteins, requirements of C-terminal signal peptide for the protein modification by GPI, and distribution of GPI-anchored proteins in living world, are described in relation to our studies.

Amino Acid Sequence↗

PubMed2001

Effects of arginine- and lysine-vasopressin on phospholipase C activity, intracellular calcium concentration and prostaglandin F2alpha secretion in pig endometrial cells.

Oxytocin and vasopressin are related peptides that have receptors in the uterus. Species from families other than Suidae produce only arginine-vasopressin; in contrast, pigs apparently express both arginine- and lysine-vasopressin. The aim of this study was to determine whether arginine- or lysine-vasopressin would activate phospholipase C, increase intracellular calcium concentration [Ca(2+)](i) and stimulate PGF(2alpha) production in enriched cultures of stromal, glandular epithelial and luminal epithelial cells from pig endometrium. Cells were obtained from gilts on day 16 after oestrus by differential enzymatic digestion and sieve separation. After 96 h in culture, the cells were treated with 0 or 100 nmol arginine- or lysine-vasopressin l(-1). The responses to 100 nmol oxytocin l(-1) and 100 nmol GnRH l(-1) were used as positive and negative controls, respectively. Consistent with previous results, oxytocin stimulated phospholipase C activity (P < 0.05), increased [Ca(2+)](i) (P < 0.05) and promoted PGF(2alpha) secretion (P < 0.05) from stromal and glandular epithelial cells. Activity of phospholipase C, [Ca(2+)](i) and PGF(2alpha) release were also increased (P < 0.05) by arginine-vasopressin in stromal cells, but the responses were less (P < 0.01) than those induced by oxytocin. An oxytocin antagonist attenuated the [Ca(2+)](i) response of stromal cells to both oxytocin and arginine-vasopressin. Sequential treatment of cells with oxytocin and arginine-vasopressin indicated that oxytocin desensitized the response to oxytocin, but arginine-vasopressin did not similarly desensitize the response to oxytocin. In glandular and luminal epithelial cells, arginine-vasopressin did not stimulate phospholipase C activity, [Ca(2+)](i) or PGF(2alpha) secretion. Neither GnRH nor lysine-vasopressin induced phospholipase C activity, increased [Ca(2+)](i) or stimulated PGF(2alpha) production in any endometrial cell type. These results indicate that oxytocin receptors can bind arginine-vasopressin more readily than they bind lysine-vasopressin. Type 1 vasopressin receptors may also exist in endometrium predominantly on cells other than stromal, glandular epithelial and luminal epithelial cells, as in previous studies both arginine-vasopressin and lysine-vasopressin stimulated phospholipase C activity in endometrial explants to a similar extent as oxytocin.

Animals↗

PubMed1993

Bradykinin and vasopressin activate phospholipase D in rat Leydig cells by a protein kinase C-dependent mechanism.

In the present study we report that bradykinin stimulated phospholipase D activity in rat Leydig cells. Bradykinin added for 8 min stimulated choline formation in a dose-dependent manner and, in the presence of ethanol, bradykinin (100 nmol/l) stimulated transphosphatidylation by phospholipase D resulting in the formation of phosphatidylethanol. This stimulation was abolished after down-regulation of protein kinase C by long-term pretreatment for 22 h with phorbol 12-myristate 13-acetate (PMA). The stimulation of phospholipase D by the simultaneous addition for 8 min of maximum concentrations of PMA and vasopressin (AVP), PMA and bradykinin, or AVP and bradykinin produced no additive phosphatidylethanol or choline response, suggesting that AVP, bradykinin and PMA stimulated phospholipase D-catalysed phosphatidylcholine hydrolysis by a similar protein kinase C-dependent mechanism. Furthermore, LH (10 ng/ml), insulin (500 nmol/l), GH (100 ng/ml), interleukin-1 beta (5 U/ml) and platelet-activating factor (200 nmol/l) were found not to activate phospholipase D, whereas the Ca2+ ionophore A23187 (10 mumol/l) stimulated phosphatidylethanol formation, suggesting that Ca2+ might be a regulator of phospholipase D in Leydig cells.

Animals↗

PubMed1996

A novel phospholipase C inhibitor, S-PLI produced by Streptomyces sp. strain no. A-6288.

S-PLI, an inhibitor of phospholipase C (PLC) produced by Streptomyces sp. strain No. 6288, was purified from the culture filtrate by salting-out with solid ammonium sulfate, column chromatography on CM-cellulose and gel filtration on Sephadex G-75. The molecular weight of S-PLI was estimated to be 65,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was found to be a glycoprotein with a composition of 609 amino acids and 19 glucose residues having an isoelectric point at 7.8. S-PLI was stable from pH 3 to 10 at 37 degrees C and up to 40 degrees at pH 6.0. The inhibitory activity showed pH- and temperature-dependence with a maximum around pH 7.0 at 50 degrees C. S-PLI inhibited phospholipase C in a competitive manner (Ki value; 9.5 x 10(-6) mM), but did not inhibit S-Hemolysin, phospholipase A2; phospholipase B, phospholipase D and phosphatases. S-PLI is the first reported example of a glycoproteinaceous inhibitor of microbial origin which is able to specifically inhibit phospholipase C.

Amino Acids↗

PubMed2006

Improving the yield of Mozzarella cheese by phospholipase treatment of milk.

Part-skim Mozzarella cheese was manufactured from milk hydrolyzed with fungal phospholipase A1 prior to renneting. The phospholipase treatment reduced fat losses in whey and cooking water and increased cheese yield as a result of improved fat and moisture retention in the cheese curd. The amount of phospholipids in the whey was reduced because of improved retention of lysophospholipids in the cheese curd. Water binding in the fresh curds and young cheeses up to 3 wk of storage was investigated by a 1H nuclear magnetic resonance spin-spin relaxation technique. In the fresh curds, 2 dominant water fractions were present, characterized by average spin-spin relaxation times (T2) of 14 and 86 to 89 ms, respectively. These 2 fractions of low- and high-molecular-mobility water were similar in all cheeses and presumed to represent water associated with the casein matrix and water present in the pores. A few hours after manufacture, cheeses made with phospholipase showed decreased T2 of the high-mobility fraction, indicating improved water-holding capacity. It is suggested that lysophospholipids released from the fat globule membranes act as surface-active agents in the cheese curd, helping emulsification of water and fat during processing and reducing syneresis. During 3 wk of storage after manufacture, the mobility of both water fractions increased in all cheeses, but was highest in the cheeses made with phospholipase. The increase in mobility during the first weeks of storage has earlier been ascribed to structural changes in the protein matrix, which in principle could be accelerated because of the higher moisture content. However, the microstructure of phospholipase-treated cheese was investigated by confocal laser scanning microscopy and found to be very similar to the control cheese during processing and up to 28 d of storage. In addition, flowability, stretchability, and browning were acceptable and similar in all the manufactured cheeses. Thus, phospholipase hydrolysis of cheese milk improved the cheese yield without changing the cheese microstructure, and resulted in cheese with functional properties that were identical to traditional Mozzarella cheese.

Animals↗

PubMed1999

The pro-apoptotic drug camptothecin stimulates phospholipase D activity and diacylglycerol production in the nucleus of HL-60 human promyelocytic leukemia cells.

It has recently been reported (T. Shimizu et al., J. Biol. Chem., 273: 8669-8674, 1998) that the pro-apoptotic drug, camptothecin, an inhibitor of topoisomerase I, induces a protein kinase C-alpha-mediated phosphorylation of lamin B in HL-60 cells, which precedes both degradation of lamin B and fragmentation of DNA. In this paper, we report that, in HL-60 cells exposed to camptothecin, there is a rapid and sustained increase of nuclear protein kinase C-alpha activity that is due to an increase in the amount of protein kinase C-alpha present in the nucleus. The enhancement of nuclear kinase C activity is preceded by an increase in the mass of nuclear diacylglycerol. As demonstrated by its sensitivity to propranolol, the nuclear diacylglycerol mass increase is due to the activation of a phospholipase D. Indeed, inhibitors of neither phosphatidylcholine-specific phospholipase C nor phosphoinositide-specific phospholipase C blocked the rise in nuclear diacylglycerol. In vitro assays also demonstrated the activation of a nuclear phospholipase D, but not of a phosphoinositide-specific phospholipase C, after treatment with camptothecin. Propranolol was also able to block the rise in nuclear protein kinase C-alpha activity, thus suggesting that the increase in diacylglycerol mass is important for the activation of the kinase at the nuclear level. Moreover, propranolol was capable of drastically reducing the number of HL-60 cells that underwent apoptosis after treatment with camptothecin. Our results show the activation during apoptosis of a phospholipase D-mediated signaling pathway operating at the nuclear level. This pathway may represent an attractive therapeutic target for the modulation of apoptotic events in human disease.

Antineoplastic Agents, Phytogenic↗

PubMed1975

Effect of phospholipase A on active transport of amino acids with membrane vesicles of Mycobacterium phlei.

Active transport of proline remained unaffected in phospholipase A-treated electron transport particles from Mycobacterium phlei. However, the steady state level of proline was reduced 50 to 60% in phospholipase A-treated depleted electron transport particles that were devoid of membrane-bound coupling factor-latent ATPase activity. The decrease in the uptake of proline in the phospholipase A-treated depleted electron transport particles was not due to a change in the apparent K-m for proline, but it was related to the amount of phospholipid cleaved from the membranes. Restoration in the level of proline transport in phospholipase A-treated depleted electron transport particles was achieved by reconstituting these vesicles with diphosphatidylglycerol and phosphatidylethanolamine liposomes. Diphosphatidylglycerol was found to be most effective in the restoration of proline uptake. In contrast to the effect of phospholipase A treatment on proline transport, similar treatement of the electron transport particles or depleted electron transport particles failed to inhibit the active transport of either glutamine or glutamic acid. Studies with phospholipase A-treated membrane vesicles confirmed earlier findings that a proton gradient is not required for active transport of amino acids.

Adenosine Triphosphatases↗

PubMed1991

Regulation of phospholipase D-induced hydrolysis of choline-containing phosphoglycerides by cyclic AMP in human neutrophils.

In human neutrophils, the chemotactic tripeptide FMLP and the protein kinase C activator PMA stimulate the breakdown of 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine ([3H]EAPC) and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid ([3H]-EAPA) and 1-O-[3H]alkyl-2-acylglycerol ([3H]EAG) via mechanism(s) that may involve the activation of phospholipase D. We have investigated the regulation of phospholipase D by determining the effects of elevation of intracellular levels of cAMP on receptor-mediated and PMA-induced breakdown of [3H]-EAPC and formation of products. Pretreatment of neutrophils with either 10(-3) M dibutyryl-cAMP or 10(-5) M PGE2, in the presence of 4 x 10(-4) M isobutylmethylxanthine (IBMX), inhibited FMLP- and leukotriene B4-induced breakdown of [3H]EAPC and formation of [3H]EAPA and [3H]EAG. Inhibition was apparent at all time points of stimulation examined (15-120 s). In addition, the mass of diradyl-phosphatidic acid was decreased in FMLP-stimulated neutrophils pretreated with PGE2 and IBMX. In contrast, pretreatment of cells with PGE2 and IBMX did not inhibit PMA-induced breakdown of [3H]EAPC and the formation of products at 3 and 10 min after stimulation. Furthermore, formation of 1-O-[3H]alkyl-2-acyl-phosphatidylethanol, produced by phospholipase D in the presence of ethanol by a transphosphatidylation reaction, was significantly inhibited by pretreatment of cells with PGE2 and IBMX in FMLP- but not PMA-stimulated neutrophils. This differential modulation by cAMP of receptor-mediated and PMA-induced activation of phospholipase D suggests agonist-dependent activation of separate pathways and/or activation of separate phospholipase D enzymes. Thus, cAMP elevation may exert inhibitory effects directly on the phospholipase D activated by FMLP and/or on sites proximal to the enzyme that are involved in signal transmission.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Autophosphorylation of the intracellular domain of the epidermal growth factor receptor results in different effects on its tyrosine kinase activity with various peptide substrates. Phosphorylation of peptides representing Tyr(P) sites of phospholipase C-gamma.

The effect of autophosphorylation on the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) is not well understood. We previously demonstrated that phospholipase C-gamma physically associates with the EGF-activated EGFR, but not with a kinase-negative mutant of the EGFR, and, moreover, that only the tyrosine-phosphorylated EGFR is able to associate with phospholipase C-gamma. We have now investigated the effect of autophosphorylation on the tyrosine kinase activity of the EGFR by employing the purified kinase-active intracellular domain of the EGFR (EGFR-IC) produced by a baculovirus expression system. Synthetic peptides, including ones which contain the individual major tyrosine phosphorylation sites of phospholipase C-gamma, were used as substrates. We found that the extensively prephosphorylated EGFR-IC exhibited similar reaction kinetics to the unphosphorylated EGFR-IC when angiotensin II was used as a nonspecific substrate. In contrast there was a clear stimulation of kinase activity due to autophosphorylation of the EGFR-IC when peptides representing either the major autophosphorylation site of the EGFR or the EGFR phosphorylation sites of phospholipase C-gamma were used as substrates. However, the modes of stimulation for these peptides differed. The binding affinity (Km) for the unphosphorylated EGFR-IC for the peptide containing Tyr-771 of phospholipase C-gamma was relatively poor compared with other peptides, but improved 5-6-fold when the EGFR-IC was prephosphorylated. On the other hand, autophosphorylation improved the reaction velocity (Vm) of the phosphorylation of other peptides by 2-3-fold, with little or no increase in affinity. These results suggest that autophosphorylation of the EGFR may induce a conformational change of its kinase domain which enhances its kinase activity with exogenous substrates and may induce association with phospholipase C-gamma by increasing its affinity to a domain containing Tyr-771.

Amino Acid Sequence↗

PubMed1991

Mechanism of inhibition of adenylate cyclase by phospholipase C-catalyzed hydrolysis of phosphatidylcholine. Involvement of a pertussis toxin-sensitive G protein and protein kinase C.

The phospholipase C-mediated hydrolysis of phosphatidylcholine has been shown recently to be activated by a number of agonists. Muscarinic receptors, which trigger various signal transduction mechanisms including inhibition of adenylate cyclase through Gi, have been shown to be potent stimulants of this novel phospholipid degradative pathway. We demonstrate here, by exogenous addition of Bacillus cereus phosphatidylcholine-hydrolyzing phospholipase C, that phosphatidylcholine breakdown mimics the ability of carbachol to inhibit adenylate cyclase. This effect is sensitive to pertussis toxin and is entirely dependent on the presence of protein kinase C. This kinase is also required for the inhibition by carbachol of adenylate cyclase. These results suggest that the activation of phosphatidylcholine breakdown by phospholipase C may play an important role linking or favoring the coupling muscarinic receptors to Gi. Results presented here also show that phospholipase C-mediated hydrolysis of phosphoinositides by exogenous addition of Bacillus thuringiensis phosphoinositide-hydrolyzing phospholipase C does not affect adenylate cyclase, despite the fact that protein kinase C is translocated to an extent similar to that produced by the hydrolysis of phosphatidylcholine. According to the results shown here, both phospholipases also differ in their ability to down-regulate protein kinase C as well as to phosphorylate p80 and to transmodulate the binding of epidermal growth factor, two well established effects of protein kinase C in Swiss 3T3 fibroblasts. This emphasizes the complexity, from a functional point of view, of protein kinase C activation "in vivo."

Adenylate Cyclase Toxin↗

PubMed1991

Cyclic AMP-elevating agents block chemoattractant activation of diradylglycerol generation by inhibiting phospholipase D activation.

Agents which elevate cellular cAMP (prostaglandin E2, theophylline, and forskolin) or mimic cAMP action (dibutyryl cAMP) are known to inhibit human neutrophil activation (superoxide generation and secretion) by receptor-linked agonists such as formyl-methionyl-leucyl-phenylalanine (fMLP). Herein, we show that these agents also markedly inhibit fMLP-stimulated diradylglycerol generation (assayed by mass methods). The magnitude of inhibition correlated with the ability of a given agent or combination of agents to elevate cAMP. Both 1,2-diacylglycerol and 1-O-alkyl,2-acyl glycerol generation were affected. Effects on the latter species, as well as a lack of effect on fMLP-stimulated inositol phosphate release, implied that cAMP affected diradylglycerol generation from a source other than phospholipase C-dependent phosphoinositide hydrolysis, since phosphatidylinositols do not contain appreciable quantities of the 1-O-alkyl linkage. In cells in which the phosphatidylcholine pool was prelabeled using 1-O-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine, prostaglandin E2 plus theophylline inhibited the fMLP-activated rapid generation of [3H]phosphatidic acid and its subsequent conversion to [3H]diradylglycerol, implying an effect at the level of phospholipase D. In the presence of ethanol, the fMLP-activated transphosphatidylation of [3H]phosphatidylcholine to generate [3H]phosphatidylethanol (a phospholipase D-dependent reaction) was also markedly inhibited. In contrast, when phorbol 12-myristate 13-acetate was used to activate cells, cAMP-related agents had no effect on phospholipase D activity, diradylglycerol generation, or superoxide generation. The data indicate an inhibitory effect of cyclic AMP on receptor-mediated phospholipase D activation at a site proximal to phospholipase D (e.g., the receptor or G protein). These studies provide a new example of "cross-talk" among signal transduction systems.

Chemotactic Factors↗