Reversible protein kinase activation of hormone-sensitive lipase from chicken adipose tissue

Hormone-sensitive lipase partially purified from adipose tissue of laying hens was markedly activated by cyclic AMP-dependent protein kinase. Activation was approximately 4-fold (ranging up to as great as 10-fold) compared with the much lower degree of activation obtained with analogous preparations from rat and human adipose tissues (59 and 86%, respectively). The partially purified preparations contained adequate endogenous protein kinase activity to effect complete activation with addition of cyclic AMP, ATP, and Mg(2+). Activation was blocked by protein kinase inhibitor (from rabbit skeletal muscle) but could be restored fully by addition of excess exogenous protein kinase (from bovine skeletal muscle). The fully activated lipase was slowly deactivated by dialysis at 4 degrees C and then rapidly and almost fully reactivated by addition of cyclic AMP and ATP-Mg(2+). Reactivation was blocked by protein kinase inhibitor. This deactivation-reactivation cycle was rapid at 23 degrees C with dialysis against charcoal and could be demonstrated repeatedly using a single preparation. The reversible deactivation of protein kinase-activated enzyme is presumed to reflect the action of a lipase phosphatase. Lipase prepared from tissue previously exposed to glucagon yielded a much smaller degree of activation than lipase prepared from tissue not exposed to the lipolytic hormone, indicating that the physiological hormone-induced activation is probably similar to or identical with the protein kinase activation demonstrated in the cell-free preparations. Under the conditions of assay used, the partially purified lipase fraction contained diglyceride, monoglyceride, and lipoprotein lipase activities. However, treatment with cyclic AMP-dependent protein kinase had virtually no effect on these lipase activities.     

Differential metabolism of diradyl glycerol molecular subclasses and molecular species by rabbit brain diglyceride kinase

Elevations in the mass of ether-linked diglycerides (i.e. 1-O-alk-1'-enyl-2-acyl-sn-glycerol (AAG) and 1-O-alkyl-2-acyl-sn-glycerol (Alkyl AG)) during cellular activation are prolonged in comparison to their 1,2-diacyl-sn-glycerol (DAG) counterparts. Since the metabolic removal of DAG is determined, in large part, by the rate of its phosphorylation by diglyceride kinase, we quantified differences in the activity of diglyceride kinase utilizing individual subclasses of diradyl glycerols as substrate. Rabbit brain microsomal diglyceride kinase activity was over 30-fold greater utilizing DAG as substrate (25.8 nmol.mg-1.min-1) in comparison to AAG (0.8 nmol.mg-1.min-1). No alterations in the affinity of microsomal diglyceride kinase for ATP were present (Km approximately 0.5 mM) utilizing each diradyl glycerol subclass. Similar subclass specificities for diglyceride kinase (i.e. DAG greater than Alkyl AG much greater than AAG) were present in brain and liver cytosol as well as in liver microsomes utilizing multiple assay conditions. In sharp contrast, Escherichia coli diglyceride kinase phosphorylated DAG, Alkyl AG, or AAG diradyl glycerol molecular subclasses at identical rates. Furthermore, although DAG was rapidly hydrolyzed by diglyceride lipase, catabolism of AAG or Alkyl AG by plasmalogenase, alkyl ether hydrolase, or diglyceride/monoglyceride lipase was undetectable. Collectively, these results demonstrate the importance of the differential catabolism of each diradyl glycerol molecular subclass as a primary determinant of their biologic half-lives. Since individual subclasses of diglycerides have distinct physical properties and physiologic functions, these results underscore the importance of lipid subclass specific metabolism in tailoring individual cellular responses during activation.     

Triglyceride, diglyceride, monoglyceride, and cholesterol ester hydrolases in chicken adipose tissue activated by adenosine 3':5'-Monophosphate-dependent protein kinase. Chromatographic resolution and immunochemical differentiation from lipoprotein lipase.

Hormone-sensitive lipase and cholesterol ester hydrolase of chicken adipose tissue were markedly activated by adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase (on the average, 235 to 275%; occasionally as much as 1000%). Diglyceride and monoglyceride hydrolases were also activated, but to a lesser extent (60 to 87%). The activation of all four hydrolases was inhibited by protein kinase inhibitor and reversed by the addition of exogenous protein kinase. Following activation by cAMP-dependent protein kinase, all four hydrolases were deactivated in a Mg2+-dependent reaction and then reactivated to or near initial levels on incubation with cAMP and Mg2+-ATP. The reversible deactivation is assumed to reflect activity of one or more protein phosphatases. The maximum activation obtainable for the four hydrolases decreased when the tissue had been previously exposed to glucagon, indicating that the glucagon-induced activation was probably similar to or identical with the activation demonstrated in cell-free preparations. The pH optima for the four hydrolase activities were similar (7.13 to 7.38). Although the absolute activities and relative degrees of kinase activation differed according to the particular emulsified substrates used, the results do not rule out the possibility that all four hydrolase activities are referable to a single hormone-sensitive hydrolase. Hormone-sensitive acyl hydrolases were separated from lipoprotein lipase by heparin-Sepharose affinity chromatography. Lipoprotein lipase was active against triolein, diolein, and monoolein, but not cholesterol oleate. Incubation of lipoprotein lipase with exogenous protein kinase, cAMP, and Mg2+ATP had no effect on any of the three hydrolase activities. Lipoprotein lipase was further purified to homogeneity and used to prepare antiserum in rabbits. The immunoglobin G fraction from these antisera completely inhibited lipoprotein lipase eluted from heparin-Sepharose columns. However, the hormone-sensitive hydrolase activities (not retained on heparin-Sepharose affinity chromatography) were not inhibited by anti-lipoprotein lipase immunoglobin G, and anti-lopoprotein lipase immunoglobin G did not affect the activation process in crude fractions. Thus, hormone-sensitive lipase and lipoprotein lipase, functionally distinct enzymes, have been physically resolved and immunochemically distinguished. Apparently lipoprotein lipase activity is not regulated, at least directly, by cAMP-dependent protein kinase.     

Regulation of protein kinase C activity by lipids

Protein kinase C is activated by the simultaneous presence of phospholipid, a diglyceride, and Ca2+. Under physiological conditions the activity of the enzyme is regulated by the availability of diglycerides, which are the products of phosphoinositide hydrolysis. The phospholipid-kinase interactions appear not to be of a highly specific nature. Phosphatidylserine (PS) is presumed to be the endogenous lipid that interacts with the kinase, but other acidic lipids can substitute. On the other hand, the kinase-diglyceride interactions are highly specific in nature, as would be expected of a physiological regulator. These interactions are stereo-specific and stoichiometric with respect to diglyceride. The specificity is directed toward the glycerol backbone and hydrophilic oxygen moieties of the diglyceride. The removal of one or more of the oxygen atoms or the addition of a single methyl group to the glycerol backbone virtually abolishes the activity of a putative diglyceride activator. The extreme specificity of the kinase toward the diglycerides, however, must be contrasted with the abilities of structurally diverse tumor promotors and irritants to activate the kinase. Specific small-molecule antagonists of protein kinase C have yet to be developed. The small-molecule antagonists that have been developed so far have been relatively nonspecific cationic lipids that appear to function by interfering with the interaction between the acidic phospholipids and Ca2+.     

Monoglyceride and diglyceride lipases from human platelet microsomes

In the present study, we have characterized the properties of both diglyceride lipase (lipoprotein lipase, EC 3.1.1.24) and monoglyceride lipases (acylglycerol lipase, EC 3.1.1.23) in an attempt to assess the potential roles of these two enzymes in the release of arachidonate in activated human platelets. Diglyceride lipase exhibited maximal activity at pH 3.5, whereas monoglyceride lipase showed optimal activity at pH 7.0. Neither of the lipases were inhibited by EDTA or stimulated by Ca2+, Mg2+ or Mn2+. Both enzymes, however, were strongly inhibited by Hg2+ and Cu2+, indicating the involvement of sulfhydryl groups in catalytic activity. This suggestion was further supported by their sensitivity toward sulfhydryl inhibitors, with monoglyceride lipase being more susceptible to inhibition. Both lipases were found to be inhibited to a different degree by a variety of antiplatelet drugs blocking aggregation and arachidonate release. Kinetic studies indicated that dichotomous metabolism of diacylglycerol to monoacylglycerol and to phosphatidic acid could occur concurrently, since the apparent Km values for diglyceride lipase and for diglyceride kinase were comparable. Further studies showed that the specific activity of monoglyceride lipase was at least 100-fold higher than that of diglyceride lipase, indicating that the rate-limiting step in the release of arachidonate was the reaction catalyzed by diglyceride lipase.     

Role of phosphoprotein phosphatases in reversible deactivation of chicken adipose tissue hormone-sensitive lipase.

The reversible deactivation of chicken adipose tissue hormone-sensitive lipase alpha(previously activated with Mg2+ ATP and adenosine 3':5'-monophosphate) required Mg2+ and was inhibited by phosphate. These results are consistent with the assumption that deactivation of the protein kinase-activated enzyme is catalyzed by a lipase phosphatase. Cholesterol ester is catalyzed by a lipase phosphatase. Cholesterol ester hydrolase similarly was activated and reversibly deactivated. The activity of endogenous lipase phosphatase in pH 5.2 precipitate fractions was reduced, and in some cases eliminated, by incubation at 50 degrees for 20 min in buffer containing 20% glycerol. Heating at 50 degrees greatly increased the apparent percentage activation of triglyceride and cholesterol ester hydrolases but this was due to a selective decrease in basal (nonactivated) hydrolase activities. Essentially all endogenous lipase phosphatase could be removed by treatment of the pH 5.2 precipitate fraction with ATP-Sepharose affinity gel. The addition of a partially purified preparation of rat liver phosphorylase phosphatase deactivated triglyceride and cholesterol ester hydrolases. The deactivation process was concentration, 5 mM) and was inhibited by 5 mM phosphate and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipase alpha was also observed with crude prepa- and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipas alpha was also observed with crude preparations of phosphoprotein phosphatases from rat and turkey hearts, and from rat epididymal fat pads. Thus, hormone-sensitive lipase is deactivated by a variety of phosphoprotein phosphatases from different tissues and different species, implying a low degree of specificity for the deactivating system.     

An in vitro model to study adipose differentiation in serum-free medium

Adipose differentiation was studied in a teratoma-derived fibroadipogenic cell line (1246) cultured in serum-free medium. The addition of dexamethasone and 1-methyl-3-isobutylxanthine to the serum-free medium induced confluent 1246 cells to differentiate into adipocyte-like cells as evidenced by triglyceride accumulation and increased levels of lipolytic enzyme activities. Hormone-sensitive lipase activity measured 5 days after the addition of dexamethasone and 1-methyl-3-isobutylxanthine increased 17-fold and was activated by cAMP-dependent protein kinase. Neutral diglyceride lipase, monoglyceride lipase, and cholesterol ester hydrolase specific activities increased 23-, 75-, and 73-fold, respectively. Among these three activities, only cholesterol ester hydrolase was activated by cAMP-dependent protein kinase. Differentiated 1246 cells expressed receptors to lipolytic hormones as shown by the stimulation of glycerol release by epinephrine (8.6-fold), glucagon (2.2-fold), and adrenocorticotrophic hormone (5.5-fold). Heparin treatment of 1246 cells in serum-free medium resulted in the release of lipoprotein lipase activity into the culture medium. Thus, 1246 cells can serve as a model for the study of adipose differentiation under defined culture conditions since they are capable of growth and survival in the absence of serum while retaining their ability to differentiate into adipocytes.     

Glyceride lipases in nerve endings of guinea-pig brain and their stimulation by noradrenaline, 5-hydroxytryptamine and adrenaline

1. Combined guinea-pig cortex and cerebellum was shown to contain triglyceride lipase, diglyceride lipase and monoglyceride lipase, which were assayed by the release of [1-(14)C]palmitate from [1-(14)C]palmitoylglycerol esters. Triglyceride lipase and diglyceride lipase were found in all particulate fractions. 2. With osmotically ruptured synaptosomes the rates of release of palmitate from glyceryl tripalmitate and glyceryl dipalmitate were 7-25mumol/h per g of protein and 0.18-0.69mmol/h per g of protein respectively. The logarithm of the rate of hydrolysis of glyceryl monopalmitate increased linearly with the logarithm of protein concentration. The pH optima of triglyceride lipase and diglyceride lipase were between 7 and 8. The pH optimum for monoglyceride lipase was approx. 8. 3. Triglyceride lipase and diglyceride lipase of osmotically ruptured synaptosomes were stimulated by noradrenaline, 5-hydroxytryptamine and adrenaline. Triglyceride lipase of isolated synaptic membranes was stimulated by 0.01-1mm-noradrenaline. Aging of membranes at 0 degrees C decreased activity, which could still be stimulated by noradrenaline. Diglyceride lipase of isolated membranes was stimulated by 1mum-1mm-noradrenaline. The activity of triglyceride lipase in isolated synaptic vesicles was diminished by 1mm-5-hydroxytryptamine.     

Cholesterol esterase in rat adipose tissue and its activation by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

A high level of cholesterol esterase activity, comparable to that of hormone-sensitive triglyceridase, has been demonstrated in rad adipose tissue. Essentially all of the activity was in the isolated adipocytes, primarily in the 100,000 times g supernatant fraction of the adipocytes. Cholesterol esterase activity in the 100,000 times g supernatant fraction was increased 40 plus or minus 16% by incubation with ATP (0.5 mM), Mg-2+ (1.25 mM), and cyclic adenosine 3':5'-monophosphate (cyclic AMP) (10 muM), conditions which also activated hormone-sensitive triglyceridase. Protein kinase inhibitor (rabbit skeletal muscle) blocked activation, and activation was restored by the addition of excess protein kinase (bovine skeletal muscle). In extracts prepared from adipocytes first incubated for 5 min with 10 muM epinephrine and 1 mM theophylline, there was no cyclic AMP-dependent cholesterol esterase activation, implying that the enzyme had been activated by a similar mechanism in the intact cell. The physiological role of this high level of cholesterol esterase activity in adipose tissue is unclear. Its relationship to hormone-sensitive triglyceride lipase, with which it extensively co-fractionates, and its possible involvement in fat mobilization remain to be determined.     

pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine

An hepatic protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) on Ser/Thr residues is markedly activated after intraperitoneal injection of cycloheximide in the rat. The enzyme has been purified greater than 10,000-fold to near homogeneity and corresponds to a 54-kDa polypeptide, based on auto-phosphorylation, renaturation of activity from sodium dodecyl sulfate gels, and gel filtration. The protein kinase activity is unaffected by prior autophosphorylation, Ca2+, diacylglycerol and phospholipids, cyclic nucleotides, staurosporine, and protein kinase inhibitor, but can be totally and specifically deactivated by the Ser/Thr protein phosphatase 2A. The enzyme is inhibited completely but reversible by transition metals and p-chloromercuribenzoate, and is strongly stimulated by poly-L-lysine toward most, but not all protein substrates. The activity of the cycloheximide-stimulated MAP-2 kinase (pp54 MAP-2 kinase) toward potential polypeptide substrates was compared to that of an insulin-stimulated MAP-2 kinase (pp42 MAP-2 kinase). Although both MAP-2 kinases exhibited little or no ability to phosphorylate histones and casein, the two kinases had a distinguishable substrate specificity. At comparable MAP-2 phosphorylating activities, pp42 MAP-2 kinase, but not pp54 MAP-2 kinase, phosphorylated and activated the Xenopus S6 protein kinase II. Moreover, pp42 MAP-2 kinase phosphorylated myelin basic protein at 10-12-fold higher rates than did pp54 MAP-2 kinase. Cycloheximide-activated pp54 MAP-2 protein kinase appears to be a previously uncharacterized protein kinase that is itself regulated through Ser/Thr phosphorylation and, perhaps, polypeptide regulators with basic domains. The identity of the upstream regulatory elements and the native substrates remain to be established.     

In vivo activation of a microtubule-associated protein kinase during meiotic maturation of the Xenopus oocyte

We have characterized a serine/threonine protein kinase from Xenopus metaphase-II-blocked oocytes, which phosphorylates in vitro the microtubule-associated protein 2 (MAP2). The MAP2 kinase activity, undetectable in prophase oocytes, is activated during the progesterone-induced meiotic maturation (G2-M transition of the cell cycle). p-Nitrophenyl phosphate, a phosphatase inhibitor, is required to prevent spontaneous deactivation of the MAP2 kinase in crude preparations; conversely, the partially purified enzyme can be in vitro deactivated by the low-Mr polycation-stimulated (PCSL) phosphatase (also termed protein phosphatase 2A2), working as a phosphoserine/phosphothreonine-specific phosphatase and not as a phosphotyrosyl phosphatase indicating that phosphorylation of serine/threonine is necessary for its activity. S6 kinase, a protein kinase activated during oocyte maturation which phosphorylates in vitro ribosomal protein S6 and lamin C, can be deactivated in vitro by PCSL phosphatase. S6 kinase from prophase oocytes can also be activated in vitro in fractions known to contain all the factors necessary to convert pre-M-phase-promoting factor (pre-MPF) to MPF. Active MAP2 kinase can activate in vitro the inactive S6 kinase present in prophase oocytes or reactivate S6 kinase previously inactivated in vitro by PCSL phosphatase. These data are consistent with the hypothesis that the MAP2 kinase is a link of the meiosis signalling pathway and is activated by a serine/threonine kinase. This will lead to the regulation of further steps in the cell cycle, such as microtubular reorganisation and S6 kinase activation.     

Interfacial structure and lipase action. Characterization of taurodeoxycholate-didecanoylglycerol monolayers by physical and kinetic methods.

Surface pressure-area isotherms for 1,3-didecanoyl-glycerol (dicaprin) were determined as a function of the concentration of taurodeoxycholate in the subphase. Analysis of these curves indicates that, from 0.05 to 0.80 mM bile salt, surface structure is dependent only on the surface concentration of the diglyceride. The limiting areas for dicaprin in the presence and absence of bile salt were about 38 A2/molecule. Subjecting the monolayers to hydrolysis by pancreatic lipase yielded kinetic data which, together with the physical studies, support a model for monolayer glyceride molecules undergoing discrete changes of state. In the absence of bile salt, the relatively expanded state exhibits an area of 75 A2/diglyceride molecule and is not a substrate for pancreatic lipase B. The more condensed state exhibits an area of 38 A2/diglyceride molecule and is hydrolyzed at a rate proportional to its concentration in the monolayer. Taurodeoxybholate at 0.05 to 0.60 mM shifts the apparent area of the expanded state to 360 A2/diglyceride molecule.     

Insulin-induced dephosphorylation of hormone-sensitive lipase. Correlation with lipolysis and cAMP-dependent protein kinase activity

The effect of insulin on the state of phosphorylation of hormone-sensitive lipase, cellular cAMP-dependent protein kinase activity and lipolysis was investigated in isolated adipocytes. Increased phosphorylation of hormone-sensitive lipase in response to isoproterenol stimulation was closely paralleled by increased lipolysis. Maximal phosphorylation and lipolysis was obtained when the cAMP-dependent protein kinase activity ratio was greater than or equal to 0.1, and this corresponded to a 50% increase in the state of phosphorylation of hormone-sensitive lipase. Insulin (1 nM) reduced cAMP-dependent protein kinase activity and also reduced lipolysis with both cAMP-dependent and cAMP-independent antilipolytic effects up to an activity ratio of approximately 0.4, above which the antilipolytic effect was lost. Insulin caused a decrease in the state of phosphorylation of hormone-sensitive lipase at all levels of cAMP-dependent protein kinase activity. Under basal conditions, with cAMP-dependent protein kinase activity at a minimum, this reflected a dephosphorylation of the basal phosphorylation site of hormone-sensitive lipase in a manner not mediated by cAMP. When the cAMP-dependent protein kinase was stimulated to phosphorylate the regulatory phosphorylation site of hormone-sensitive lipase, the insulin-induced dephosphorylation occurred both at the basal and regulatory sites. At low levels of cAMP-dependent protein kinase activity ratios (0.05-0.1), dephosphorylation of the regulatory site correlated with reduced cAMP-dependent protein kinase activity, but not at higher activity ratios (greater than 0.1). Stimulation of cells with isoproterenol produced a transient (1-5 min) peak of cAMP-dependent protein kinase activity and of phosphorylation of hormone-sensitive lipase. The state of phosphorylation also showed a transient peak when the protein kinase was maximally and constantly activated. In the presence of raised levels of cellular cAMP, insulin (1 nM) caused a rapid (t1/2 approximately 1 min) dephosphorylation of hormone-sensitive lipase. In unstimulated cells the reduction in phosphorylation caused by insulin was distinctly slower (t1/2 approximately 5 min). These findings are interpreted to suggest that insulin affects the state of phosphorylation of hormone-sensitive lipase and lipolysis through a cAMP-dependent pathway, involving reduction of cAMP, and through a cAMP-independent pathway, involving activation of a protein phosphatase activity that dephosphorylates both the regulatory and basal phosphorylation sites of hormone-sensitive lipase.     

Activation of myocardial neutral triglyceride lipase and neutral cholesterol esterase by cAMP-dependent protein kinase

Lipolysis of intracellular triglycerides in the heart has been shown to be regulated by hormones. However, activation of myocardial triglyceride lipase in a cell-free system has not been directly demonstrated. In the present studies, initial attempts to demonstrate cAMP-dependent activation of triglyceride lipase using the 1,000 X g supernatant fraction (S1) of mouse heart homogenate were unsuccessful, presumably due to the masking effects of high levels of lipoprotein lipase activity even when assayed at pH 7.4 and in the absence of apolipoprotein C-II. Myocardial lipoprotein lipase in the 40,000 X g supernatant fraction was then removed by heparin-Sepharose affinity chromatography. The lipoprotein lipase-free fractions were shown to contain neutral triglyceride lipase and neutral cholesterol esterase of about equal activities. The triglyceride lipase and cholesterol esterase activities fell progressively during preincubation in the presence of 5 mM Mg2+. Additions of cAMP and ATP resulted in 40-70% activation of both triglyceride lipase and cholesterol esterase. The activation was blocked by protein kinase inhibitor and was restored by the addition of exogenous cAMP-dependent protein kinase. Since lipoprotein lipase has no activity toward cholesteryl oleate, activation of cholesterol esterase in untreated S1 was readily demonstrable. Both triglyceride lipase and cholesterol esterase activities were present in homogenates prepared from isolated rat heart myocytes. We conclude that the myocardium contains a hormone-sensitive lipase that is regulated in a fashion similar to that of the adipose tissue enzyme.     

The regulatory and basal phosphorylation sites of hormone-sensitive lipase are dephosphorylated by protein phosphatase-1, 2A and 2C but not by protein phosphatase-2B

The activity of hormone-sensitive lipase, the rate-limiting enzyme in adipose tissue lipolysis, is controlled by cAMP-mediated phosphorylation at a specific regulatory phosphorylation site. The lipase is also phosphorylated at a site, termed basal, without any effects on its activity [Str?lfors et al. (1984) Proc. Natl Acad. Sci. USA 81, 3317-3321]. The capacity of protein phosphatase-1, 2A, 2B and 2C to dephosphorylate the lipase, selectively phosphorylated by glycogen synthase kinase-4 and cAMP-dependent protein kinase at the basal and regulatory phosphorylation sites, was compared with that towards glycogen phosphorylase and phosphorylase kinase (alpha subunit). Protein phosphatase-1, 2A and 2C were found to dephosphorylate both phosphorylation sites of hormone-sensitive lipase, while protein phosphatase-2B had no measureable activity towards any of the sites. When the activities of protein phosphatase-1, 2A and 2C were normalized with respect to the reference substrates, they were found to dephosphorylate the lipase regulatory site in the approximate relations of 1:4:3 and the basal site in the approximate relations of 1:6:4. Protein phosphatase-1 showed 20% higher and protein phosphatase-2A and 2C 80% higher activity towards the basal site compared to the regulatory site. The two phosphorylation sites of the lipase were comparable to good substrates for protein phosphatase-2A and 2C, but relatively poor substrates for protein phosphatase-1. Protein phosphatase-2C activity towards the lipase was completely dependent on Mg2+ with a half-maximal effect at 3 mM. Protamine increased the lipase dephosphorylation by protein phosphatase-1 3-5-fold with half-maximal effect at 0.6 microgram/ml, and by protein phosphatase-2A about 2-fold with half-maximal effect at 3-5 micrograms/ml, thus illustrating the potential for control of these lipase phosphatase activities.     

Protein phosphorylation associated with the stimulation of neutrophils. Modulation of superoxide production by protein kinase C and calcium

Neutrophils and other phagocytic cells of the immune system possess a superoxide-generating oxidase system which is essential for the efficient killing of microbes. The system is activated by a wide variety of stimuli, some of which operate through pathways involving protein kinase C (PKC), while others appear not to. The PKC-dependent pathway is probably the major signal transduction route for most of the stimuli. Alterations in cellular Ca²⁺ and diglyceride levels can have a pronounced stimulatory effect on this pathway by their ability to synergistically activate PKC. This review discusses PKC, the different interactions of this kinase with the plasmalemma that are important in superoxide production, the synergy between Ca²⁺ and diglyceride, and the nature of the phosphoproteins involved. Evidence supporting the existence of the PKC-independent pathway is also reviewed.     

Complement receptor-mediated phagocytosis is associated with accumulation of phosphatidylcholine-derived diglyceride in human neutrophils. Involvement of phospholipase D and direct evidence for a positive feedback signal of protein kinase.

Complement receptor (CR)-mediated phagocytosis is associated with an increased accumulation of diglyceride (sn-1,2-diacylglycerol and/or 1-O-alkyl-2-acyl-glycerol) in human neutrophils. The C3bi-mediated increase in diglyceride (5-20 min) was only partially impaired when phosphoinositide-specific phospholipase C (PLC) activity was abolished by reduction of cytosolic free Ca2+. At an early time point (1 min), however, diglyceride production was barely detectable in control cells, whereas production was considerable in cells with a reduced cytosolic free Ca2+ concentration. C3bi stimulation of 32P-labeled neutrophils caused a rapid and significant breakdown of [32P]phosphatidylcholine (PC) which was not affected by inhibition of Ca(2+)-dependent phosphoinositide-specific PLC. Thus, PC hydrolysis could be involved in C3bi-induced diglyceride formation. Stimulation of cells labeled with [3H]1-O-alkyl-lyso-PC ([3H]alkyl-lyso-PC), resulted in an increased formation of [3H]1-O-alkyl-phosphatidic acid ([3H]alkyl-PA) and a later and slower formation of [3H]1-O-alkyl-diglyceride ([3H]alkyl-diglyceride); this suggests activation of phospholipase D (PLD). When these labeled cells were stimulated in the presence of 0.5% ethanol a marked accumulation of [3H]1-O-alkyl-phosphatidylethanol ([3H]alkyl-PEt) was observed in both controls and calcium-reduced cells, further strengthening the suggested involvement of PLD activity. In parallel with the sustained increase in diglyceride formation, CR-mediated phagocytosis was also associated with phosphorylation of a cellular protein kinase C substrate (MARCKS). Therefore it seems reasonable to suggest a causal relationship between C3bi-induced PLD activation, which results in diglyceride formation, and activation of protein kinase C. In electropermeabilized cells which were incapable of ingesting particles, C3bi particles were still able to activate PLD and induce formation of diglyceride. This signaling event must therefore be triggered by binding of particles to the cell and not by the engulfment process. Most importantly, introduction of the protein kinase C inhibitor peptides, PKC(19-36) and PKC(19-31), into these permeabilized cells resulted in a clear reduction of the C3bi-induced production of diglyceride, indicating that CR-mediated activation of protein kinase C directly triggers a positive feedback mechanism for additional diglyceride formation. Taken together, these data further clarify the mechanisms of CR-mediated diglyceride formation and give added support to the concept that protein kinase C plays an important role in the phagocytic process.     

Obtaining monoglycerides by esterification of glycerol with palmitic acid using some high activity preparations of Candida antarctica lipase B

Cross-linked enzyme aggregates (CLEAs), protein coated microcrystals (PCMCs), cross-linked protein coated microcrystals (CLPCMCs) of Candida antarctica lipase B (CALB) were used for esterification of glycerol with palmitic acid in acetone under low water condition. With CLEAs, 81% monoglyceride (MG) along with 4.5% diglyceride (DG) were produced at 1% (v/v) water content in 24 h. The water content in the medium was managed by stepwise addition of the molecular sieves at appropriate time intervals. With PCMCs (potassium sulfate as a core material), 82% monoglyceride along with 4.0% diglyceride were obtained, with 0.5% water (v/v) added initially to anhydrous acetone with molecular sieves present in the reaction medium. With CLPCMC (prepared by cross-linking with 200 mM glutaraldehyde), 87% monoglyceride and 3.3% diglyceride were produced in 24 h in presence of 1% (v/v) water (added initially) and with appropriate amount of molecular sieves added in the reaction medium. The results offer a comparative study on the performance of three high activity preparations of CALB for preparation of monopalmitin with ≤10% of the diglyceride content.     

Okadaic acid activates microtubule-associated protein kinase in quiescent fibroblastic cells

Okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A, and is a strong tumor promoter that is not an activator of protein kinase C. Treatment of quiescent cultures of rat fibroblastic 3Y1 cells with okadaic acid induced marked activation of a kinase activity that phosphorylated microtubule-associated protein (MAP) 2 and myelin basic protein, but not histone or casein, in vitro. This activated kinase eluted at approximately 0.15 M NaCl on a DEAE-cellulose column and its apparent molecular mass was determined to be approximately 40 kDa by gel filtration. Detection of the kinase activity in polyacrylamide gels containing substrate proteins after sodium dodecyl sulfate gel electrophoresis revealed that the okadaic-acid-activated kinase activity resided mainly in two closely related polypeptides with apparent molecular mass approximately 40 kDa. The characteristics of this kinase were indistinguishable from those of the mitogen-activated MAP kinase in the same cells. The okadaic-acid-activated MAP kinase was deactivated by protein phosphatase 2A treatment in vitro. These results suggest that MAP kinase is negatively regulated by protein phosphatases 1 and/or 2A in quiescent cells and therefore can be activated by inhibiting these protein phosphatases. Interestingly, the okadaic-acid-induced activation of MAP kinase was transient and epidermal-growth-factor-induced activation was also transient, even in the presence of okadaic acid. These data may imply that protein phosphatases 1 and 2A are not involved in the deactivation of MAP kinase in cells.     

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.