Effects of protein kinase CK2, extracellular signal-regulated kinase 2, and protein phosphatase 2A on a phosphatidic acid-preferring phospholipase A1.

A soluble, phosphatidic acid-preferring phospholipase A1, expressed in mature bovine testes but not in newborn calf testes, may contribute to the formation or function of sperm. Here we incubated a recombinant preparation of the phospholipase in vitro with several enzymes including protein kinase CK2 (CK2), extracellular signal-regulated kinase 2 (ERK2), and protein phosphatase 2A (PP2A) to identify effects that might be of regulatory importance in vivo. Major findings were that 1) CK2 phosphorylated the phospholipase on serines 93, 105, and 716; 2) ERK2 phosphorylated the enzyme on serine 730; 3) there was cross-antagonism between the reactions that phosphorylated serines 716 and 730; 4) PP2A selectively hydrolyzed phosphate groups that were esterified to serines 716 and 730; 5) CK2alpha formed a stable, MgATP/MgGTP-dependent complex with the phospholipase by a novel mechanism; and 6) the complex showed reduced phospholipase activity and resembled a complex identified in homogenates of macaque testis. These results provide the first available information about the effects of reactions of phosphorylation and dephosphorylation on the behavior of the phospholipase, shed light on properties of CK2alpha that may be required for the formation of complexes with its substrates, and raise the possibility that a complex containing CK2alpha and the phospholipase may play a special biological role in the testis.

Membrane lipids have multiple effects on interfacial catalysis by a phosphatidic acid-preferring phospholipase A1 from bovine testis.

We previously purified a cytosolic phospholipase A1 that could catalyze the preferential hydrolysis of phosphatidic acid in mixed-micelle assays. Here we studied the enzyme's interactions with unilamellar lipid membranes and examined effects of the lipids on enzyme binding, stability, and catalysis. A major finding was that membrane lipids could influence the stability, activity, and specificity of the enzyme under conditions where enzyme binding to the membranes was likely to be saturated. Thus, the enzyme was unstable at 37 degrees C in the absence of membranes but bound to membranes that contained anionic phosphoglycerides and could be stabilized by these membranes in the presence of albumin. The overall activity of the bound enzyme toward membrane phosphoglycerides, assayed in the presence of albumin, increased when phosphatidylethanolamine was substituted for phosphatidylcholine. Furthermore, the enzyme's catalytic preference for phosphatidic acid increased when cholesterol and diacylglycerol were included in the membranes, sn-1-stearoyl-2-arachidonoylphosphatidylethanolamine was substituted for sn-1-palmitoyl-2-oleoylphosphatidylethanolamine, and the concentration of phosphatidic acid was increased from 0 to 10 mol % of the total membrane phosphoglycerides. Finally, changes in the relative contents of phosphatidylcholine and phosphatidylserine in the membranes influenced the enzyme's catalytic preference for different molecular species of phosphatidic acid. These results provide the first available information about the enzyme's ability to interact with membranes and identify conditions that yield high enzyme activity toward membrane-associated phosphatidic acid.

Disruption of Rab3-calmodulin interaction, but not other effector interactions, prevents Rab3 inhibition of exocytosis.

Rab GTPases regulate membrane traffic between the cellular compartments of eukaryotic cells. Rab3 is associated with secretory vesicles of neuronal and endocrine cells and controls the Ca(2+)-triggered release of neurotransmitters and hormones. To clarify the mode of action of Rab3 we generated mutants of the GTPase that do not interact efficiently with its putative effectors Rabphilin and RIM. Surprisingly, these mutants transfected in PC12 cells were still capable of inhibiting Ca(2+)-evoked secretion. Rab3 was shown previously to bind to calmodulin in a Ca(2+)-dependent manner. By replacing two arginines conserved between Rab3 isoforms, we generated a mutant with a reduced affinity for calmodulin. This mutant retained the capacity to interact with the Rab3 regulatory proteins, Rabphilin, RIM, Mss4 and RabGDI, and was correctly targeted to dense-core secretory granules. However, replacement of the two arginines abolished the ability of the GTP-bound form of Rab3 to inhibit exocytosis of catecholamine- and insulin-secreting cells. We propose that a Rab3-calmodulin complex generated by elevated Ca(2+) concentrations mediated at least some of the effects of the GTPase and limited the number of exocytotic events that occurred in response to secretory stimuli.

Multiple factors influence the binding of a soluble, Ca2+-independent, diacylglycerol kinase to unilamellar phosphoglyceride vesicles.

We studied the influence of membrane lipids, MgCl2, and ATP on the ability of a soluble diacylglycerol kinase to bind to 100-nm lipid vesicles. The enzyme did not bind detectably to vesicles that contained phosphatidylcholine alone or to vesicles that contained 50 mol % phosphatidylcholine + 50 mol % phosphatidylethanolamine. But it did bind to vesicles that contained anionic phosphoglycerides, and maximal binding occurred (in the presence of MgCl2) when the vesicles contained anionic phosphoglycerides alone. When increasing amounts of phosphatidylcholine were included in phosphatidylserine-containing vesicles, enzyme binding to the vesicles decreased by as much as 1000-fold. However, when increasing amounts of phosphatidylethanolamine were included in phosphatidylserine-containing vesicles, little change in binding occurred until the concentration of phosphatidylserine was reduced to below 25 mol %. These results and results obtained with vesicles that contained various mixtures of anionic phosphoglycerides, phosphatidylcholine, phosphatidylethanolamine, and unesterified cholesterol provided evidence that anionic phosphoglycerides were positive effectors of binding, phosphatidylcholine was a negative effector, and phosphatidylethanolamine and unesterified cholesterol were essentially neutral diluents. Other experiments showed that diacylglycerol and some of its structural analogues also were important, positive effectors of enzyme binding and that addition of ATP to the medium increased their effects. The combined results of the study suggest that the enzyme may bind to vesicles via at least two types of binding sites: one type that requires anionic phospholipids and is enhanced by Mg2+ but inhibited by phosphatidylcholine, and one type that requires diacylglycerol and is enhanced by ATP.

Affinity purification and catalytic properties of a soluble, Ca2+-independent, diacylglycerol kinase.

We used a new procedure that involved selective enzyme binding to lipid vesicles to partially purify a soluble diacylglycerol kinase, then studied the relation between enzyme-vesicle binding and activity in vesicle-based assays. The vesicle-binding procedure required about 2 h, increased the enzyme's specific activity 50-fold with a 50% yield of activity, and combined well with additional purification steps. Studies of the activity of the partially purified diacylglycerol kinase toward vesicle-associated diacylglycerols revealed linear reaction kinetics that reflected enzyme binding to the vesicles; factors known to influence enzyme binding to the vesicles affected enzyme activity only indirectly, not by influencing the diacylglycerol kinase reaction itself. On the other hand, special incubation experiments that caused both substrate depletion in vesicles and enzyme stalling provided evidence that the diacylglycerol kinase could desorb from these vesicles, adsorb to freshly added, substrate-containing vesicles, and resume catalysis of phosphorylation reactions. The molecular basis for this enzyme-vesicle "hopping" behavior remains to be clarified. But enzyme-catalyzed conversion of diacylglycerol to phosphatidic acid may not have been a contributing factor because separate, enzyme-vesicle binding experiments showed that the enzyme had only a marginally higher affinity for diacylglycerol-containing vesicles than it did for vesicles that contained comparable amounts of phosphatidic acid. The combined results of our experiments suggest that the linear rates of diacylglycerol phosphorylation observed in standard assays with diacylglycerol-containing vesicles may have been combined functions of both the rate of enzyme hopping among vesicles and the rate of diacylglycerol phosphorylation by enzyme that was bound transiently on substrate-containing vesicles.

Cloning of a phosphatidic acid-preferring phospholipase A1 from bovine testis.

We report the molecular cloning and expression of a phosphatidic acid-preferring phospholipase A1 from bovine testis. The open reading frame encoded an 875-amino acid protein with a calculated molecular mass of 97,576 daltons and a pI of 5.61. The sequence included a region similar to a lipase consensus sequence containing the putative active site serine and also included a potential, coiled-coil-forming region. Expression of the open reading frame in COS1 cells resulted in a 20-44-fold increase in phosphatidic acid phospholipase A1 activity over that of control cells. Mutation of the putative active site serine (amino acid 540) demonstrated that it was essential for this increase in enzyme activity. Northern blot analysis revealed at least five different messages with the highest overall message levels in mature testis, but detectable message in all tissues examined. Two possible alternately spliced regions in the open reading frame also were identified. Finally, a search of the data base identified six related proteins: a potential counterpart of the phospholipase A1 in Caenorhabditis elegans, two putative lipases in yeast, and three proteins separately encoded by the Drosophila retinal degeneration B gene and its mouse and human homologues.

On the relation between a stearoyl-specific transacylase from bovine testis membranes and a copurifying acyltransferase.

Bovine testis membranes contain a coenzyme A-dependent transacylase that can catalyze the preferential transfer of stearoyl groups from phosphoglycerides to sn-2-acyl molecular species of lysophosphatidic acid and lysophosphatidylinositol [Itabe et al., (1992) J. Biol. Chem. 267, 15319-15325]. We have now purified this enzyme 1000-fold and shown that it copurifies with an acyltransferase. The purified transacylase can use phosphatidic acid, phosphatidylinositol, or phosphatidylinositol-4-phosphate as an acyl donor and catalyzes the transfer of stearoyl groups in preference to palmitoyl groups or oleoyl groups. In contrast, the purified acyltransferase uses acyl-coenzyme A as an acyl donor and shows no such preference for stearoyl group transfer. Furthermore, phosphatidylinositol-4, 5-bisphosphate inhibits the two enzymes to different extents and by different mechanisms. Nevertheless, the enzymes are similar in several respects: they use the same acyl acceptors and, when assayed together, compete for the acyl acceptor, sn-2-oleoyl lysophosphatidic acid; they lose activity in parallel unless stabilized by the addition of an anionic phosphoglyceride or stearoyl-coenzyme A; and they show similar sensitivities to heat and pH. One way to explain these results is to postulate that the transacylase reaction occurs in two successive steps: a stearoyl-specific first step in which a stearoyl group is transferred from an sn-1-stearoyl-2-acyl phosphoglyceride to coenzyme A, and a relatively non-acyl-chain-specific second step in which a stearoyl group is transferred from stearoyl-coenzyme A to an sn-2-acyl lysophosphoglyceride. According to this line of reasoning, the transacylase assay that we have used measures the net effect of both steps, whereas the acyltransferase assay measures only the effect of the second step.

Ca2+/calmodulin causes Rab3A to dissociate from synaptic membranes.

The GTPase Rab3A has been postulated to cycle on and off synaptic membranes during the course of neurotransmission. Moreover, a Rab guanine nucleotide dissociation inhibitor has been shown to cause Rab3A to dissociate from synaptic membranes in vitro. We demonstrate here that Ca2+/calmodulin also can cause Rab3A to dissociate from synaptic membranes in vitro. Like Rab guanine nucleotide dissociation inhibitor, it forms a 1:1 complex with Rab3A that requires both the lipidated C terminus of Rab3A and the presence of bound guanine nucleotide. In addition, a synthetic peptide corresponding to the Lys62-Arg85 sequence of Rab3A can prevent the dissociating effect of each protein and disrupt complexes between each protein and Rab3A. However, Ca2+/calmodulin's effect differs from that of Rab guanine nucleotide dissociation inhibitor not only in being Ca2+-dependent but also in having a less stringent requirement for GDP as opposed to GTP and in involving a less complete dissociation of Rab3A. The functional significance in vivo of Ca2+/calmodulin's effect remains to be determined; it may depend in part on the relative amounts of Ca2+/calmodulin and Rab guanine nucleotide dissociation inhibitor that are available for binding to Rab3A in individual, activated nerve termini.

Purification and properties of a phosphatidic acid-preferring phospholipase A1 from bovine testis. Examination of the molecular basis of its activation.

We recently identified a cytosolic phospholipase A1 activity in bovine brain and testis that preferentially hydrolyzes phosphatidic acid substrates. We also showed that the enzyme displays sigmoidal kinetics toward phosphatidic acid substrates in Triton X-100 mixed micelle assay system (Higgs, H.N., and Glomset J.A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9574-9578). In the present work we purified the bovine testis enzyme 14,000-fold and used a combination of size exclusion chromatography, labeling with the phospholipase A inhibitor, methyl arachidonyl fluorophosphonate, and SDS-polyacrylamide gel electrophoresis to provide evidence that it is a homotetramer of 110-kDa subunits. Studies of the molecular basis of the enzyme reaction in Triton micelles revealed that (a) a nonhydrolyzable sn-1-alkyl-2-oleoyl-analogue of phosphatidic acid activated the enzyme 30-fold in a sigmoidal fashion (Hill coefficient 3.2, EC50 4 mol %) without substantially affecting its preference for specific diacyl phosphoglyceride substrates, (b) the activator promoted tight binding of the enzyme to micelles, and (c) the enzyme's activity toward unsaturated phosphatidic acid substrates was affected by the location and nature of the fatty acyl chain double bonds.

Arachidonoyl-diacylglycerol kinase. Specific in vitro inhibition by polyphosphoinositides suggests a mechanism for regulation of phosphatidylinositol biosynthesis.

We previously described the purification of a membrane-bound diacylglycerol kinase highly selective for sn-1-acyl-2-arachidonoyl diacylglycerols (Walsh, J. P., Suen, R., Lemaitre, R. N., and Glomset, J. A. (1994) J. Biol. Chem. 269, 21155-21164). This enzyme appears to be responsible for the rapid clearance of the arachidonate-rich pool of diacylglycerols generated during stimulus-induced phosphoinositide turnover. We have now shown phosphatidylinositol 4,5-bisphosphate to be a potent and specific inhibitor of arachidonoyl-diacylglycerol kinase. Kinetic analyses indicated a Ki for phosphatidylinositol 4,5-bisphosphate of 0.04 mol %. Phosphatidic acid also was an inhibitor with a Ki of 0.7 mol %. Other phospholipids had only small effects at these concentrations. A series of multiply phosphorylated lipid analogs also inhibited the enzyme, indicating that the head group phosphomonoesters are the primary determinants of the polyphosphoinositide effect. However, these compounds were not as potent as phosphatidylinositol 4,5-bisphosphate, indicating some specificity for the polyphosphoinositide additional to its total charge. Five other diacylglycerol kinases were activated to varying degrees by phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, suggesting that inhibition by acidic lipids may be specific for the arachidonoyl-DAG kinase isoform. Given the presumed role of arachidonoyl-diacylglycerol kinase in the phosphoinositide cycle, this inhibition may represent a mechanism for polyphosphoinositides to regulate their own synthesis.

Rab geranylgeranyl transferase catalyzes the geranylgeranylation of adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A.

Rab proteins are Ras-related small GTPases that are geranylgeranylated on cysteine residues located at or near their C termini. They differ from other geranylgeranylated small GTPases in several important respects. (i) Most Rab proteins contain two adjacent cysteine residues within one of the following C-terminal sequence motifs: -XXCC, -XCXC, or -CCXX; (ii) a Rab protein that ends in a -XCXC motif has been shown to be geranylgeranylated on both adjacent cysteine residues; and (iii) Rab proteins are substrates of a unique Rab-specific geranylgeranyltransferase. Whether this enzyme catalyzes the geranylgeranylation of both cysteines is unknown. We addressed this question by direct structural analysis of in vitro prenylated proteins. We incubated recombinant Rab geranylgeranyltransferase, Rab escort protein, and [1-3H]geranylgeranyl pyrophosphate with recombinant wild-type Rab1A (-XXCC), Rab3A (-XCXC), or Rab5A (-CCXX) and treated each labeled protein with trypsin. We then analyzed the resulting peptides by HPLC and electrospray mass spectrometry and found that for each protein both C-terminal adjacent cysteines were geranylgeranylated. These results indicate that Rab geranylgeranyltransferase/Rab escort protein catalyzes the geranylgeranylation of both cysteines in Rab proteins with three distinct C-terminal motifs and suggest that other Rab proteins with these motifs may be similarly modified.

Identification of a phosphatidic acid-preferring phospholipase A1 from bovine brain and testis.

Recent experiments in several laboratories have provided evidence that phosphatidic acid functions in cell signaling. However, the mechanisms that regulate cellular phosphatidic acid levels remain obscure. Here we describe a soluble phospholipase A1 from bovine testis that preferentially hydrolyzes phosphatidic acid when assayed in Triton X-100 micelles. Moreover, the enzyme hydrolyzes phosphatidic acid molecular species containing two unsaturated fatty acids in preference to those containing a combination of saturated and unsaturated fatty acyl groups. Under certain conditions, the enzyme also displays lysophospholipase activity toward lysophosphatidic acid. The phospholipase A1 is not likely to be a lysosomal enzyme because its optimum pH is 7.5-8.5. Furthermore, it is probably not a general lipid metabolic enzyme because high levels of activity are found in mature testis and brain but no measurable activity is seen in liver, spleen, or heart. The fact that the activity of the phospholipase A1 in mature bovine testis is > 10-fold higher than that in newborn calf testis raises the possibility that the enzyme may play a regulatory role in spermatogenesis or sperm function.

Arachidonoyl-diacylglycerol kinase from bovine testis. Purification and properties.

Previous work in our laboratory demonstrated the existence of a membrane-bound diacylglycerol kinase highly selective for diacylglycerols containing arachidonate as the sn-2 fatty acyl moiety (MacDonald, M. L., Mack K. F., Richardson, C. N., and Glomset, J. A. (1988) J. Biol. Chem. 263, 1575-1583). We now report the purification of arachidonoyl-diacylglycerol kinase 34,400-fold to apparent homogeneity from bovine testis. High concentrations of both salt and detergent were required to extract the enzyme from membranes and stabilize its activity, suggesting that in vivo the enzyme is part of a complex with other membrane or cytoskeletal proteins. Arachidonoyl-diacylglycerol kinase had an apparent M(r) of 58,000 both on SDS-polyacrylamide gels and by size exclusion chromatography. The enzyme appeared to be an integral membrane protein. In a mixed micellar assay, arachidonoyl-diacylglycerol kinase followed surface dilution kinetics with respect to diacylglycerol. The purified enzyme retained the arachidonate selectivity observed previously in membranes. Kinetic analyses indicated a Km for sn-1-stearoyl-2-arachidonoylglycerol of 2.4 mol %, as compared to 43 mol % for sn-1-palmitoyl-2-oleoylglycerol. Calcium, an activator of some other diacylglycerol kinases, had no apparent effect on the arachidonate-specific enzyme. Guanosine triphosphate could effectively substitute for ATP as the phosphoryl donor and Mg2+ could be replaced by Mn2+ or Ca2+. Phosphatidylserine and, to a lesser extent, phosphatidylinositol inhibited the purified enzyme. Phosphatidylcholine and phosphatidylethanolamine had only small effects.

Spreading of differentiating human monocytes is associated with a major increase in membrane-bound CDC42.

In a search for a model cell system that might allow studies of the function of the Rho-related GTPase CDC42Hs in human cells, we measured the content and distribution of CDC42Hs in monocytes that were differentiating into macrophages. The total content of this protein increased 5- to 6-fold in phorbol ester-treated human monocytic THP-1 and U-937 cells and increased 13-fold in normal human blood monocytes. Moreover, membrane-associated CDC42Hs in these cells increased 13-fold and 30-fold, respectively, while cytosolic CDC42Hs increased only 3- and 6-fold. Measurements made specifically in U-937 cells showed that the increase in membrane CDC42Hs correlated closely with an increase in cell spreading. The changes in CDC42Hs in U-937 cells probably depended on increased mRNA translation and/or decreased protein degradation, since no change in CDC42Hs mRNA could be detected. Finally, the changes in CDC42Hs were relatively specific, since contents of the CDC42Hs-binding protein Rho-GDI and the Rho-related protein Rac2 were unaffected and no change in CDC42Hs occurred when the cells were stimulated by agonists that induce monocytes to differentiate into nonadherent cells. These findings show that marked changes in content and distribution of CDC42Hs occur when monocytes differentiate into macrophages, suggesting that membrane CDC42Hs may play a role in cell spreading.

Substrate specificity of a CoA-dependent stearoyl transacylase from bovine testis membranes.

We identified a CoA-dependent stearoyl transacylase activity in bovine testis membranes, then examined the enzyme's specificity in mixed micelle systems containing the neutral detergent Triton X-100. The enzyme transferred stearoyl groups from a variety of phospholipids to sn-2-arachidonoyl lysophosphatidic acid (lysoPA), but showed very little palmitoyl transacylase activity. Its ability to transfer stearoyl groups was both donor- and acceptor-dependent. For example, it used weakly acidic phospholipids, such as sn-1-stearoyl-2-acyl species of phosphatidylinositol (PI), as donors, but did not use phosphatidylinositol-4,5-bisphosphate or sn-1-stearoyl-2-arachidonoyl phosphatidylcholine. Moreover, it used sn-2-acyl species of lysoPA and sn-2-arachidonoyl lysoPI as acceptors but did not use sn-2-arachidonoyl species of lysophosphatidylserine, lysophosphatidylethanolamine, or lysophosphatidylcholine. When taken together, our results raise the possibility that sn-1-stearoyl-2-acyl species of PI may be the primary acyl donors in the transacylase reaction in vivo, while sn-2-acyl species of lysoPA may be the primary acyl acceptors. Available evidence suggests that the PA that is formed may subsequently be converted into PI, but the metabolic fate of the other reaction product, sn-2-acyl lysoPI, remains to be determined.