Molecular characterization of cytosolic phospholipase A2-beta.

We have isolated a cDNA encoding a 1012-amino acid polypeptide cPLA2-beta, that has significant homology with cPLA2-alpha in both the calcium-dependent lipid binding domain as well as in the catalytic domain. Transient expression of cPLA2-beta cDNA in COS cells results in an increase in calcium-dependent phospholipase A1 (PLA1) and PLA2 activities compared with vector-transfected cells. cPLA2-beta is markedly less selective for cleavage at sn-2 as compared with cPLA2-alpha and cPLA2-gamma. Northern analysis reveals a cPLA2-beta transcript of 8 kilobase pairs that is expressed in all the human tissues examined. With the identification of cPLA2-beta, the newly defined cPLA2 family now comprises three members that may have dramatically different mechanisms for regulation of expression and enzymatic activation.

A novel calcium-independent phospholipase A2, cPLA2-gamma, that is prenylated and contains homology to cPLA2.

We report the cloning and characterization of a novel membrane-bound, calcium-independent PLA2, named cPLA2-gamma. The sequence encodes a 541-amino acid protein containing a domain with significant homology to the catalytic domain of the 85-kDa cPLA2 (cPLA2-alpha). cPLA2-gamma does not contain the regulatory calcium-dependent lipid binding (CaLB) domain found in cPLA2-alpha. However, cPLA2-gamma does contain two consensus motifs for lipid modification, a prenylation motif (-CCLA) at the C terminus and a myristoylation site at the N terminus. We present evidence that the isoprenoid precursor [3H]mevalonolactone is incorporated into the prenylation motif of cPLA2-gamma. Interestingly, cPLA2-gamma demonstrates a preference for arachidonic acid at the sn-2 position of phosphatidylcholine as compared with palmitic acid. cPLA2-gamma encodes a 3-kilobase message, which is highly expressed in heart and skeletal muscle, suggesting a specific role in these tissues. Identification of cPLA2-gamma reveals a newly defined family of phospholipases A2 with homology to cPLA2-alpha.

Phospholipase C gamma-2 (Plcg2) and phospholipase C gamma-1 (Plcg1) map to distinct regions in the human and mouse genomes.

The phospholipase C gamma-2 (Plcg2) gene encodes an enzyme that plays a crucial role in intracellular signal transduction pathways. This enzyme is important because of its role in the generation of second messengers following the hydrolysis of phosphatidylinositol 4,5-bisphosphate. We have now determined the chromosomal location of this gene in the mouse and human genomes. An interspecific backcross involving AEJ/Gn and Mus spretus mice was used to localize the gene in mouse. A rodent/human somatic cell hybrid panel was used to map PLCG2 in the human genome. Our results position Plcg2 in the central region of mouse chromosome 8. We also show that PLCG2 maps to the long arm of human chromosome 16, in the region q22-qter. Plcg2 does not map near its most closely related family member, Plcg1, in either genome, indicating that the mammalian Plcg genes belong to a dispersed family.

Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory Ca(2+)-dependent lipid-binding domain and a Ca(2+)-independent catalytic domain.

Cytosolic phospholipase A2 (cPLA2) associates with natural membranes in response to physiological increases in Ca2+, resulting in the selective hydrolysis of arachidonyl phospholipids. The isolation and sequence analysis of cPLA2 cDNA clones from four different species revealed several highly conserved regions. The NH2-terminal conserved region is homologous to several other Ca(2+)-dependent lipid-binding proteins. Here we report that the first 178 residues of cPLA2, containing the homologous Ca(2+)-dependent lipid-binding (CaLB) motif, and another recombinant protein containing the cPLA2(1-178) fragment placed at the COOH terminus of the maltose-binding protein (MBP-CaLB) associate with membranes in a Ca(2+)-dependent manner. cPLA2 and MBP-CaLB also bind to synthetic liposomes at physiological Ca2+ concentrations, demonstrating that accessory proteins are not required. In contrast, delta C2, a truncated cPLA2 lacking the CaLB domain, fails to associate with membranes and fails to hydrolyze liposomal substrates. However, both delta C2 and cPLA2 hydrolyze monomeric 1-palmitoyl-2-lysophosphatidylcholine at identical rates in a Ca(2+)-independent fashion. These results delineate two functionally distinct domains of cPLA2, the Ca(2+)-independent catalytic domain, and the regulatory CaLB domain that presents the catalytic domain to the membrane in response to elevated Ca2+.

cPLA2 is phosphorylated and activated by MAP kinase.

Treatment of cells with agents that stimulate the release of arachidonic acid causes increased serine phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). Here we report that cPLA2 is a substrate for mitogen-activated protein (MAP) kinase. Moreover, phosphorylation by MAP kinase increases the enzymatic activity of cPLA2. The site of cPLA2 phosphorylation by MAP kinase, Ser-505, is identical to the major site of cPLA2 phosphorylation observed in phorbol ester-treated cells. Replacement of Ser-505 with Ala resulted in a mutant cPLA2 that is not a substrate for MAP kinase and causes little or no enhanced agonist-stimulated arachidonate release from intact cells. Taken together, these data indicate that MAP kinase mediates, at least in part, the agonist-induced activation of cPLA2.

Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid.

Cytosolic phospholipase A2 (cPLA2) binds to natural membrane vesicles in a Ca(2+)-dependent fashion, resulting in the selective release of arachidonic acid, thus implicating cPLA2 in the hormonally regulated production of eicosanoids. Here we report that the treatment of Chinese hamster ovary (CHO) cells overexpressing cPLA2 with ATP or thrombin resulted in an increased release of arachidonic acid as compared with parental CHO cells, demonstrating the hormonal coupling of cPLA2. In contrast, CHO cells overexpressing a secreted form of mammalian PLA2 (sPLA2-II) failed to show any increased hormonal responsiveness. Interestingly, we have noted that the activation of cPLA2 with a wide variety of agents stimulates the phosphorylation of cPLA2 on serine residues. Pretreatment of cells with staurosporin blocked the ATP-mediated phosphorylation of cPLA2 and strongly inhibited the activation of the enzyme. Increased cPLA2 activity was also observed in lysates prepared from ATP-treated cells and was sensitive to phosphatase treatment. These results suggest that in addition to Ca2+, the phosphorylation of cPLA2 plays an important role in the agonist-induced activation of cPLA2.

A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP.

We report the cloning and expression of a cDNA encoding a high molecular weight (85.2 kd) cytosolic phospholipase A2 (cPLA2) that has no detectable sequence homology with the secreted forms of PLA2. We show that cPLA2 selectively cleaves arachidonic acid from natural membrane vesicles and demonstrate that cPLA2 translocates to membrane vesicles in response to physiologically relevant changes in free calcium. Moreover, we demonstrate that an amino-terminal 140 amino acid fragment of cPLA2 translocates to natural membrane vesicles in a Ca(2+)-dependent fashion. Interestingly, we note that this 140 amino acid domain of cPLA2 contains a 45 amino acid region with homology to PKC, p65, GAP, and PLC. We suggest that this homology delineates a Ca(2+)-dependent phospholipid-binding motif, providing a mechanism for the second messenger Ca2+ to translocate and activate cytosolic proteins.

Purification of a 110-kilodalton cytosolic phospholipase A2 from the human monocytic cell line U937.

The major dithiothreitol-resistant phospholipase A2 activity present in the cytosol of U937 cells has been purified greater than 200,000-fold by sequential chromatography on phenyl-5PW, heparin-Sepharose CL-6B, high-performance hydroxylapatite, TSK-gel G3000-SW, and Mono Q columns. This 110-kDa cytosolic phospholipase A2 is distinct from the relatively small (14-kDa) dithiothreitol-sensitive phospholipases A2 that are secreted from many cell types. This additional phospholipase A2 selectively hydrolyzes fatty acid at the sn-2 position of the glycerol and favors phospholipids containing arachidonic acid, which is the rate-limiting precursor for prostaglandin and leukotriene production. Interestingly, a greater than 5-fold increase in phospholipase A2 activity is noted as the calcium concentration increases from the levels found in resting cells to those observed in activated macrophages. We suggest that this enzyme and not the previously described secretory phospholipase A2 is activated by cytosolic effectors such as GTP-binding regulatory proteins and protein kinases to initiate the production of prostaglandins, leukotrienes, and platelet-activating factor. To distinguish this cytosolic enzyme from the previously described secretory ones, we suggest referring to it as cPLA2 for cytosolic phospholipase A2 and collectively referring to the secretory phospholipases A2 as sPLA2s.

Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-specific phospholipase C activities.

Protein sequence analysis of a bovine brain phosphoinositide-specific phospholipase C (PI-PLC; PLC-154) has permitted the isolation of a cDNA that appears to code for this protein. Transient expression of this cDNA in COS-1 cells demonstrates that the cDNA encodes a functional phospholipase C that migrates at approximately 150,000 daltons. A transcript of approximately 7 kb is observed in RNA derived from bovine brain and a related transcript of the same size is present in certain human cell lines. Southern blot analysis indicates that one or possibly two genes hybridize with a PLC-154 probe. Regions of homology between PLC-154 and the previously described PLC-148 allow the assignment of a putative catalytic domain to the central region of PLC-154.

Sequence similarity of phospholipase C with the non-catalytic region of src.

The production of the second messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate is mediated by activated phosphatidylinositol-specific phospholipase C (PLC) enzymes. Here we report the cloning of a bovine brain complementary DNA encoding an enzyme PLC-148 that is characterized by calcium-dependent and phosphatidylinositol-specific phospholipase C activity when expressed in mammalian cells. Bovine brain messenger RNA contains a 7.5-kilobase transcript corresponding to the isolated cDNA; a related transcript of the same size is present in mRNA from some but not all human cell lines tested. Southern blot analysis of the bovine genome indicated that one or possibly two genes hybridize to the cloned PLC-148 cDNA. There is a striking sequence similarity between specific regions of PLC-148 and the non-catalytic domain of the non-receptor tyrosine kinases. The newly characterized crk transforming gene of the avian sarcoma virus CT10 also contains extensive sequence similarities with PLC-148.

Immunochemical identification of protein kinase C isozymes as products of discrete genes.

Immunocytochemical studies of rat cerebellum using specific antibodies against type I, II, and III PKC revealed the presence of the type I PKC in the Purkinje cells where transcripts of tau cDNA were localized, the type II PKC in the granule cells where transcripts of beta cDNA were detected, and the type III PKC in both the Purkinje and granule cells. Immunoblot analysis revealed that the expressed PKC in COS cells transfected with either alpha, beta, or tau cDNA of PKC were recognized by specific antibodies against the type III, II, and I PKC isozymes, respectively. These results prove that the type I, II, and III PKC are products of PKC genes, tau, beta, and alpha, respectively. With these specific antibodies we have identified the presence of multiple species of PKC in a variety of cell types.

Cloning and expression of multiple protein kinase C cDNAs.

Three different protein kinase C related cDNA clones were isolated from a rat brain cDNA library and designated PKC-I, PKC-II, and PKC-III. These each encode very similar, but distinct, polypeptides that contain a region homologous with other protein kinases. COS cells transfected with either PKC-I or PKC-II specifically bind at least 5-fold more 3H-PDBu (phorbol ester) than control cells. An increase in Ca2+, phosphatidylserine, and diacylglycerol/phorbol-ester-dependent protein kinase activity is also observed in COS cells transfected with either PKC-I or PKC-II. The physiological implications of the discovery of three protein-kinase-C-related cDNAs are discussed.

Molecular cloning of a cDNA encoding human antihaemophilic factor.

A complete copy of the mRNA sequences encoding human coagulation factor VIII:C has been cloned and expressed. The DNA sequence predicts a single chain precursor of 2,351 amino acids with a relative molecular mass (Mr) 267,039. The protein has an obvious domain structure, contains sequence repeats and is structurally related to factor V and ceruloplasmin.

Differential, multihormonal regulation of the mouse major urinary protein gene family in the liver.

The hormonal requirements for the regulation of the major urinary protein (MUP) mRNA levels in mouse liver have been examined. Previous experiments have shown that administration of testosterone to female or castrated male mice increases MUP mRNA levels approximately fivefold to normal male levels. We have found that thyroxine and the peptide hormone, growth hormone, each had a pronounced effect on MUP mRNA levels. MUP mRNA was reduced 150-fold in growth-hormone-deficient mutant mice (little). The administration of growth hormone and thyroxine induced MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. testosterone administration. When administered separately to these mice, growth hormone and thyroxine induced with MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. Testicular feminized mice, which lack a functional major testosterone receptor protein, can also be induced to male levels by treatment with both growth hormone and thyroxine. In addition, we present evidence which indicates that growth hormone, thyroxine, and testosterone differentially regulate the levels of distinct MUP mRNA species.

A DNA polymorphism, consistent with gene duplication, correlates with high renin levels in the mouse submaxillary gland.

The renin regulatory locus (Rnr) is a genetic element governing mouse submaxillary gland (SMG) renin levels. A 45,000 dalton polypeptide detectable after in vitro translation of mouse SMG mRNA has been identified by genetic and physical criteria as SMG renin. A cDNA recombinant clone specific for SMG renin has been isolated and used to demonstrate that the previously described genetic regulation of SMG renin levels is manifest at the level of renin mRNA concentration. The renin cDNA clone has also been used in Southern blot analyses to study the organization of homologous DNA sequences in strains carrying different alleles at the Rnr locus. Restriction digest patterns of high renin strains (Rnrs) are characteristically distinct from patterns observed for low renin strains (Rnrb) and are suggestive of a structural gene duplication at the chromosome 1 locus in high renin strains. However, gene dosage cannot account for the increased levels in high renin strains, since SMG renin levels in Rnrs and those in Rnrb may differ up to 100-fold.