Characterization of lipocortin I and an immunologically unrelated 33-kDa protein as epidermal growth factor receptor/kinase substrates and phospholipase A2 inhibitors

Reversible calcium-dependent association with a particulate fraction from human placenta was used as the first step in the purification of substrates for the epidermal growth factor-stimulated protein kinase. A protein with apparent Mr of 35,000 was purified to homogeneity, and the sequence was determined for approximately one-fourth of the protein. These residues could be aligned exactly with the previously published sequence of lipocortin I derived from the cDNA from a human lymphoma. Two other proteins that appear to be formed by proteolytic removal of 12 or 26 of the amino acids from the NH2 terminus of the protein also were isolated. Placental lipocortin I was phosphorylated in Tyr-21 in an epidermal growth factor-dependent manner by the kinase activity in a particulate fraction from A431 cells; half-maximal phosphorylation occurred at 50 nM lipocortin I. Lipocortin I phosphorylated on Tyr-21 was approximately 10-fold more sensitive to tryptic cleavage at Lys-26 than was the native protein. Placental lipocortin I and its two truncated forms were potent inhibitors of pancreatic phospholipase A2 activity. Another 33-kDa protein that was not related immunologically to lipocortin I or lipocortin II (calpactin I) also was purified from the EGTA extract of placenta. The unidentified protein inhibited phospholipase A2 but was not a substrate for the epidermal growth factor-stimulated kinase. The mechanism by which these proteins inhibit phospholipase A2 activity was investigated. Attempts to detect direct interaction between these proteins and the enzyme were unsuccessful. However, both the unidentified protein, lipocortin I, and 32P-labeled lipocortin I bound in a Ca2+-dependent manner to the [3H]oleic acid-labeled Escherichia coli membranes used as substrate in the phospholipase A2 assay. Heparin, which is known to block lipocortin I inhibition of phospholipase A2, also blocked binding of lipocortin I to E. coli membranes. The results of these and other experiments raise the possibility that placental lipocortin I inhibits phospholipase A2 activity in this assay by coating the phospholipid and thereby blocking interaction of enzyme and substrate.     

Persistent Ca2(+)-induced activation of erythrocyte membrane Ca2(+)-ATPase unrelated to calpain proteolysis

Preincubation of human erythrocyte membranes with calcium in the submillimolar to millimolar concentration range resulted in an increase of the Ca2+ affinity and apparent maximum velocity of the Ca2(+)-stimulated Mg2(+)-dependent ATPase (Ca2(+)-ATPase). The activation was persistent, as it was not reversed when the Ca2(+)-preincubated membranes were washed with ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid-containing buffers. Magnesium was not required for the activation, whereas greater than 2 mM Mg2+ partially antagonized the activation by Ca2+. In some membrane preparations ATP was required in addition to Ca2+ for activation of the Ca2(+)-ATPase, but nonhydrolyzable analogs of ATP had the same effect. Calmodulin prevented the activation by Ca2+ over the same concentration range in which it interacts with the Ca2(+)-ATPase. Taken together the results obtained provided strong evidence that the Ca2+ activation of the enzyme was not due to proteolytic cleavage by endogenous calpain. Thus, activation by Ca2+ was not blocked by leupeptin (100-200 microM), did not require dithiothreitol, and occurred at Ca2+ concentrations greater than those required for activation of calpain I. Furthermore, Ca2+ activation did not result in change in the mobility the native 136-kDa species of the Ca2(+)-ATPase on SDS-gel electrophoresis. Moreover, solubilization of the Ca2(+)-pretreated membranes with Triton X-100 reversed the Ca2+ activation of the Ca2(+)-ATPase. On the other hand, Ca2(+)-pretreatment of the membranes modified the susceptibility of the Ca2(+)-ATPase to both cleavage and activation by exogenously added calpain I. We conclude that pretreatment of Ca2(+)-ATPase in erythrocyte membranes with millimolar Ca2+ activates the enzyme by inducing a persistent conformational change of the enzyme which is, however, subsequently reversed by detergent solubilization.     

Novel missense mutations in red/green opsin genes in congenital color-vision deficiencies

Lipocortin I-S100 (calcyclin) heterotetramer exhibited ATPase activity in the presence of dsDNA but not ssDNA. To demonstrate its helicase activity, an 80-mer polynucleotide complementary to the replication origin of M13mp18 was synthesized, and the oligonucleotide, (dC)(20), was ligated to either its 5'- or 3'- end for binding to lipocortin. Lipocortin I heterotetramer displaced chains of the partially Y-shaped duplexes with a dC-tail at either the 5'- or 3'- end. The chain displacement required ATP and Mg(2+). Nonhydrolyzable ATP analogues were not effective. Lipocortin I heterotetramer also catalyzed annealing of the polynucleotides to M13mp18. Ca(2+) and phospholipids but not ATP and Mg(2+) were essential for this reaction. Since the chain displacing and annealing reactions were inhibited by monospecific anti-lipocortin I or anti-S100 antibodies, the present observations suggest that the lipocortin I heterotetramer regulates unwinding and annealing of DNA by Mg(2+) (plus ATP) and Ca(2+) (and phospholipids), respectively.     

Lipocortin (Annexin) I heterotetramer binds to purine RNA and pyrimidine DNA.

Lipocortin I-like protein with a molecular weight of 94,000 Da as judged by Western analysis was found to bind to ssDNA rather than to dsDNA in a Ca(2+)-dependent manner. This protein was also bound to [(32)P]poly(rA) and [(32)P]poly(rG) as measured by EMSA. Poly(rG), poly(rA), poly(dC), and poly(dT) were competitive against binding of either [(32)P]poly(rA) or [(32)P]poly(rG), while poly(rC), poly(rU), and poly(dA) were less effective binding competitors. The binding of this protein to poly(rA) or poly(rG) was inhibited by immunoprecipitable anti-lipocortin I (calpactin II) and anti-S100 protein antibodies, but not by an anti-Ig antibody. Phospholipids such as phosphatidylserine and phosphatidylinositol enhanced the binding of lipocortin I to poly(rA). Taken together, our present observations suggest that the lipocortin I-S100 protein heterotetramer binds to either purine RNAs or pyrimidine ssDNAs in a Ca(2+)- and phospholipid-dependent manner.     

Purification of annexin I and annexin II from human placental membranes by high-performance liquid chromatography.

Annexin I and annexin II were extracted from human placental membranes with ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) and purified by high-performance liquid chromatography by measuring their ability to inhibit phospholipase A2 activity in vitro. Neither protein was capable of binding to a DEAE-5PW HPLC column at neutral pH; however, they were resolved through binding to a Mono S column and passage through size-exclusion HPLC columns. Annexin I and its covalently linked dimer (36 and 66 kDa, respectively, by sodium dodecyl sulfate (SDS)-gel electrophoresis) reacted in one-dimensional immunoblots with monoclonal antibodies to annexin I and calpactin II, and with monoclonal and polyclonal antibodies to lipocortin I, confirming that annexin I, calpactin II, and lipocortin I are the same or closely related proteins. Milligram amounts of monomeric annexin I containing negligible amounts of the cross-linked dimeric annexin I were selectively isolated from placental membranes by using buffers containing the sulfhydryl reagent iodoacetic acid. Milligram amounts of cross-linked annexin I were selectively isolated when placental membranes were initially treated with buffers that did not contain iodoacetic acid and then extracted with Triton X-100, suggesting that sulfhydryl-dependent transglutaminase activity contributes to the selective isolation of this protein. A third phospholipase A2-inhibitory protein (35 kDa by SDS-gel electrophoresis) that reacted in immunoblots with monoclonal antibodies to calpactin I and annexin II, indicating their similar identity, was isolated. The procedure employed allows the rapid purification of annexins I and II in milligram amounts from placental membranes within 2 days.     

Purification and characterization of phospholipase A2 inhibitory proteins from pig thyroid gland

A 32 kDa phospholipase A2 inhibitory protein was isolated from pig thyroid gland after calcium precipitation and fast protein liquid anion-exchange chromatography. SDS-polyacrylamide gel electrophoresis revealed the purity of the protein. The protein activity was assessed by the inhibition of pancreatic phospholipase A2 on [3H]oleic acid-labelled Escherichia coli membranes as substrate and on the prostaglandin E2 production of cultured thyroid cells. The amino acid composition and the isoelectric point were quite similar to those of endonexin previously described in other tissues or cells. The cross-reactivity of a polyclonal antibody against a 32 kDa lipocortin from human peripheral blood mononuclear cells with our thyroidal 32 kDa protein confirmed its lipocortin nature. Before the purification by fast protein liquid chromatography, the Ca2+ pellet contained lipocortin I (35 kDa and its core protein 33 kDa) identified by its cross-reactivity with a polyclonal antibody.     

Cross-linking of lipocortin I and enhancement of its Ca2+ sensitivity by tissue transglutaminase

The stimulation of human epidermoid carcinoma A431 cells with the calcium ionophore A23187 resulted in the formation of high-molecular-weight lipocortins I, having apparent molecular weights of 75 kDa and 160 kDa as detected with specific anti-lipocortin I antibody. These immunoreactive proteins were identified to be covalently cross-linked multimers of lipocortin I, since essentially the same cross-linked multimers were observed when purified lipocortin I was incubated with tissue transglutaminase (TGase) in vitro. Classical amine substrates for TGase, such as dansylcadaverine and putrescine, were also incorporated stoichiometrically into lipocortin I. Cross-linking or amine incorporation was not observed with lipocortin II. Des 1-26 lipocortin I did not serve as a substrate for TGase, indicating that the N-terminal region of lipocortin I plays an important role in the formation of lipocortin I multimers. The cross-linking of lipocortin I by TGase resulted in a remarkable enhancement of calcium sensitivity for phospholipid binding; i.e., the free calcium concentration required for the cross-linked lipocortin I to attain 50% maximal binding to phosphatidylserine vesicles was as little as 3 microM, while that required for intact monomeric lipocortin I was 20 microM.     

Effect of dexamethasone on prostaglandin synthesis and on lipocortin status in human endothelial cells. Inhibition of prostaglandin I2 synthesis occurring without alteration of arachidonic acid liberation and of lipocortin synthesis

Glucocorticoids have been shown to decrease prostaglandin I2 synthesis in human endothelial cells, suggesting the possible involvement of lipocortin in the inhibition of arachidonic acid liberation achieved by phospholipase A2 (De Caterina, R., and Weksler, B. B. (1986) Thromb. Haemostasis 55, 369-374). To test this hypothesis, human endothelial cells labeled with [14C]arachidonic acid were stimulated with thrombin (2 units/ml, 10 min), resulting in the secretion of free arachidonic acid together with various 14C-labeled metabolites, mainly 6-keto-prostaglandin F1 alpha, the stable derivative of prostaglandin I2. Under conditions where prior incubation of cells with dexamethasone reduced by 51% 6-keto-prostaglandin F1 alpha production, phospholipid hydrolysis induced by thrombin remained unaffected. Using three rabbit polyclonal antibodies directed against endonexin I, lipocortin I, and lipocortin II, evidence was obtained for the presence in human endothelial cells of equivalent amounts of lipocortin I and an immunologically unrelated 33-kDa protein, together with lower quantities of 67-kDa calelectrin/calcimedin. These Ca2+- and phospholipid-binding proteins were selectively extracted with [ethylene-bis(oxyethylene-nitrilo)]tetraacetic acid (EGTA) from cell membranes precipitated in the presence of Ca2+, and they displayed an inhibitory activity against pig pancreas phospholipase A2. However, the amounts of the three proteins were not changed by cell treatment with 2.5 microM dexamethasone, as detected upon polyacrylamide gel electrophoresis by silver staining, immunoblotting, or autoradiography following [35S]methionine in vivo labeling. Since the antiphospholipase A2 activity of EGTA extracts was hardly modified, it was concluded that an increased synthesis of lipocortin cannot account for the inhibition of prostaglandin synthesis brought about by dexamethasone, suggesting other biological functions for these proteins.     

Calcium-induced intracellular cross-linking of lipocortin I by tissue transglutaminase in A431 cells. Augmentation by membrane phospholipids.

Covalently cross-linked multimers of lipocortin I are shown to be present in human epidermoid carcinoma A431 cells treated with epidermal growth factor or the calcium ionophore A23187. This intracellular cross-linking of lipocortin I is suggested to be mediated by the action of tissue transglutaminase, a Ca2(+)-dependent protein cross-linking enzyme. Cross-linking of lipocortin I competes with proteolytic digestion of the protein, and pretreatment of the cells with inhibitors for calpain (Ca2(+)-dependent intracellular protease) markedly enhanced the cross-linking of lipocortin I. Cross-linked lipocortin I is shown to be present in the soluble fraction of A431 cells as well as in the particulate fraction; a 34-kDa fragment of lipocortin I was solubilized successfully by plasmin digestion of the latter fraction. Immunofluorescence microscopy using specific antilipocortin-I antibody showed that cross-linked lipocortin I forms an envelope-like structure, which is not extracted with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) or Triton X-100. In vitro incubation of purified lipocortin I with tissue transglutaminase resulted in the formation of covalently cross-linked lipocortin I dimer, tetramer, and so on. Amine incorporation and cross-linking studies using lipocortin I and its N-terminal truncated derivatives indicated that the cross-linking site is localized within the plasmin-susceptible N-terminal 29 amino acids of lipocortin I. The cross-linking of lipocortin I is shown to be accelerated more than 10 times by the addition of phosphatidylserine vesicles, on which lipocortin I molecules are most likely aligned in a conformation suitable for cross-linking. Collectively, these findings suggest that an increase of intracellular calcium concentration results in the attachment of lipocortin I onto the plasma membrane phospholipids through the C-terminal domain of the molecule where the membrane-bound lipocortin I is cross-linked by the action of tissue transglutaminase through the N-terminal domain.     

Common domain structure of Ca2+ and lipid-binding proteins

The phospholipase A2 inhibitor, lipocortin, shares common sequences with three abundant Ca2+-regulated membrane binding proteins of unknown function which are present in many cell and tissue types. A two-domain model for the structure of lipocortin is described and it is suggested that the new Ca2+-regulated proteins each contain at least one lipocortin domain. The structural and biochemical properties of each protein indicate that they all directly interact with phospholipids. Potential sites of interaction with the lipocortin domain are identified on the basis of homology with phospholipid transfer proteins and phospholipase A2.     

Purification and characterization of a lipocortin-like 33 kDa protein from guinea pig neutrophils

A lipocortin-like, phospholipase A2 inhibitory 33 kDa protein was purified from guinea pig neutrophils. From amino acid composition and sequence data, this protein was found to have a high degree of homology to human lipocortin I. This protein inhibited porcine pancreatic phospholipase A2 activity in the presence of [3H]oleic acid-labeled Escherichia coli membranes as substrate. Maximal inhibition amounted to 65% whereas 50% inhibition occurred at 83.5 nM. This protein showed F-actin-binding ability in a Ca2+-dependent manner.     

The binding of monoclonal and polyclonal antibodies to the Ca2(+)-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules.

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.     

Isolation of two 67 kDa calcium-binding proteins from pig lung differing in affinity for phospholipids and in anti-phospholipase A2 activity

Two 67 kDa proteins adsorbed to membranes in the presence of Ca2+ have been purified to homogeneity from pig lung using conventional procedures, followed by calcium-dependent affinity chromatography on polyacrylamide-immobilized phosphatidylserine. The two proteins were, respectively, excluded (67E) and retained (67R) on the column in the presence of Ca2+. On the basis of amino acid composition and isoelectric point, 67R was identified as 67 kDa calelectrin/calcimedin, whereas 67E could be differentiated from albumin, calregulin, 67 kDa fragment of protein kinase C and surfactant-associated proteins. Only 67R was slightly phosphorylated by protein kinase C, reacted with an antibody raised against 32.5 kDa endonexin and inhibited pig pancreas phospholipase A2 in a way similar to that of lipocortin or endonexin. These data bring further support to the view that inhibition of phospholipase A2 by lipocortin or other related proteins involves interaction with the lipid/water interface. They also provide evidence for a new kind of Ca2+-binding protein (67E), whose role still remains to be determined.     

Characterizations of two distinct Ca2+-dependent phospholipid-binding proteins of 68-kDa isolated from human placenta

Two distinct 68-kDa proteins, named 68K-I (pI 6.4) and 68K-II (pI 5.6), were solubilized from human placenta by treatment with 5 mM EGTA. On DE52 cellulose column chromatography at pH 7.4, 68K-I in the EGTA eluate was recovered in the unadsorbed fractions, whereas 68K-II was retained on the column and eluted with 0.2 M NaCl. The 68K-I protein was obtained in more than 95% purity by further hydroxylapatite and cation exchange chromatographies, while the 68K-II protein was purified further by gel filtration and hydroxylapatite chromatographies. Partial amino acid sequence data showed that 68K-I protein was a novel protein which shared the same sequences as lipocortin I and that 68K-II was the same as human p68/67-kDa calelectrin (Crompton, M. R., Owens, R. J., Totty, N. F., Moss, S. E., Waterfield, M.D., and Crumpton, M. J. (1988) EMBO J. 7, 21-27; Südhof, T. C., Slaughter, C. A., Leznicki, I., Barjon, P., and Reynolds, G. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 664-668). The two proteins bound to acidic phospholipids, phosphatidylserine, and/or phosphatidylinositol, but not to phosphatidylcholine, in the presence of micromolar levels of Ca2+. 68K-I bound to phosphatidylinositol preferentially to phosphatidylserine, whereas 68K-II bound only to phosphatidylserine. Both 68K-I and 68K-II inhibited phospholipase A2 activity, and the inhibition by 68K-II was detectable only in the presence of 100 mM KCl. 68K-I, but not 68K-II, was found to bind to F-actin in a Ca2+-dependent (1 mM) manner. Moreover 68K-I, but not 68K-II, was phosphorylated in vitro at tyrosine residues by fps kinase and by epidermal growth factor receptor/kinase, the latter reaction being dependent on Ca2+ and epidermal growth factor. Western blot analysis with affinity purified anti-68K-I and anti-68K-II antibodies showed that 68K-I was located in only certain tissues, especially human placenta, whereas 68K-II was present in many human and rat tissues.     

Monoclonal antibodies recognizing epitopes of calretinins: dependence on Ca2+-binding status and differences in antigen accessibility in colon cancer cells

Monoclonal antibodies are very helpful tools to investigate the localization and sometimes even the function of specific proteins in cells and tissues. By generating monoclonal antibodies against calretinin-22k (CR-22k), a C-terminally truncated isoform of calretinin (CR) as a result of alternative splicing of the CR mRNA, we envisaged that screening multiple monoclonal antibodies would allow the identification of CR-22k as well as CR. Both proteins share the first 178 amino acids, but have different C-termini. All three antibodies 10C10, 6B3 and 2H4 recognize recombinant CR-22k and the specificity to also recognize CR was demonstrated in brain extracts of different species and human tumour cells, which express CR. All monoclonal antibodies did not crossreact with the closely related protein calbindin D-28k. Antibody binding was depending on the Ca2+-binding status of both forms of calretinin. Generally, the Ca2+-bound form was better recognized than the Ca2+-free form. Carboxy- and amino-terminally truncated CR proteins were expressed in E. coli in order to characterize the epitopes recognized by the three antibodies. Additionally, tryptic and cyanogen bromide fragments were produced to further narrow down the sequences recognized by the three antibodies. 10C10 recognizes an epitope consisting of the linker region between EF-hand domains I and II and the N-terminal part of EF-hand II, while the others (6B3, 2H4) bind to a region including the linker between EF-hand domains III and IV. These antibodies are valuable tools to further investigate the distribution and eventually the specific function of these two proteins in the nervous tissue and under pathological conditions, e.g. in colon tumours and mesotheliomas.     

Reconstitution of the skeletal sarcoplasmic reticulum Ca2(+)-pump: influence of negatively charged phospholipids.

The Ca2(+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in the presence of phosphatidyl choline using the freeze-thaw sonication technique. The effect of incorporation of negatively charged phospholipids, phosphatidylserine and phosphatidylinositol phosphate, into the phosphatidylcholine proteoliposomes was investigated. Various ratios of phosphatidylserine or phosphatidylinositol phosphate to phosphatidylcholine were used, while the total amount of phospholipid in the reconstituted vesicles was kept constant. Enrichment of phosphatidylcholine proteoliposomes by phosphatidylserine or phosphatidylinositol phosphate was associated with activation of Ca2(+)-uptake and Ca2(+)-ATPase activities. The highest activation was obtained at a 50:50 molar ratio of phosphatidylserine:phosphatidylcholine and at a 10:90 molar ratio of phosphatidylinositol phosphate:phosphatidylcholine. The initial rates of Ca2(+)-uptake obtained at 1 microM Ca2+ were 2.6 +/- 0.1 mumol/min per mg of phosphatidylserine:phosphatidylcholine proteoliposomes and 1.5 +/- 0.1 mumol/min per mg of phosphatidylinositol phosphate:phosphatidylcholine proteoliposomes, compared to 0.9 +/- 0.05 mumol/min per mg of phosphatidylcholine proteoliposomes. These findings suggest that negatively charged phospholipids may be involved in the activation of the reconstituted skeletal muscle sarcoplasmic reticulum Ca2(+)-pump.     

Production of lipocortin-like proteins by cultured human tracheal submucosal gland cells

Evidence is obtained for the presence of lipocortin-like proteins in human tracheal gland cells in culture. Using polyclonal antibodies to lipocortin I, indirect immunofluorescence studies demonstrate that lipocortin I is mainly confined to the tracheal gland cell surface. From cell membranes, four Ca2(+)-dependent proteins (35, 40, 45 and 67 kDa) were identified as lipocortin related proteins by using immunoblotting and fluorography following [35S]methionine metabolic labeling experiments. A strong immunoreactivity for the 35 kDa protein was observed. In addition, lipocortin-like proteins with apparent Mr33, 35, 37 and 67 kDa, respectively, were released in the apical culture medium by tracheal gland cells cultured on microporous membrane of a double chamber culture system.     

Tumor promoter-dependent phosphorylation of a Triton X-100 extractable form of lipocortin I in T51B rat liver cells

The phosphorylation of lipocortin (a substrate of EGF-receptor kinase, and a putative phospholipase A2 inhibitor) was examined in T51B cells. By using Western blot procedures and antisera specific to lipocortin I, we found that most immunoreactive lipocortin I was located in the cytosol (lipocortin(cvt] of cells extracted in Ca2+-free buffers These cells however had another pool of immunoreactive lipocortin I located in the particulate fraction that was Triton X-100 extractable (lipocortin(mem]. Increasing Ca2+ concentrations in the extraction buffer resulted in more lipocortin(mem) recovered. In vitro phosphorylation of endogenous proteins demonstrated that lipocortin I became phosphorylated in a Ca2+ and phosphatidylserine-dependent manner, suggesting an involvement of protein kinase C. Treatment of cells with 100 ng/ml 12-0-tetradecanoylphorbol-13-acetate (TPA) but not with 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) resulted in the in vitro phosphorylation of lipocortin(mem) by protein kinase C. TPA also increased the phosphorylation of lipocortin(mem) in [32P]phosphate-labeled cells.     

Ca2(+)-binding site of carp parvalbumin recognized by monoclonal antibody.

Monoclonal antibody 235 which was used for immunohistochemical staining of parvalbumin in tissue sections partially protects Lys-54 of carp muscle parvalbumin from reaction with acetic anhydride in the parvalbumin-antibody complex. Lys-54 is located in the CD-loop of parvalbumin and is flanked by the Ca2(+)-ligands Asp-53 and Ser-55 of the Ca2(+)-site I. Another monoclonal antibody against carp parvalbumin, mca 239, partially protects lysine residues 27, 32, 87 and 107, indicating that this antibody is directed against a discontinuous epitope distant from the two Ca2(+)-binding sites of parvalbumin.     

Enhancement of calcium sensitivity of lipocortin I in phospholipid binding induced by limited proteolysis and phosphorylation at the amino terminus as analyzed by phospholipid affinity column chromatography

A phospholipid column was prepared by coating siliconized porous glass beads with phospholipids. The analysis of the Ca2+ requirement of lipocortin I and its derivatives in the binding to phospholipids was carried out with this column. The Ca2+ concentration required for 50% binding to the phospholipid column at room temperature was about 30 microM for lipocortin I, while that was reduced to 15 microM when lipocortin I was phosphorylated by the epidermal growth factor receptor/kinase, and a further reduction in the Ca2+ requirement was observed with proteolytic cleavage at the N-terminal region. Cathepsin D and calpain I (low calcium-requiring form of calcium-activated neutral protease) rapidly cleaved human placental lipocortin I at Trp-12 and Lys-26, respectively. These N-terminal-truncated proteins required only 5 microM Ca2+ for 50% binding to the phospholipid column. This enhancement of Ca2+ sensitivity by limited proteolysis was also observed for porcine lung lipocortin I. Essentially the same results were obtained when the Ca2+ sensitivities of the modified lipocortins I were analyzed using dispersed phospholipid vesicles instead of the phospholipid affinity column. Equilibrium dialysis indicated that the release of the N-terminal region markedly increased the affinity of lipocortin I for Ca2+ in the presence of phosphatidylserine, without any appreciable change of the number of Ca2+-binding sites. Limited proteolysis by endogenous proteases such as calpain may be an important regulatory mechanism for the Ca2+ sensitivity of lipocortin I in phospholipid binding.