Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The tyrosine kinase c-src associates with growth factor receptors, focal contacts and cytoskeletal proteins and is involved in signaling events. The aim of this study was to investigate the role of src in the regulation of mesangial cell (MC) proliferation and differentiation in three-dimensional (3D) culture in collagen gels. Using retroviral gene transfer we have overexpressed wild-type c-src, a kinase-negative c-src mutant (c-src295) and transforming v-src in MC. The MC differentiation in 3D culture was characterized by the formation of a nonproliferating multicellular network in control cells and in cells expressing wild-type c-src. Immunoblotting demonstrated a rapid down-regulation of the alpha-smooth muscle actin expression. The kinase-negative MC (c-src295) failed to differentiate, maintained a significant proliferative rate, and the alpha-smooth muscle actin expression remained stable during 3D culture. MC transformed with v-src showed a high level of tyrosine phosphorylation and proliferation in 3D culture. Analyses of proteins involved in cell cycle regulation demonstrated dephosphorylation of the retinoblastoma protein (Rb) during 3D culture in control and c-src transfected cells. Expression of v-src resulted in sustained Rb phosphorylation. Zymographic analysis of plasminogen activator (u-PA) revealed an inhibition of u-PA secretion in MC transfected with c-src295. These results indicate that c-src exerts regulatory effects on MC proliferation, cytoskeletal organization, matrix proteases and differentiation. Targeted manipulation of the c-src kinase may be useful in modulating MC behavior in vivo.
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PMID:pp60c-src is required for the induction of a quiescent mesangial cell phenotype. 899 24

Erythrina variegata trypsin inhibitor ETIa belongs to the Kunitz inhibitor family, but is unique in its ability to bind and inhibit tissue-type plasminogen activator (tPA). A cDNA clone encoding ETIa was isolated from the lambda gt11 cDNA library using specific antiserum as a probe and characterized by nucleotide sequencing. The cloned ETIa cDNA consists of 762 nucleotides and includes an open reading frame encoding a polypeptide of 198 amino acids. Comparison of the deduced protein sequence and the determined protein sequence indicated the presence of two signal peptides composed of 24 and 2 amino acids at the N- and C-termini, respectively. The cDNA encoding mature ETIa was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3). The recombinant ETIa (rETIa) was expressed in E. coli as inclusion bodies; it was purified to homogeneity by gel filtration on Sephadex G-75. The rETIa exhibited almost the same inhibitory activity toward trypsin and tPA as ETIa. Six mutants, in which the amino acids Arg61, Leu62, Arg63, and Ala65 were replaced by Pro, Phe, Leu/Asp, and Tyr, respectively, were constructed by site-specific mutagenesis and expressed in E. coli. The site-specific mutation of Arg63 to Leu (aR63L) or Asp (aR63D) in ETIa resulted in abolition of the inhibitory activities toward both trypsin and tPA. The mutants aR61P and aL62F showed significantly reduced tPA-inhibitory activity, and furthermore the double mutant aR61P/L62F lacked tPA-inhibitory activity, despite retaining the trypsin-inhibitory activity. In contrast, the mutant aA65Y exhibited tPA-inhibitory activity to the same extent as rETIa. This result suggests that Arg61 and Leu62 in ETIa, in addition to Arg63, may play an important role in the interaction with tPA.
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PMID:The tissue-type plasminogen activator inhibitor ETIa from Erythrina variegata: structural basis for the inhibitory activity by cloning, expression, and mutagenesis of the cDNA encoding ETIa. 913 14

We characterized a new iodinated, high affinity, linear V1a vasopressin antagonist, phenylacetylD-Tyr(Et)Phe-Gln-Asn-Lys-Pro-Arg-Tyr-NH2. The antagonist bound specifically to the V1a vasopressin receptor in crude rat liver membranes with an apparent Kd value of 0.168 nM. This affinity is approximately 1 order of magnitude greater than that of the natural agonist, vasopressin. The inhibitory activity of the antagonist can be demonstrated by its inability to elicit activation and uncoupling of G proteins from the receptor. Thus, after occupancy of receptor sites in rat liver membranes with labeled antagonist and detergent solubilization, the labeled receptor (approximately 60 kDa) was eluted as a stable 400-kDa complex on size-exclusion chromatography. In contrast, when the receptor sites were occupied by the agonist [3H]vasopressin, the receptor eluted as a 60-kDa peak. Coincubation of membranes with iodinated antagonist and an excess of unlabeled vasopressin caused both reduced antagonist binding and a complete shift from the 400-kDa to the 60-kDa peak. The addition of vasopressin to unliganded 400-kDa fractions resulted in a 75% increase in [35S]guanosine-5'-O-(3-thio)triphosphate binding activity, indicating that the 400-kDa fraction contains complexes between the V1a receptor and G proteins. The vasopressin-elicited increase was inhibited by antagonist. Using specific antibodies and immunoadsorption to protein A/Sepharose columns, we found that G protein isotypes G(alpha q/11), G(alpha i3), and G(alpha s), and effector enzymes PLC-beta1, PLC-gamma2 and PLA-2 were associated with the antagonist-labeled receptor in the 400-kDa fraction. Because the 400-kDa complex was found in the absence of ligand, the V1a receptor and the appropriate G proteins and effector enzymes are likely preassociated with each other and do not aggregate after antagonist addition. The association of V1a receptor with the different specific G proteins and effector enzymes is consistent with the multiple actions of vasopressin on liver cells. Antibodies directed against a portion of the carboxyl-terminal domain of the V1a receptor interacted with 60-kDa antagonist-occupied receptor but not with receptor in the 400-kDa complex. These results suggest that the carboxyl-terminal region of the receptor is sterically hindered when coupled to G proteins. The iodinated linear vasopressin antagonist therefore allows stable receptor/G protein complexes and can be an important tool (along with the antisera) for use in the study of factors that control V1a receptor/G protein coupling.
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PMID:A new linear V1A vasopressin antagonist and its use in characterizing receptor/G protein interactions. 920 26

The effects of different flavonoids isolated from the roots of Scutellaria baicalensis Georgi on the production of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) induced by thrombin and thrombin receptor agonist peptide, Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe, have been examined in cultured human umbilical vein endothelial cells (HUVECs). Thrombin and thrombin receptor agonist peptide induced production of both t-PA and PAI-1 and the elevation of intracellular free calcium concentration ([Ca2+]i). Baicalein isolated from Scutellariae Radix dose-dependently inhibited PAI-1 production induced by thrombin and thrombin receptor agonist peptide; its concentrations for 50% inhibition (IC50) were 6.8 and 3.5 microM, respectively. Other flavonoids had no effect. In contrast, flavonoids isolated from Scutellariae Radix had no effect on production of t-PA induced by thrombin and thrombin receptor agonist peptide. Baicalein inhibited the elevation of [Ca2+]i induced by thrombin and thrombin receptor agonist peptide and, at a concentration of 1000 microM, slightly increased t-PA production. These findings suggest that the mechanism by which baicalein inhibits PAI-1 production induced by thrombin and thrombin receptor agonist peptide might be by reduction of [Ca2+]i elevation. The results suggest that baicalein in Scutellariae Radix might be active as a drug in the treatment of arteriosclerosis and thrombosis.
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PMID:Effects of flavonoids isolated from scutellariae radix on the production of tissue-type plasminogen activator and plasminogen activator inhibitor-1 induced by thrombin and thrombin receptor agonist peptide in cultured human umbilical vein endothelial cells. 937 63

Vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) are structurally related growth factors for endothelial cells. VEGF binds to the related receptor tyrosine kinases Flt 1 and KDR/Flk 1 with high affinity, whereas PlGF binds only to Flt 1. Ligand-stimulated KDR is known to transduce signals for cellular activity such as proliferation and migration, whereas weak or no responses have been recorded for Flt 1. We examined VEGF and PlGF for their capacity to stimulate signal transduction in porcine aortic endothelial cells expressing Flt 1 or KDR. VEGF had essentially no effect on Flt 1 expressing cells, but induced DNA synthesis and migration of KDR expressing cells. PIGF on the other hand induced DNA synthesis but not migration of the Flt 1 cells. In agreement, MAP kinase, examined as a marker for DNA synthesis, was activated both by VEGF-stimulation of the KDR cells and by PlGF-stimulation of the Flt 1 cells. In contrast, phospholipase C-gamma (PLC-gamma), was tyrosine phosphorylated only in VEGF stimulated KDR cells, and not in the PlGF-stimulated Flt 1 cells, which is in agreement with a role for PLC-gamma in cellular migration. We furthermore examined induction of protein levels of plasminogen activator (PA), which was evident in the PlGF-stimulated Flt 1 cells, but not in the VEGF-stimulated KDR cells. These data show that Flt 1 is able to mediate an array of biological signals when appropriately stimulated and that the pattern of responses of PlGF-stimulation of Flt 1 is distinct from the pattern of responses to VEGF-stimulation of KDR.
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PMID:Placenta growth factor stimulates MAP kinase and mitogenicity but not phospholipase C-gamma and migration of endothelial cells expressing Flt 1. 946 61

Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate-treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate-treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no effect on binding to UPAR on human ECs. The functional integrity of the growth factor domain in the non-receptor binding Asn22Tyr mutant is suggested by the fact that binding of this mutant to a murine UPAR can be restored after additional mutations in the growth factor domain, Asn27,His29,Trp30 to Arg27,Arg29,Arg30. We conclude that Asn22 and Asn27,His29,Trp30 in human UPA are key determinants in the species-specific binding of the enzyme to its receptor and that changing Asn22 into Tyr results in a UPA mutant with strongly reduced binding to UPAR.
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PMID:Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding. 959 26

Staphylokinase (Sak) forms an inactive 1:1 stoichiometric complex with plasminogen which requires both conversion of plasminogen to plasmin and hydrolysis of the Lys10-Lys11 peptide bond of Sak to become a potent plasminogen activator (Schlott, B., Guhrs, K.-H., Hartmann, M., Rocker, A., and Collen, D. (1997) J. Biol. Chem. 272, 6067-6072). Exposure of a positively charged NH2-terminal amino acid after hydrolysis of Sak is a major determinant of the plasminogen-activating potential, but in itself is neither necessary nor sufficient. Here, the structural motifs of the NH2-terminal region Lys11-Gly-Asp-Asp-Ala-Ser16-Tyr-Phe-Glu of processed Sak, required for plasminogen activating potential, were studied by deletion and substitution mutagenesis. Expression in Escherichia coli of variants with deletion of 11, 14, 15, or 16 NH2-terminal amino acids yielded correctly processed but inactive molecules. Expression of their homologues with the NH2-terminal amino acid substituted with Lys-generated derivatives from which the NH2-terminal initiation Met was no longer removed, yielding inactive (</= 10%) Sak42DDeltaN11(M),G12K, active (>50%) Sak42DDeltaN14(M), A15K and Sak42DDeltaN15(M),S16K, and inactive Sak42DDeltaN16(M),Y17K. Lys variants without NH2-terminal Met, generated from fusion proteins in which a His6 tag and a factor Xa recognition sequence were linked to the NH2 terminus of the Sak variants, were indistinguishable from their NH2-terminal Met-containing counterparts. All variants studied had intact affinities for plasminogen as measured by biospecific interaction analysis. The activity of Sak42DDeltaN11(M),G12K could be restored by additional substitution of both Asp13 and Asp14 with Asn, yielding active Sak42DDeltaN11(M),G12K, D13N, D14N, whereas substitution in Sak42DDeltaN16(M),Y17K of Phe18 and Glu19 with Asn yielded inactive Sak42DDeltaN16(M),Y17K,F18N,E19N. These data, in combination with the recent finding that the 20 NH2-terminal amino acids of Sak lack secondary structure, suggest that the NH2-terminal region of Sak is not required for binding to plasmin/plasminogen, but that a positively charged amino acid in the ultimate or penultimate NH2-terminal position corresponding to amino acids 11-16 of this flexible region participates in the reconfiguration of the active site of the plasmin molecule to endow it with plasminogen-activating potential.
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PMID:NH2-terminal structural motifs in staphylokinase required for plasminogen activation. 971 54

Thrombin can regulate the-fibrinolytic system by increasing the endothelial production of both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1). The thrombin receptor transducts signals through the GTP-binding protein system, the classical pathway being the Galpha q-protein. The purpose of the present study was to examine the roles of Galpha i-protein and tyrosine kinases in the thrombin signal transduction of t-PA and PAI-1 production from human adult vein endothelial cells (HAVEC). t-PA and PAI-1 antigen were analysed in conditioned medium from cultured HAVEC after 16 h incubation. Data are expressed as percentages of basal release (100%), means +/- 95% confidence intervals. Thrombin increased t-PA and PAI-1 production (234 +/- 42% and 211 +/- 42%, respectively). Pertussis toxin (PTX) (inhibiting Galpha i-pathway) reduced basal PAI-1 (66 +/- 8%), but had only a weak influence on basal t-PA production. Pertussis toxin and genistein (inhibiting tyrosine kinase) significantly reduced the thrombin induction of both t-PA and PAI-1 (PTX: 142 +/- 23% and 146 +/- 19%, respectively, genistein: 156 +/- 42% and 76 +/- 24%, respectively). The present study demonstrated that thrombin can increase the production of t-PA and PAI-1 by transducting signals through the Galpha i and tyrosine kinase pathway, in addition to the Galpha q/protein kinase C pathway as has been found previously.
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PMID:Thrombin signal transduction of the fibrinolytic system in human adult venous endothelium in vitro. 974 23

Aberrant expression or activity of the epidermal growth factor (EGF) receptor family of tyrosine kinases has been associated with tumor progression and an invasive phenotype. In this study, we utilized 4 ovarian cancer cell lines, OVCA 432, DOV 13, OVEA6 and OVCA 429, to determine the effects of EGF on the regulation of proteolytic enzymes and their inhibitors, cellular migration and in vitro invasion. Induction of urinary-type plasminogen activator (u-PA) activity and tissue inhibitor of matrix metalloproteinase (TIMP)-1 was observed in all 4 cell lines. OVCA 432 cells showed strong PAI-1 induction; however, the other 3 lines displayed substantial baseline PAI-1 expression that was not induced by EGF. EGF-dependent stimulation of migration and induction of matrix metalloproteinase (MMP)-9 (gelatinase B) was observed in OVEA6 and OVCA 429 cells only. Upon EGF receptor activation, DOV 13, OVEA6 and OVCA 429 cells were induced to invade through an artificial basement membrane (Matrigel); however, no invasion was detected in OVCA 432 cells. Cell lines displaying induction of migration and MMP-9 (OVEA6 and OVCA 429) demonstrated robust EGF-induced invasion (5- to 20-fold), and cell invasion was substantially reduced in the presence of anti-catalytic MMP-9 antibody. Addition of anti-catalytic u-PA antibody inhibited the modest (<2-fold) EGF-induced invasion in a cell line that did not express MMP-9 (DOV 13) and in OVEA6 cells that displayed the highest baseline u-PA activity. Together, our findings indicate that multiple proteinases are important in ovarian cell invasion and implicate EGF induction of MMP-9 and migration as key components of more aggressive ligand-induced invasion.
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PMID:Proteinase requirements of epidermal growth factor-induced ovarian cancer cell invasion. 976 68

A membrane proteinase from Pseudomonas aeruginosa, called insulin-cleaving membrane proteinase (ICMP), was located in the outer membrane leaflet of the cell envelope. The enzyme is expressed early in the logarithmic phase parallel to the bacterial growth during growth on peptide rich media. It is located with its active center facing to the outermost side of the cell, because its whole activity could be measured in intact cells. The very labile membrane proteinase was solubilized by non-ionic detergents (Nonidet P-40, Triton X-100) and purified in its amphiphilic form to apparent homogeneity in SDS-PAGE by copper chelate chromatography and two subsequent chromatographic steps on Red-Sepharose CL-4B (yield 58.3%, purification factor 776.3). It consisted of a single polypeptide chain with a molecular mass of 44.6 kDa, determined by mass spectrometry. ICMP was characterized to be a metalloprotease with pH-optimum in the neutral range. The ICMP readily hydrolyzed Glu(13)-Ala(14) and Tyr(16)-Leu(17) bonds in the insulin B-chain. Phe(25)-Tyr(26) and His(10)-Leu(11) were secondary cleavage sites suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at P'(1)-position. The ICMP differed from elastase, alkaline protease and LasA in its cleavage specificity, inhibition behavior and was immunologically diverse from elastase. The amino acid sequence of internal peptides showed no homologies with the known proteinases. This outer membrane proteinase was capable of specific cleavage of alpha and beta fibrinogen chains. Among the p-nitroanilide substrates tested, substrates of plasminogen activator, complement convertase and kallikrein with arginine residues in the P(1)-subsite were the substrates best accepted, but they were only cleaved at a very low rate.
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PMID:Characterization and purification of an outer membrane metalloproteinase from Pseudomonas aeruginosa with fibrinogenolytic activity. 1045 58


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