Biomedical subjects

L Covic

Publications and source records attributed to L Covic.

8 recordsLinked to original sources

PubMed2000

Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets.

Thrombin activates platelets in an ordered sequence of events that includes shape change, increase in cytoplasmic Ca(2+), activation of the alphaIIbbeta3 integrin, granule secretion, aggregation, and formation of a stable hemostatic plug. Activation of this process has also been implicated in the pathogenesis of atherosclerosis, stroke, and thrombosis. There are two identified thrombin-activated receptors on the surface of human platelets. PAR1 is a high-affinity thrombin receptor, and PAR4 is a low apparent affinity thrombin receptor of uncertain function. The goal of these studies is to determine the kinetics of thrombin activation of PAR1 and PAR4 and to relate the individual inputs from each receptor to platelet Ca(2+) signaling, secondary autocrine stimulation, and aggregation. Using a combination of PAR-specific peptide ligands and anti-PAR1 reagents, we separated the biphasic thrombin Ca(2+) response of platelets into two discrete components-a rapid spike response caused by PAR1, followed by a slower prolonged response from PAR4. Despite having a 20-70-fold slower rate of activation, PAR4 produces the majority of the integrated Ca(2+) signal that is sustained by the continuous presence of catalytically active thrombin. Surprisingly, PAR4 activation is much more effective than PAR1 activation in mounting secondary autocrine Ca(2+) signals from secreted ADP. The strong ADP response due to activated PAR4, however, requires prior activation of PAR1 as would normally occur during treatment of platelets with thrombin. Thus, the late signal generated by activated PAR4 is not redundant with the early signal from PAR1 and instead serves to greatly extend the high intracellular Ca(2+) levels that support the late phase of the platelet aggregation process.

Adenosine Diphosphate↗

PubMed2000

PAR1 thrombin receptor-G protein interactions. Separation of binding and coupling determinants in the galpha subunit.

Signal transfer between the protease-activated PAR1 thrombin receptor and membrane-associated heterotrimeric G proteins is mediated by protein-protein interactions. We constructed a yeast signaling system that resolves domain-specific functions of binding from coupling in the Galpha subunit. The endogenous yeast Galpha subunit, Gpa1, does not bind to PAR1 and served as a null structural template. N- and C-terminal portions of mammalian G(i2) and G(16) were substituted back into the Gpa1 template and gain-of-function assessed. The C-terminal third of G(16), but not of G(i2), provides sufficient interactions for coupling to occur with PAR1. The N-terminal two-thirds of G(i2) also contains sufficient determinants to bind and couple to PAR1 and overcome the otherwise negative or missing interactions supplied by the C-terminal third of Gpa1. Replacement of the N-terminal alpha-helix of G(i2), residues 1-34, with those of Gpa1 abolishes coupling but not binding to PAR1 or to betagamma subunits. These data support a model that the N-terminal alphaN helix of the Galpha subunit is physically interposed between PAR1 and the Gbeta subunit and directly assists in transferring the signal between agonist-activated receptor and G protein.

Amino Acid Sequence↗

PubMed1999

Functional characterization of ARAKIN (ATMEKK1): a possible mediator in an osmotic stress response pathway in higher plants.

The Arabidopsis thaliana ARAKIN (ATMEKK1) gene shows strong homology to members of the (MAP) mitogen-activated protein kinase family, and was previously shown to functionally complement a mating defect in Saccharomyces cerevisiae at the level of the MEKK kinase ste11. The yeast STE11 is an integral component of two MAP kinase cascades: the mating pheromone pathway and the HOG (high osmolarity glycerol response) pathway. The HOG signal transduction pathway is activated by osmotic stress and causes increased glycerol synthesis. Here, we first demonstrate that ATMEKK1 encodes a protein with kinase activity, examine its properties in yeast MAP kinase cascades, then examine its expression under stress in A. thaliana. Yeast cells expressing the A. thaliana ATMEKK1 survive and grow under high salt (NaCl) stress, conditions that kill wild-type cells. Enhanced glycerol production, observed in non-stressed cells expressing ATMEKK1 is the probable cause of yeast survival. Downstream components of the HOG response pathway, HOG1 and PBS2, are required for ATMEKK1-mediated yeast survival. Because ATMEKK1 functionally complements the sho1/ssk2/ssk22 triple mutant, it appears to function at the level of the MEKK kinase step of the HOG response pathway. In A. thaliana, ATMEKK1 expression is rapidly (within 5 min) induced by osmotic (NaCl) stress. This is the same time frame for osmoticum-induced effects on the electrical properties of A. thaliana cells, both an immediate response and adaptation. Therefore, we propose that the A. thaliana ATMEKK1 may be a part of the signal transduction pathway involved in osmotic stress.

Arabidopsis↗

PubMed1999

Plasmin desensitization of the PAR1 thrombin receptor: kinetics, sites of truncation, and implications for thrombolytic therapy.

It has been hypothesized that protease-activated receptors may be activated and attenuated by more than one protease. Here, we explore a desensitization mechanism of the PAR1 thrombin receptor by anticoagulant proteases and provide an explanation to the enigma of why plasmin/tissue plasminogen activator (t-PA) can both activate and deactivate platelets prior to thrombin treatment. By using a soluble N-terminal exodomain (TR78) as a model for the full-length receptor, we were able to unambiguously compare cleavage rates and specificities among the serum proteases. Thrombin cleaves TR78 at the R41-S42 peptide bond with a kcat of 120 s-1 and a KM of 16 microM to produce TR62 (residues 42-103). We found that, of the anticoagulant proteases, only plasmin can rapidly truncate the soluble exodomain at the R70/K76/K82 sites located on a linker region that tethers the ligand to the body of the receptor. Plasmin cleavage of the TR78 exodomain is nearly equivalent to that of thrombin cleavage at R41 with similar rates (kcat = 30 s-1) and affinity (KM = 18 microM). Specificity was demonstrated since there is no observed cleavage at the five other potential plasmin-cleavage sites. Plasmin also cleaves the TR78 exodomain at the R41 thrombin-cleavage site generating transiently activated exodomain. We directly demonstrated that plasmin cleaves these same sites in full-length membrane-embedded receptor expressed in yeast and COS7 fibroblasts. The rate of plasmin truncation is similar between the extensively glycosylated COS7-expressed receptor and the nonglycosylated yeast-produced receptor. Mutation of the R70/K76/K82 sites to A70/A76/A82 eliminates plasmin truncation and desensitization of thrombin-dependent Ca2+ signaling and converts PAR1 into a plasmin-activated receptor with full agonist activity for plasmin. Plasmin does not desensitize the Ca2+ response of platelets or COS7 cells to SFLLRN consistent with intermolecular ligand-binding sites being located to the C-terminal side of K82. Truncation of the wild-type receptor at the C-terminal plasmin-cleavage sites removes the N-terminal tethered ligand or preligand, thereby providing an effective pathway for PAR1 desensitization in vivo.

Amino Acid Sequence↗

PubMed1996

Arabidopsis thaliana cDNA isolated by functional complementation shows homology to serine/threonine protein kinases.

The yeast Saccharomyces cerevisiae ste6 mutant is defective in transport of a-mating factor, resulting in an inability of ste6 a cells to mate with alpha cells. The gene encodes an ATP-binding cassette, ABC transporter. We used functional complementation of a yeast ste6 mutant with an Arabidopsis thaliana expression library in an attempt to clone an Arabidopsis homolog. Sequence analysis of the isolated Arabidopsis complementing cDNA however showed no homology to the STE6 gene. High sequence similarity was detected to members of the mitogen-activated serine/threonine protein (MAP) kinase family involved in signal transduction: STE20, STE11, BCK1, Byr2 and p65PAK. The Arabidopsis clone failed to complement a fus3/kss1 mutant of S. cerevisiae, but did complement a defect in ste11, ste20, as well as ste6. The isolated clone encodes a protein that is truncated at its amino-terminal, and might function in a similar way as a dominant STE11 truncation allele. These results suggest that the Arabidopsis cDNA encodes a putative serine/threonine kinase that can function in the mating response pathway upstream of FUS3/KSS1 in S. cerevisiae, at the level of STE11 gene. Interestingly, this clone is able to restore the ability of the ste6 yeast mutant to export a-factor.

ATP-Binding Cassette Transporters↗

PubMed1992

Novel ion channels in the protists, Mougeotia and Saprolegnia, using sub-gigaseals.

Protoplasts of the filamentous alga, Mougeotia, and the filamentous fungal oomycete, Saprolegnia ferax, exhibit two K+ ion channels (2-6 pA) using the patch-clamp technique when the seals are less than 1 G omega (about 100 M omega). The membrane potential of the protoplasts was near 0 mV as measured intracellularly with double-barreled micropipettes; thus, inward K+ flux is due solely to concentration differences. Although conductances are in the range expected for K+ channels, the activity at 0 mV is not seen in other organisms under gigaseal conditions. This paper draws attention to the usefulness of this subsidiary patch-clamp technique and the novel characteristics of ion channels in Mougeotia and Saprolegnia.

Chlorophyta↗

PubMed1991

Association of p60c-src with polyoma virus middle-T antigen abrogating mitosis-specific activation.

Polyoma middle-T antigen is required for tumorigenesis in animals and for viral transformation of a variety of cells in culture (reviewed in ref. 1). Middle-T associates with and thereby activates p60c-src, a cellular tyrosine kinase homologous to the oncogene product of Rous sarcoma virus. Activation of p60c-src by middle-T is accompanied both by dephosphorylation of tyrosine 527, a site which negatively regulates src kinase src kinase activity (reviewed in refs 4-6) and by autophosphorylation on tyrosine 416 (refs 7-10). Phosphoprotein p60c-src is subject to cell cycle-specific regulation. It is most active during mitosis and repressed in interphase. Here we report that mitotic p60c-src is dephosphorylated at tyrosine 527. We also show that in cells expressing middle-T, src kinase activity is high both in mitosis and during interphase. An oncogenic mutant src protein, p60c-src(527F), where tyrosine 527 is substituted by phenylalanine, is also highly active in all phases of the cell cycle.

Animals↗

PubMed1985

Plasmalogenase in hamster heart.

In this study, the presence of plasmalogenase for the hydrolysis of the alk-1-enyl bond at the C-1 position of 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (ethanolamine plasmalogens) in the hamster heart was examined. A new spectrophotometric assay was developed for this study, in which the aldehyde released by the hydrolysis of the plamalogenase was oxidized to carboxylic acid by the action of aldehyde dehydrogenase, with the production of the molar equivalent of NADH. The results obtained from the spectrophotometric assay were comparable to those obtained by determining the rate of ethanolamine plasmalogens utilized during the reaction. However, the sensitivity of the spectrophotometric assay for plasmalogenase was shown to be 25-fold higher than with the methods described previously and enzyme activity could be detected with 1 micrograms of microsomal protein. Hamster heart plasmalogenase activity was located exclusively in the microsomal fraction, and the enzyme displayed a pH optimum at 8.5. The enzyme showed no absolute requirement for divalent metallic cations.

Aldehydes↗