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C Couture

Publications and source records attributed to C Couture.

At least 37 records · Page 2Linked to original sources
PubMed1996

Involvement of Src-homology-2-domain-containing protein-tyrosine phosphatase 2 in T cell activation.

Activation of resting T lymphocytes by ligands to the complex of T cell antigen receptor (TCR) and CD3 is initiated by a series of critical tyrosine phosphorylation and dephosphorylation events. Protein-tyrosine kinases of the Syk, Src and Csk families and the CD45 protein-tyrosine phosphatase (PTPase) are known to be involved in these early biochemical reactions. We have found that one of the two T-cell-expressed SH2-domain-containing PTPases, SHPTP2, is rapidly phosphorylated on tyrosine upon addition of anti-CD3 mAbs. This response was absent in cells lacking the Src family kinase Lck. Concomitantly with tyrosine phosphorylation, SHPTP2 co-immunoprecipitated with two unphosphorylated cellular proteins; phosphatidylinositol 3-kinase p85 and Grb2. Binding of SHPTP2 to Grb2 occurred through the SH2 domain of Grb2, while the association between SHPTP2 and p85 seemed to be mediated through Grb2 as an intermediate. In addition, many other molecules associate with Grb2 and may thereby become juxtaposed to SHPTP2. Our results indicate that SHPTP2 participates actively at an early stage in TCR signaling and that its phosphorylation on tyrosine may direct a Grb2-dependent association with selected substrates.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Vav: function and regulation in hematopoietic cell signaling.

Vav, a 95 kDa proto-oncogene product expressed specifically in hematopoietic cells, was originally isolated as a transforming human oncogene. Vav contains an array of functional domains that are involved in interactions with other proteins and, possibly, with lipids. These include, among others, a putative guanine nucleotide exchange domain, a cysteine-rich region similar to the phorbol ester/diacylglycerol-binding domain of protein kinase C, a pleckstrin-homology domain, and Src-homology 2 and 3 (SH2 and SH3, respectively) domains. The presence of these domains, the transforming activity of the vav oncogene, and the rapid increase in tyrosine phosphorylation of Vav induced by triggering of diverse receptors indicate that it plays an important role in hematopoietic cell signaling pathways. Such a role is supported by recent studies using "knockout" mice and transiently transfected T cells, in which Vav deletion or overexpression, respectively, had marked effects on lymphocyte development or activation. The presence of a putative guanine nucleotide exchange domain, the prototype of which is found in the dbl oncogene product, implies that Vav functions as a guanine nucleotide exchange factor (GEF) for one (or more) members of the Ras-like family of small GTP-binding proteins. In support of such a role, Vav preparations were found in some (but not other) studies to mediate in vitro-specific GEF activity for Ras. Additional studies are required to identify the physiological regulators and targets of Vav, and its exact role in hematopoietic cell development and signaling.

Amino Acid Sequence↗
PubMed1996

Functional and physical interactions of Syk family kinases with the Vav proto-oncogene product.

Syk family kinases are essential for lymphocyte development and activation. Therefore the identification of their direct effectors is of critical importance. Here, we report that Syk interacts in the yeast two-hybrid system with Vav, a proto-oncogene product exclusively expressed in hematopoietic cells. This interaction was direct, required the catalytic activity of Syk, the SH2 domain of Vav, and tyrosine residues in the linker domain of Syk. Vav also associated with Syk and Zap in antigen receptor-stimulated B or T cells, respectively. Functionally, Vav was phosphorylated by Syk family kinases both in vivo and in vitro. Furthermore, Syk and Vav cooperated to activate NF-AT synergistically. These results indicate that the interaction between Syk family kinases and Vav plays an important role in coupling immune recognition receptors to signaling pathways involved in lymphokine production.

B-Lymphocytes↗
PubMed1995

Linkage results on 11Q21-22 in Eastern Quebec pedigrees densely affected by schizophrenia.

The 11q21-22 region is of interest for schizophrenia because several candidate genes are located in this section of the genome. The 11q21-22 region, including DRD2, was surveyed by linkage analysis in a sample (N = 242) made of four large multigenerational pedigrees densely affected by schizophrenia (SZ) and eight others by bipolar disorder (BP). These pedigrees were ascertained in a large area of Eastern Quebec and Northern New Brunswick and are still being extended. Family members were administered a "consensus best-estimate diagnosis procedure" (DSM-III-R criteria) blind to probands and relatives' diagnosis and to pedigree assignment (SZ or BP). For linkage analysis, 11 microsatellite polymorphism (CA repeat) markers, located at 11q21-22, and comprising DRD2, were genotyped. Results show no evidence of a major gene for schizophrenia. However, a maximum lod score of 3.41 at the D11S35 locus was observed in an affected-only analysis of one large SZ family, pedigree 255. Whether or not the positive linkage trend in pedigree 255 reflects a true linkage for a small proportion of SZ needs to be confirmed through the extension of this kindred and through replication.

Base Sequence↗
PubMed1995

Regulation of the p70zap tyrosine protein kinase in T cells by the CD45 phosphotyrosine phosphatase.

Two classes of protein tyrosine kinases (PTK) are utilized by the T cell antigen receptor (TcR)/CD3 complex for initiation of the signaling cascade, the Src-family PTK p56lck and p59fyn, and the Syk-family PTK p70zap and p72syk. In addition, the CD45 phosphotyrosine phosphatase (PTPase) is required for the induction of tyrosine phosphorylation by the TcR/CD3, presumably by positively regulating Src-family PTK. Here we report that CD45 also regulates the Syk-family PTK p70zap (or ZAP-70). In CD45-negative T cells, p70zap was constitutively phosphorylated on tyrosine and co-immunoprecipitated with the TcR-zeta chain. In resting wild-type CD45-positive cells, p70zap was mainly unphosphorylated, but it was rapidly phosphorylated on tyrosine upon treatment of the cells with anti-CD3 or PTPase inhibitors. Finally, p70zap co-distributed with CD45 in intact T cells, and tyrosine phosphorylated p70zap was dephosphorylated by CD45 in vitro. These findings suggest that CD45 plays an important role, direct or indirect, in the regulation of p70zap and its function in TcR/CD3 signaling.

Cell Line, Transformed↗
PubMed1994

p56lck-independent activation and tyrosine phosphorylation of p72syk by T-cell antigen receptor/CD3 stimulation.

Activation of resting T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex is initiated by rapid tyrosine phosphorylation of cellular proteins. Protein-tyrosine kinases (PTKs) of the src family are known to be important, but the mechanism of their recruitment and their interactions with PTKs of other families are incompletely understood. We show that a member of another family of PTKs, the p72syk kinase, is constitutively bound to the TCR/CD3 complex and becomes tyrosine phosphorylated and activated within 1 min after TCR/CD3 stimulation. This activation did not depend on the presence of p56lck in T cells and in transfected COS cells. In both cases, however, the phosphorylation of cellular substrates was augmented by src family PTKs. We propose that p72syk may act as an immediate receptor-activated kinase upstream of the related p70zap PTK and the src family PTKs p56lck and p59fyn in T cells and that these src family PTKs act as signal amplifiers.

Amino Acid Sequence↗
PubMed1994

Immunoselection of GRP94/endoplasmin from a KNRK cell-specific lambda gt11 library using antibodies directed against a putative heparanase amino-terminal peptide.

Induction of an invasive phenotype by metastatic tumour cells results in part from inappropriate expression of extracellular matrix-degrading enzymes normally involved in embryonic morphogenesis, tissue remodelling, angiogenesis and wound healing. Such enzymes include endoglycosidases that degrade heparan sulfate (HS) in endothelial basement membrane, as well as better characterized proteases. Heparanase, an endo-beta-D-glucuronidase initially detected in B16 melanoma cells, has been described as a M(r) 96,000 glycoprotein with pI of 5.2, and has been immunolocalized to the cell surface and cytoplasm. We have utilized a polyacrylamide-gel-based HS degradation assay to demonstrate that KNRK, a rat kidney fibroblast cell line transformed by v-K-ras, exhibits HS-degrading activity similar to that of B16F10 mouse melanoma cells. To immunoselect heparanase-expressing clones from a KNRK-cell-specific lambda gt11 cDNA library, we have also prepared a rabbit anti-serum directed against a putative amino-terminal peptide of B16F10 cellular heparanase. Lysogens from one clone expressed a beta-galactosidase fusion protein whose staining with peptide anti-serum was inhibited by competition with excess peptide. Dideoxy-mediated sequencing of the insert termini of this recombinant revealed that it represents a rat homologue of M(r) 94,000 glucose-regulated protein (GRP94/endoplasmin), a molecular chaperone that contains the exact amino-terminal sequence previously attributed to heparanase. Our results call into question the specificity of this peptide sequence, as well as previous immunolocalization studies of heparanase carried out using such anti-sera.

Amino Acid Sequence↗
PubMed1994

Activation of phosphatidylinositol-3-kinase in Jurkat T cells depends on the presence of the p56lck tyrosine kinase.

Activation of resting T lymphocytes by ligands to the T cell receptor (TcR)/CD3 complex is initiated by phosphorylation of a number of key regulatory proteins on specific tyrosine residues. One such protein is the heterodimeric enzyme phosphatidylinositol-3-kinase (PI3K). We recently found that this enzyme is also rapidly activated following TcR/CD3 triggering and that immunoprecipitated PI3K was activated in vitro by direct tyrosine phosphorylation. Here we show that TcR/CD3-induced tyrosine phosphorylation and activation of PI3K in Jurkat T leukemia cells depend on the presence of the p56lck tyrosine kinase: in a variant of the Jurkat T cell line lacking p56lck, JCaM1, these responses were absent. We also show that p56lck directly activates PI3K purified from transfected COS-1 cells, indicating that other T cell-specific proteins are not required for the process. Finally, tryptic peptide maps show that p56lck phosphorylates three tyrosine residues in the p85 alpha subunit of PI3K and two in p110 of PI3K. Our results suggest that p56lck is required for activation of PI3K in Jurkat T cells and can itself directly activate it by phosphorylating one or several stimulatory sites.

Enzyme Activation↗
PubMed1994

Triggering of human natural killer cells through CD16 induces tyrosine phosphorylation of the p72syk kinase.

Activation of protein tyrosine kinases (PTK) is an initial and obligatory event for the triggering of human natural killer (NK) cells to cytotoxicity. Of the different PTK detected in NK cells, only p56lck has previously been shown to participate in NK cell activation. Here we present evidence that another PTK, p72syk, is involved in activation of NK cells. Stimulation with a monoclonal antibody to to the Fc gamma RIII receptor (CD16) induced an increased tyrosine phosphorylation of p72syk. This phosphorylation correlated with an increased tyrosine kinase activity of p72syk towards a synthetic peptide substrate. A severalfold increase in the catalytic activity of p72syk was also seen after treatment of NK cells with an inhibitor of phosphotyrosine phosphatases, pervanadate. We conclude that triggering of the cytotoxic response in NK cells is associated with activation of p72syk.

Amino Acid Sequence↗
PubMed1994

Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation.

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

Amino Acid Sequence↗
PubMed1994

An efficient expression, purification and immunodetection system for recombinant gene products.

We describe a modification of the mammalian expression vector pRc/CMV, which drives expression of inserted genes from either the human cytomegalovirus (CMV) immediate-early promoter or the bacteriophage T7 RNA polymerase promoter. The modification is designed to allow expression, simple purification and specific immunodetection of recombinant fusion proteins. The modified plasmid, termed pTag/CMV-neo, encodes a Kozak consensus ribosome-binding site (RBS) and a 30-amino acid fusion tag peptide. This peptide consists of a metal ion-binding site, (His)6, for single-step affinity purification using Ni(2+)-chelating resin and a multi-purpose HIV-1-derived peptide (p18HIV). This viral epitope can be used to identify, detect and characterize target fusion proteins in conjunction with a specific monoclonal antibody H902 that does not display cross-reactivity with cellular proteins. The H902 production hybridoma cell line is reagent #521 from the NIH AIDS Research and Reference Program.

Amino Acid Sequence↗
PubMed1994

TCR/CD3-triggering causes increased activity of the p50csk tyrosine kinase and engagement of its SH2 domain.

The majority of known protein tyrosine kinases are integral membrane proteins or bound to cellular membranes via a covalently attached myristic acid. An exception is the newly described p50csk tyrosine kinase, which is believed to control the activity of the src-family of protein tyrosine kinases. This small kinase does not contain a myristylation signal or other known membrane attachment motifs, and indeed appears to be cytosolic. Recently, we found that p50csk specifically phosphorylates the negative regulatory Tyr-505 of the T cell-specific src-family kinase p56lck, and thereby suppresses its catalytic activity. Here we show that p50csk is activated in Jurkat T cells within one minute after stimulation of the cells with anti-CD3 MAbs. In parallel with this activation, p50csk formed a stable complex with one major 72 kDa tyrosine phosphorylated protein and minor polypeptides at 90 and 110 kDa. The isolated SH2 domain of p50csk specifically bound the 72 kDa protein in lysates from activated, but not resting, T or B cells. By several criteria, the 72 kDa protein was not a tyrosine kinase itself and it did not react with antibodies to a panel of known proteins of this molecular size. Our results suggest that p50csk is rapidly engaged via its SH2 domain and participates in the earliest events of T cell activation.

CSK Tyrosine-Protein Kinase↗
PubMed1993

Two developmentally regulated mRNAs encoding actin-binding proteins in Physarum polycephalum.

A cDNA library from amoebae of Physarum polycephalum was screened by differential hybridization. Two clones contained inserts for mRNAs present in amoebae and absent in plasmodia. The LAV3-4 cDNA encodes a 402 aa protein (ABP-46) that shows sequence similarity to the actin binding site in the N-terminal region of the alpha-actinin family. The LAV3-5 cDNA is 76% identical to the Dictyostelium actin bundling protein, which cross-links and stabilizes actin filaments in amoebal filopodia.

Actinin↗
PubMed1993

Identification of a 16-kDa thymocyte membrane glycoprotein involved in the thymocyte/thymic medullary epithelial cell interaction.

We have previously described a type of lymphoepithelial interaction involving CD4+ CD8+ thymocytes and a medullary epithelial cell line (E-5). This interaction is mediated by the recently described gp23/45 epithelial adhesion molecule and an as yet unknown thymocyte receptor. The present work describes a thymocyte surface glycoprotein of 16 kDa which binds both to E-5 cells and to the purified gp23/45 adhesion molecule. In addition, a thymic lymphoma cell line (Ti-6), which interacts with the E-5 cells via the gp23/45 receptor, also present a 16-kDa glycoprotein on its surface. Taken together, the data suggest that the 16-kDa thymocyte surface glycoprotein participates in the binding between these cells and the thymic epithelium.

Animals↗
PubMed1992

Purification and characterization of a mammalian endo-exonuclease.

An endo-exonuclease has been purified from cultured monkey (CV-1) cells. The enzyme which was purified to near homogeneity to be a 65 kDa monomeric protein. The single-strand DNase activity is endonucleolytic and nonprocessive, whereas the double-strand DNase activity is exonucleolytic and processive. The enzyme was also found to have RNase activity using poly-rA as substrate. The pH optimum for ss-DNase is 8 and for ds-DNase it is 7.5. Both DNase activities require a divalent metal ion (Mg2+, Mn2+, Ca2+, Zn2+) for activity and exhibit the same kinetics of heat inactivation. The purified protein binds to and cleaves a synthetic Holliday junction substrate. The overall enzymatic characteristics of the mammalian protein are very similar to the putative recombination endo-exonucleases purified from Neurospora crassa, Aspergillus nidulans and Saccharomyces cerevisiae.

Animals↗
PubMed1992

Tyrosine kinase activation in thymic epithelial cells: necessity of thymocyte contact through the gp23/45/90 adhesion complex.

Interactions between thymocytes and thymic stromal cells are necessary for T cell differentiation, maturation and proliferation. The signals required for these events to occur often necessitate close contact, and indeed adhesion, between the cell types involved. While the transmission of signals from stromal cells to thymocytes has been well documented, there is little evidence that binding of thymocytes to stromal cells can result in stromal cell activation. We have recently identified a novel thymic epithelial adhesion complex composed of three non-covalently associated glycoproteins (gp23, gp45 and gp90). While gp23 and gp45 are jointly required for adhesion to thymocytes, the function of gp90 is unknown. In the present work, we show that gp23/45-mediated contact with thymocytes induces de novo tyrosine phosphorylation of gp90. Furthermore, the protein tyrosine kinase responsible for gp90 neophosphorylation is itself an integral part of the adhesion complex.

Animals↗