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Biomedical subjects

A Veillette

Publications and source records attributed to A Veillette.

At least 55 records · Page 3Linked to original sources
PubMed1995

Regulation of Zap-70 by Src family tyrosine protein kinases in an antigen-specific T-cell line.

To further understand the interactions between Zap-70, Src family kinases, and other T-cell proteins, we have examined the regulation of Zap-70 in the antigen-specific T-cell line BI-141. By analyzing derivatives containing an activated version of either p56lck or p59fynT, it was observed that the two Src-related enzymes augmented T-cell receptor (TCR)-mediated tyrosine phosphorylation of Zap-70, as well as its association with components of the antigen receptor complex. Importantly, the accumulation of TCR.Zap-70 complexes quantitatively and temporally correlated with the induction of tyrosine phosphorylation of the CD3 and zeta chains of TCR. Using a CD4-positive variant of BI-141, we also found that the ability of Zap-70 to undergo tyrosine phosphorylation and associate with TCR was enhanced by aggregation of TCR with the CD4 co-receptor. Further studies allowed the identification of two distinct pools of tyrosine-phosphorylated Zap-70 in activated T-cells. While one population was associated with TCR, the other was co-immunoprecipitated with a 120-kDa tyrosine-phosphorylated protein of unknown identity. In addition to supporting the notion that Src-related enzymes regulate the recruitment of Zap-70 in TCR signaling, these data added further complexity to previous models of regulation of Zap-70. Furthermore, they suggested that p120 may be an effector and/or a regulator of Zap-70 in activated T-lymphocytes.

Animals↗
PubMed1995

The Src and Csk families of tyrosine protein kinases in hemopoietic cells.

There is increasing evidence that the Src family of cytoplasmic tyrosine protein kinases is involved in the signal transduction of antigen receptor- and Fc receptor-mediated cellular activation. This function relates at least in part to the ability of Src-related enzymes to phosphorylate conserved tyrosine-based motifs in the cytoplasmic domains of the antigen and Fc receptors. The catalytic function of Src-like products is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue, which is mediated by another cellular tyrosine protein kinase, p50csk. Based on this property, it is postulated that Csk is a potent negative regulator of antigen and Fc receptor signalling. The balance between the actions of Src-related kinases and the p50csk is likely a major determinant of immune responsiveness.

Animals↗
PubMed1995

Downregulation of Lck-mediated signal transduction by tip of herpesvirus saimiri.

A protein, called tip, of herpesvirus saimiri associates with Lck in transformed T cells. To investigate the effects of complex formation on cellular signal transduction, we constructed human Jurkat-T-cell lines expressing tip. The expression of tip in Jurkat-T cells dramatically suppressed cellular tyrosine phosphorylation and surface expression of lymphocyte antigens. The expression of tip also blocked the induction of tyrosine phosphorylation by anti-CD3 stimulation. The expression of tip in fibroblast cells suppressed the transforming activity of oncogenic F505 Lck. Binding assays showed that the SH3 domain of Lck is sufficient to form a stable complex with tip in vitro. These results demonstrate that tip acts at an early stage of the T-cell signal transduction cascade by associating with Lck and downregulating Lck-mediated activation. Inhibition of Lck-mediated signal transduction by tip in T cells appears to be analogous to the inhibition of Lyn/Syk-mediated signal transduction in B cells by LMP2A of the B-cell-tropic Epstein-Barr virus.

Base Sequence↗
PubMed1995

Requirement of the SH3 and SH2 domains for the inhibitory function of tyrosine protein kinase p50csk in T lymphocytes.

Previous studies from our laboratory have shown that the cytosolic tyrosine protein kinase p50csk is involved in the negative regulation of T-cell activation (L.M. L. Chow, M. Fournel, D. Davidson, and A. Veillette, Nature [London] 365:156-160, 1993). This function most probably reflects the ability of Csk to phosphorylate the inhibitory carboxy-terminal tyrosine of p56lck and p59fynT, two Src-related enzymes abundantly expressed in T lymphocytes. Herein, we have attempted to better understand the mechanisms by which Csk participates in the inhibitory phase of T-cell receptor signalling. Our results demonstrated that the Src homology 3 (SH3) and SH2 domains of p50csk are crucial for its negative impact on T-cell receptor-mediated signals. As these two sequences were not essential for phosphorylation of the carboxy-terminal tyrosine of a Src-like product in yeast cells, we postulated that they mediate protein-protein interactions allowing the recruitment of p50csk in the vicinity of activated Lck and/or FynT in T cells. In complementary studies, it was observed that linkage of a constitutive membrane targeting signal to the amino terminus of Csk rescued the deleterious impact of a point mutation in the SH2 domain of p50csk. This observation suggested that the SH2 sequence is in part necessary to translocate p50csk from the cytoplasm to the plasma membrane, where Src-related enzymes are located. Nevertheless, constitutive membrane localization was unable to correct the effect of complete deletion of the SH3 or SH2 sequence, implying that these domains provide additional functions necessary for the biological activity of p50csk.

Animals↗
PubMed1995

The unique amino-terminal domain of p56lck regulates interactions with tyrosine protein phosphatases in T lymphocytes.

The catalytic activity of p56lck is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue (tyrosine 505). Accumulating data show that this phosphorylation is mediated by the tyrosine protein kinase p50csk and that it is reversed by the transmembrane tyrosine protein phosphatase CD45. Recent studies have indicated that dephosphorylation of tyrosine 505 in resting T cells is necessary for the initiation of antigen-induced T-cell activation. To better understand this phenomenon, we have characterized the factors regulating tyrosine 505 phosphorylation in an antigen-specific T-cell line (BI-141). As is the case for other T-cell lines, Lck molecules from unstimulated BI-141 cells exhibited a pronounced dephosphorylation of the inhibitory carboxyl-terminal tyrosine. This state could be corrected by incubation of cells with the tyrosine protein phosphatase inhibitor pervanadate, suggesting that it reflected the unrestricted action of tyrosine protein phosphatases. In structure-function analyses, mutation of the site of Lck myristylation (glycine 2) partially restored phosphorylation at tyrosine 505 in BI-141 cells. Since the myristylation-defective mutant also failed to stably associate with cellular membranes, this effect was most probably the consequence of removal of p56lck from the vicinity of membrane phosphatases like CD45. Deletion of the unique domain of Lck, or its replacement by the equivalent sequence from p59fyn, also increased the extent of tyrosine 505 phosphorylation in vivo. This effect was unrelated to changes in Lck membrane association and therefore was potentially related to defects in crucial protein-protein interactions at the membrane. In contrast, deletion of the SH3 or SH2 domain, or mutation of the phosphotransfer motif (lysine 273) or the site of autophosphorylation (tyrosine 394), had no impact on phosphate occupancy at tyrosine 505. In combination, these results indicated that the hypophosphorylation of the inhibitory tyrosine of p56(lck) in T lymphocytes is likely the result of the predominant action of tyrosine protein phosphatases. Moreover, they showed that both the amino-terminal myristylation signal and the unique domain of p56(lck) play critical roles in this process.

3T3 Cells↗
PubMed1994

Intramolecular and extramolecular mechanisms repress the catalytic function of p56lck in resting T-lymphocytes.

Accumulating data show that the catalytic function of the Src-related tyrosine protein kinase p56lck is repressed by phosphorylation of a conserved carboxyl-terminal tyrosine residue (tyrosine 505). However, previous findings (Abraham, N., Miceli M.C., Parnes, J.R., and Veillette, A. (1991) Nature 350, 62-66) suggest that mechanisms unrelated to tyrosine 505 phosphorylation repress the catalytic function of p56lck in resting T-cells. In keeping with this view, we report herein that the Src homology 3 (SH3) and SH2 domains negatively regulate the catalytic activity of p56lck, by a process independent of carboxyl-terminal tyrosine phosphorylation. While the exact mechanism of this inhibition are not established, its structural requirements in the SH2 domain are distinct from those allowing recruitment of Lck in T-cell receptor signaling. In addition, we obtained evidence that the elevated tyrosine protein phosphatase activity present in T-cells also contributes to inhibit the enzymatic function of p56lck. Such an effect is seemingly mediated by dephosphorylation of tyrosine 394, the site of positive regulation of p56lck. Collectively, these results indicate that the catalytic function of p56lck in resting T-cells is repressed by a complex set of processes, which involves both intramolecular and extramolecular mechanisms.

3T3 Cells↗
PubMed1994

Ntk: a Csk-related protein-tyrosine kinase expressed in brain and T lymphocytes.

The activity of Src-related protein-tyrosine kinases is repressed by the phosphorylation of a conserved carboxyl-terminal tyrosine by another cytoplasmic protein-tyrosine kinase termed p50csk. In this study, we characterize Ntk, a protein-tyrosine kinase bearing striking similarities to p50csk. Like p50csk, Ntk possesses Src homology 3 and Src homology 2 domains and lacks the consensus tyrosine phosphorylation and myristoylation sites found in members of the Src family. Expression of ntk transcripts was maximal in brain, and was observed at significant levels in thymus and spleen. ntk RNA levels were dramatically reduced upon mitogenic stimulation of normal T lymphocytes and were minimal in transformed T-cell populations. Firm evidence that Ntk is a Csk-related enzyme was provided by the observation that it phosphorylated a Src-related polypeptide on the inhibitory carboxyl-terminal tyrosine. These findings indicate that Ntk is a Csk-related enzyme that may play an inhibitory role in the control of T-cell proliferation.

Amino Acid Sequence↗
PubMed1994

Oncogenic activation of p59fyn tyrosine protein kinase by mutation of its carboxyl-terminal site of tyrosine phosphorylation, tyrosine 528.

As a result of alternative splicing, the Src-related tyrosine protein kinase p59fyn consists of two distinct isoforms termed FynB and FynT. Whereas the first product accumulates principally in brain, the second is expressed in hemopoietic cells, especially in T-lymphocytes. There is increasing evidence that the Fyn proteins are critical for normal functions of neuronal and lymphoid cells. To better understand the regulation of the catalytic function of p59fyn, we have tested the effects of mutating the major site of in vivo tyrosine phosphorylation, tyrosine 528, on the biological and biochemical properties of this enzyme. Our studies showed that a tyrosine 528-->phenylalanine (Y528F) mutation converted either Fyn isoform into a dominant oncoprotein, capable of full transformation of rodent fibroblasts. However, while both Y528F p59fynT and Y528F p59fynB were able to transform NIH 3T3 cells, activated FynT molecules were consistently more efficient at this process. It was also found that expression of wild-type p59fyn or kinase-defective Y528F Fyn molecules failed to provoke transformation of NIH 3T3 cells, implying that the transforming capabilities of Y528F Fyn relied on deregulated catalytic activity. Contrary to an earlier study (Cheng, S. H., Espino, P. C., Marshall, J., Harvey, R., Merrill, J., and Smith, A. E. (1991) J. Virol. 65, 170-179), these findings showed that mutation of the conserved carboxyl-terminal tyrosine residue markedly stimulated the catalytic function of p59fyn in vivo, implying that dephosphorylation of tyrosine 528 is sufficient to produce biologically relevant activation of the Fyn kinase. Moreover, our results provided further indication that the two Fyn isoforms possess distinct biochemical activities that may dictate functional differences in normal cell physiology.

3T3 Cells↗
PubMed1994

Unique catalytic properties dictate the enhanced function of p59fynT, the hemopoietic cell-specific isoform of the Fyn tyrosine protein kinase, in T cells.

As a result of alternative splicing, the fyn gene encodes two different tyrosine protein kinase isoforms. While one protein (p59fynB) is abundantly expressed in the brain, the alternative product (p59fynT) is contained only in cells of hemopoietic lineages, especially T lymphocytes. Sequence analyses have revealed that these two isoforms differ exclusively within a stretch of 52 amino acids which overlaps the end of the Src homology 2 (SH2) motif and the beginning of the catalytic domain. Consistent with the idea that FynT provides a specialized function in hemopoietic cells, we have previously shown that expression of activated FynT molecules, but not that of activated FynB polypeptides, enhanced the antigen responsiveness of a mouse T-cell line (BI-141) (D. Davidson, L. M. L. Chow, M. Fournel, and A. Veillette, J. Exp. Med. 175:1483-1492, 1992). In this study, we examined the basis for the distinct signalling capabilities of the two Fyn isoforms in T lymphocytes. Our biochemical analyses revealed that FynT is more adept than FynB at promoting antigen receptor-triggered calcium fluxes. This phenomenon likely contributes to the improved biological function of FynT during antigen stimulation, as the calcium ionophore ionomycin partially rescued the inability of FynB to enhance antigen-induced lymphokine secretion. To establish the structural basis for these observations, we also created and analyzed a series of chimeras of FynT and FynB. These studies demonstrated that the distinct catalytic domain of FynT, and not its altered SH2 motif, is responsible for the improved ability to augment antigen responsiveness. Similarly, this sequence enhances the ability to mobilize cytosolic calcium in response to antigen receptor stimulation. Taken together, these data show that the distinct biological impacts of FynT and FynB in T cells are related to limited structural differences in the amino-terminal portion of their catalytic domains and that they reflect, at least in part, the greater ability of FynT to mobilize cytoplasmic calcium.

Alternative Splicing↗
PubMed1994

Two distinct protein isoforms are encoded by ntk, a csk-related tyrosine protein kinase gene.

Recently, we and others have cloned cDNAs encoding a second member of the Csk family of inhibitory tyrosine protein kinases, which we have termed Ntk. Intriguingly, the mouse ntk cDNA sequences published by two independent groups differed by the presence or absence of a 136 nucleotide-insert near their 5' ends. In this report, we demonstrate that this 136 nucleotide-sequence likely corresponds to a complete exon in the ntk gene (termed exon 2), and that the two types of cDNAs/transcripts are produced by alternative splicing. Using ribonuclease protection assays, it was also established that brain and lymphoid organs, as well as most hemopoietic cells, predominantly expressed ntk transcripts lacking exon 2. In contrast, selected hemopoietic cell lines, such as the immature myeloid cell lines 32D cl3(G) and WEHI-3B, exclusively possessed exon 2-bearing RNAs. Interestingly, exon 2 introduced a novel in-frame upstream AUG in the ntk transcript, which is in the appropriate context for translation initiation. Evidence was obtained that this AUG is utilized in vivo, and that it extends the amino-terminal sequence of Ntk by 40 amino acids. Indeed, while exon 2-deficient ntk RNAs were translated into a 52 kilodalton (kDa) polypeptide (p52ntk), those bearing exon 2 produced a 56 kDa protein (p56ntk). Furthermore, p56ntk, but not p52ntk, was recognized by an antiserum directed against the novel amino-terminal sequence encoded by exon 2. Additional biochemical characterizations showed that p52ntk and p56ntk were localized to the cytoplasm, and that they partially accumulated in the detergent-insoluble cellular fraction. This last finding suggested that the Ntk proteins can associate with the cytoskeleton. Finally, through linkage analysis of two multilocus crosses, the ntk gene was mapped to Chromosome 10 in the mouse. Taken together, these data showed that ntk, a csk-related tyrosine protein kinase gene, encodes two protein isoforms expressed in distinct cell types. Moreover, they raised the possibility that Ntk may be involved in the regulation of Src-like enzymes in detergent-insoluble cellular compartments.

3T3 Cells↗
PubMed1993

Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk.

Tyrosine protein phosphorylation is necessary for antigen receptor-mediated activation of T lymphocytes. This signal is generated at least in part by the Src-related tyrosine protein kinases p56lck and p59fynT (refs 2, 3). The activity of these two enzymes is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory phosphorylation may be caused by p50csk (for C-terminal Src kinase), a tyrosine protein kinase which accumulates most abundantly in thymus and spleen. To investigate the function of Csk in T lymphocytes and characterize the processes regulating T-cell receptor (TCR) signalling, we examined the effects of overexpression of Csk on the physiology of an antigen-specific mouse T-cell line. We report here that p50csk negatively regulates TCR-induced tyrosine protein phosphorylation and lymphokine production. This provides evidence for the involvement of Csk in the regulation of T-cell activation.

Animals↗
PubMed1993

Conserved cysteine residues are critical for the enzymatic function of the lymphocyte-specific tyrosine protein kinase p56lck.

We have evaluated the possibility that conserved cysteine residues are critical for the enzymatic function of p56lck. Through oligonucleotide-directed mutagenesis, 5 Lck residues (cysteines 217, 224, 378, 464, and 475) were individually mutated to alanines, and the effects of these substitutions were tested in various in vitro and in vivo assays. We found that mutation of either of 2 cysteines located in the carboxyl portion of the kinase domain (cysteines 464 and 475) abolished the catalytic function of Lck. In addition, it was noted that alteration of cysteine 475 resulted in a dramatic reduction of the half-life of p56lck. These cysteine residues are highly conserved throughout the tyrosine protein kinase family, suggesting that they may play important functions in catalysis and/or substrate recognition.

Alanine↗
PubMed1993

The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site.

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.

3T3 Cells↗
PubMed1993

Interactions of the SH2 domain of lymphocyte-specific tyrosine protein kinase p56lck with phosphotyrosine-containing proteins.

We have previously demonstrated that the non-catalytic Src homology 2 (SH2) domain is required for both positive and negative regulation of the catalytic function of the lymphocyte-specific tyrosine protein kinase p56lck. Indeed, the ability of activated p56lck molecules (tyrosine 505 to phenylalanine 505 mutants) to enhance T-cell receptor (TCR)-induced tyrosine protein phosphorylation is dramatically reduced by deletion of the SH2 domain. Paradoxically, removal of the SH2 sequence also results in constitutive elevation of the catalytic function of wild-type Lck polypeptides, rendering them capable of oncogenic transformation of rodent fibroblasts. As SH2 sequences can mediate binding to phosphotyrosine-containing peptides, the ability of the Lck SH2 domain to interact with tyrosine-phosphorylated proteins was tested. We found that the SH2 sequence of p56lck can bind several of the TCR-regulated tyrosine phosphorylation substrates in vitro. One of the substrates, an 80-kilodalton (kDa) phosphoprotein (p80) showed the tightest binding to the SH2 domain of Lck. Additionally, it was observed that the SH2 domain of Lck can bind a synthetic peptide containing the phosphorylated carboxy-terminal tyrosine 505 of p56lck. Indirect evidence indicating that the SH2 region interacts with the tyrosine-phosphorylated carboxy terminus of Lck in vivo was also obtained. As deletion of the SH2 domain or mutation of tyrosine 505 results in p56lck activation in vivo, it is conceivable that interactions between these two regions impose a conformation that is unfavorable to phosphorylation of intracellular substrates. Collectively, these findings suggest that the SH2 domain modulates the catalytic function of Lck through complex interactions with phosphotyrosine-containing proteins.

Animals↗
PubMed1992

Association of tyrosine kinase p56lck with CD4 inhibits the induction of growth through the alpha beta T-cell receptor.

The membrane glycoprotein CD4 enhances antigen-mediated activation of T cells restricted by class II molecules of the major histocompatibility complex (MHC). This positive function has been attributed to the protein tyrosine kinase p56lck (ref. 4), which is noncovalently associated with the cytoplasmic portion of CD4, and is activated on CD4 aggregation. Antigen presentation by MHC class II molecules coaggregates CD4 and the T-cell antigen receptor (TCR alpha beta-CD3). Thus, the mutual specificity of CD4 and TCR alpha beta for the MHC-antigen complex results in the juxtaposition of p56lck and TCR alpha beta-CD3. In contrast, anti-CD4 antibodies can abrogate antigen-induced, as well as anti-TCR-induced T-cell activation, indicating that CD4 might also transduce negative signals. The molecular basis for this opposing function remains unclear. Here we show that the CD4-p56lck complex prohibits the induction of activation signals through the TCR-CD3 complex when not specifically included in the signalling process. This negative effect does not require anti-CD4 treatment, indicating that the induction of distinct negative signals is probably not involved. Rather, the results demonstrate that the CD4-p56lck complex provides prerequisite signals for antigen-receptor-induced T-cell growth and thus characterize a molecular mechanism for functional constraints imposed on T-cell activation by the MHC.

Animals↗
PubMed1992

Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn.

Recent observations suggest that the src-related tyrosine protein kinase p59fyn may be involved in antigen-induced T lymphocyte activation. As a result of alternative splicing, p59fyn exists as two isoforms that differ exclusively within a short sequence spanning the end of the Src Homology 2 (SH2) region and the beginning of the tyrosine protein kinase domain. While one p59fyn isoform (fynB) is highly expressed in brain, the alternative product (fynT) is principally found in T lymphocytes. To further understand the role of p59fyn in T cell activation and to test the hypothesis that p59fynT serves a tissue-specific function in T lymphocytes, we have examined the effects of expression of activated versions (tyrosine 528 to phenylalanine 528 mutants) of either form of p59fyn on the physiology of an antigen-specific mouse T cell hybridoma. Our results demonstrated that the two forms of fyn, expressed in equivalent amounts, efficiently enhanced antibody-induced T cell receptor (TCR)-mediated signals. In contrast, only p59fynT increased interleukin 2 production in response to antigen stimulation. This finding implies that the distinct p59fyn isoform expressed in T lymphocytes regulates the coupling of TCR stimulation by antigen/major histocompatibility complex to lymphokine production.

Animals↗
PubMed1992

Profound block in thymocyte development in mice lacking p56lck.

The protein Lck (p56lck) has a relative molecular mass of 56,000 and belongs to the Src family of tyrosine kinases. It is expressed exclusively in lymphoid cells, predominantly in thymocytes and peripheral T cells. Lck associates specifically with the cytoplasmic domains of both CD4 and CD8 T-cell surface glycoproteins and interacts with the beta-chain of the interleukin-2 receptor, which implicates Lck activity in signal transduction during thymocyte ontogeny and activation of mature T cells. Here we generate an lck null mutation by homologous recombination in embryonic stem cells to evaluate the role of p56lck in T-cell development and activation. Lck-deficient mice show a pronounced thymic atrophy, with a dramatic reduction in the double-positive (CD4+CD8+) thymocyte population. Mature, single-positive thymocytes are not detectable in these mice and there are only very few peripheral T cells. These results illustrate the crucial role of this T-cell-specific tyrosine kinase in the thymocyte development.

Animals↗