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PubMed1988

Peptide antibodies to the human c-fyn gene product demonstrate pp59c-fyn is capable of complex formation with the middle-T antigen of polyomavirus.

The c-fyn proto-oncogene is a member of a family of closely related genes of which c-src is the prototype. Using peptide antibodies which had been raised against sequences predicted to be specific for the human c-fyn gene product, the c-fyn protein was identified. It is a tyrosine kinase with apparent mol. wt of 59 kd that is also phosphorylated and myristylated. Like pp60c-src and pp62c-yes, pp59c-fyn is able to form a stable complex with middle-T antigen, the transforming protein of polyomavirus. The transformation-defective middle-T mutant NG59, which is unable to associate stably with pp60c-src does not associate with pp59c-fyn. In contrast to pp60c-src, complex formation with middle-T antigen does not lead to a significant increase in the tyrosine kinase activity of pp59c-fyn. These findings lead us to suggest that middle-T mediated transformation may be a consequence of the deregulation of several members of the src-family of protein tyrosine kinases.

Animals↗
PubMed1994

Interactions of Lyn with the antigen receptor during B cell activation.

Signaling through the B cell antigen receptor requires a complex set of interactions involving transmembrane components of the IgM receptor complex and cytosolic protein-tyrosine kinases. We have focused on the nature of these protein-protein interactions, the requirements for their occurrence, as well as the temporal sequence of events during the activation process. We found that cross-linking B cell antigen receptors at 0 degree C resulted in the rapid association of the Src-family protein-tyrosine kinase, Lyn, with the antigen receptor complex as judged by the presence of Lyn in anti-IgM and anti-phosphotyrosine immune complexes and the presence of MB-1 in anti-Lyn immune complexes. Receptor engagement also resulted in the rapid association of Lyn with the phosphotyrosine phosphatase, CD45. This association of Lyn with receptor components was stable in the detergent Brij 96, but was readily disrupted by Nonidet P-40, suggesting the involvement of hydrophobic interactions in stabilizing formation of the Lyn-receptor complex. The protein-tyrosine kinase, Syk, was also found associated with activated receptor complexes. This association of Syk with components of the antigen receptor complex was stable to Nonidet P-40. Antibodies directed against the carboxyl teminus of Syk, but not against the amino-terminal SH2 domain, co-immunoprecipitated MB-1 from activated cells, consistent with the binding of Syk through an SH2 domain-phosphotyrosine interaction.

Animals↗
PubMed1992

CD28-induced T cell activation. Evidence for a protein-tyrosine kinase signal transduction pathway.

CD28 is a 44-kDa homodimeric receptor expressed on the majority of T cells. Engagement of the CD28 receptor by soluble anti-CD28 mAb in conjunction with PMA causes the induction of lymphokine/cytokine production and proliferation in resting T cells via signal transduction pathways independent of the TCR. The precise nature of the biochemical events that occur after perturbation of the CD28 receptor remain unclear. We report evidence for the coupling of CD28 to a protein-tyrosine kinase pathway. Multivalent cross-linking of the CD28 receptor or stimulation by soluble CD28 mAb plus PMA, but not PMA or soluble CD28 mAb alone, reproducibly caused the rapid (within 2 min) tyrosine phosphorylation of a 100-kDa cellular substrate. In some experiments, additional cellular substrates of 110, 85, 74, 68, 56, 43, and 29 kDa were also observed. The tyrosine phosphorylation of these substrates was completely inhibited by 12 h pretreatment of T cells with herbimycin A, a selective inhibitor of src-family protein-tyrosine kinases. Pretreatment of T cells with herbimycin was without effect on CD28 surface expression but did inhibit CD28 mAb plus PMA-induced IL-2 mRNA levels, IL-2R(CD25) up-regulation, and cell proliferation. The inhibition of IL-2 mRNA levels was likely at the level of transcription, because herbimycin inhibited NF-AT, AP-1, and CD28RC but not NF-kappa B or OCT-1 binding activities to their respective IL-2 enhancer region sequences. Herbimycin did not inhibit PMA-dependent events including CD69 surface expression, NF-kappa B nuclear binding activity or the level of CD25 induced by PMA alone, supporting the notion that herbimycin is acting to inhibit a CD28 initiated or regulated protein-tyrosine kinase pathway(s).

Antigens, CD↗
PubMed1989

A function for the lck proto-oncogene.

Proto-oncogenes encode products that comprise a select group of cellular regulatory proteins whose mutation or aberrant expression can result in oncogenic transformation. With the exception of certain growth factors and their receptors, the definition of normal functions for most proto-oncogene products has been elusive. The discovery that a member of the src-family of tyrosine protein kinases (p56lck) is associated with both the CD4 and CD8 T-lymphocyte surface glycoproteins provides the first clue to understanding the potential physiological functions of this family of proto-oncogenes.

Amino Acid Sequence↗
PubMed1994

Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein.

Tyrosine phosphorylation of cellular proteins is the earliest identifiable event following T-cell antigen receptor (TCR) stimulation and is essential for activating downstream signaling machinery. Two Src-family protein-tyrosine kinases, the TCR-associated p59fyn (Fyn) and the CD4/8-associated p56lck (Lck), have emerged as the likely mediators of early tyrosine phosphorylation in T cells. Here, we show direct binding of a 120-kDa TCR-induced phosphotyrosyl polypeptide, p120, to glutathione S-transferase fusion proteins of the Src homology 3 (SH3) domains of Fyn, Lck, and p60src (Src) but not other proteins. While binding of p120 to Fyn SH2 domain was phosphotyrosine-dependent as expected, its binding to the SH3 domain was independent of tyrosine phosphorylation, as shown by lack of competition with a phosphotyrosyl competitor peptide. In contrast, an SH3-specific proline-rich peptide completely abolished p120 binding to SH3. p120 was tyrosine-phosphorylated within 10 sec following stimulation of Jurkat cells with anti-CD3 monoclonal antibody, with maximal phosphorylation at 30 sec. Importantly, p120 was found associated with Fyn and Lck proteins in unstimulated Jurkat cells and served as an in vitro substrate for these kinases. These results provide evidence for a role of the SH3 domains of Fyn and Lck in the recruitment of early tyrosine-phosphorylation substrates to the TCR-associated tyrosine kinases.

Amino Acid Sequence↗
PubMed1994

CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development.

CD45 is one of the most abundant leukocyte cell surface glycoproteins and is expressed exclusively upon cells of the hematopoietic system. Different isoforms of CD45 are generated by alternative splicing and are expressed in cell type-specific patterns on functional subpopulations of lymphocytes. In a major advance, CD45 was identified as one of the first members of a novel class of enzymes, the protein tyrosine phosphatases (PTPs). This serendipitous discovery linked CD45 to the process of reversible protein tyrosine phosphorylation, a key regulatory mechanism for controlling the growth and division of eukaryotic cells, and provided the impetus for most of the studies described in this review. CD45 is now established as a critical component of the signal transduction machinery of lymphocytes. In particular, evidence from genetic experiments indicates that CD45 plays a pivotal role in antigen-stimulated proliferation of T lymphocytes and in thymic development. Two members of the Src-family of protein tyrosine kinases (PTKs), the p56lck and p59fyn proteins, have been implicated as physiological substrates of CD45, providing an important clue to how the action of this leukocyte-specific PTP might influence signaling by the T cell antigen receptor. Structure-function analysis of CD45 and other PTPs has identified structural features of PTP catalytic domains required for enzymatic activity. However, despite intensive efforts, little is known about how the activity of CD45 is regulated. The external domain of CD45 does not appear to be absolutely required for signal transduction by the T cell receptor, and there is currently no evidence that ligand binding modulates CD45 activity. Analysis of CD45 isoform expression has revealed a hitherto unrecognized plasticity in isoform usage by T cells and other leukocytes, adding to the regulatory complexity of isoform expression.

Animals↗
PubMed1992

Comparison of src-family cDNAs reveals distinct mechanisms underlying focus formation in transfected fibroblasts.

Despite the intensive study of both cellular transformation and src-family protein-tyrosine kinases, there have been no direct comparisons of transforming potency for normal members of this gene-family. In this study, the focus-forming activity of normal c-src, fyn, and lck cDNAs were compared in NIH 3T3 cell transfection assays. Focus formation was studied quantitatively, and individual foci were analyzed for phosphotyrosine content and expression of appropriate translational products. Each foci arising from c-src transfectants had a marked increase in phosphotyrosine content, and the majority of these foci expressed a c-src protein with an aberrant carboxyl terminus. Foci derived from lck transfectants also had a marked increase in phosphotyrosine content, and some foci expressed a lck protein with an aberrant carboxyl terminus. In contrast, foci from fyn-transfected cells were not distinguished from G418-selected mass cultures in terms of total phosphotyrosine content or expression of p59fyn. These studies support the previously published concept that overexpression of the normal fyn protein contributes to focus formation in transfected NIH 3T3 cells but suggest that the focus-forming activity observed after c-src or lck transfections is frequently attributable to mutational events. Because lck mutations have not been previously described in transformed foci, we characterized the lck transcript expressed in two foci and identified a novel point mutation that encodes a lck protein with increased in vivo kinase and focus-forming activity.

3T3 Cells↗
PubMed1992

Phenotypic changes induced by interleukin-2 (IL-2) and IL-3 in an immature T-lymphocytic leukemia are associated with regulated expression of IL-2 receptor beta chain and of protein tyrosine kinases LCK and LYN.

We have previously reported the establishment of an interleukin-3 (IL-3)-dependent and phenotypically myeloid cell line (TALL-103/3), obtained by culturing cells from an immature T-lymphoblastic leukemia in the presence of IL-3. These cells differentiated into a T-lymphoid cell line (TALL-103/2) upon removal of IL-3 and incubation in IL-2. Despite the different phenotype, the two cell lines remained karyotypically and genotypically identical. Here, we have analyzed the phenotypic changes and the signaling events induced by these two lymphokines in TALL-103/3 cells by switching them to temporary growth in IL-2 and returning them to IL-3. All four sublines obtained (the myeloid in IL-3 and the lymphoid in IL-2) expressed RNA for CD3, IL-2 receptor (R) alpha, and T-cell receptor (TCR)-gamma and -delta chains. However, cells cultured in IL-3 failed to express detectable levels of the IL-2R beta chain at both the protein and RNA levels, whereas cells exposed to IL-2 always expressed IL-2R beta. In parallel with the changes in IL-2R beta expression, the SRC-like protein tyrosine kinase (PTK) p56 LCK could not be detected in IL-3-dependent cells, but was abundant in the IL-2-dependent cells and underwent markedly increased autophosphorylation in response to IL-2. In contrast, p53/p56 LYN was highly expressed in IL-3-dependent cells, and greatly decreased when these cells were switched to growth in IL-2. LYN kinase autophosphorylation modestly increased in response to IL-3. None of the other kinases in the SRC family that were tested underwent increased autophosphorylation after lymphokine stimulation, indicating the specificity of IL-2 for LCK and of IL-3 for LYN. The TALL-103 cell lines provide a unique system to study the interaction between lymphokines and SRC-family PTKs in signal transduction pathways leading to hematopoietic cell differentiation.

Cell Division↗
PubMed1992

Interleukin-3 regulates the activity of the LYN protein-tyrosine kinase in myeloid-committed leukemic cell lines.

The lymphokine interleukin-3 (IL-3) promotes the growth and survival of immature hematopoietic cells. Previous studies have shown that IL-3 induces rapid increases in protein-tyrosine kinase (PTK) activity in IL-3--dependent cells. Unlike some other hematopoietic growth factor receptors (eg, c-fms and c-kit), however, the known subunits of the IL-3 receptor (IL-3R) lack intrinsic kinase activity. Recently, it was reported that the IL-2R (whose p75 beta-subunit shares sequence homology with a known murine IL-3R subunit and a common beta-subunit of the human IL-3R and granulocyte-macrophage colony-stimulating factor [GM-CSF] receptors) can physically associate with and regulate the activity of the SRC-family PTK, p56-LCK. Because most IL-3--dependent cells contain p53/56-LYN, but not p56-LCK, we explored the effects of IL-3 on the activities of LYN and other SRC-like PTKs in two human leukemic cell lines, AML-193 and TALL-101, which are phenotypically myeloid, and whose in vitro growth is dependent on IL-3. These cells expressed four of the eight known SRC-family proto-oncogenes: lyn, fyn, yes, and hck. When these factor-dependent leukemic cell lines were deprived of lymphokine to achieve cellular quiescence and then restimulated with IL-3, rapid increases (detectable within 1 minute and maximal by 10 minutes) were observed in the activity of the p53/56-LYN kinase, as assessed by in vitro kinase assays. In contrast, no alteration in the activities of other SRC-family PTKs present in these cells was detected after restimulation with IL-3 under the same conditions. This effect of IL-3 reflected an increase in the specific activity of the LYN kinase, because levels of the 53-Kd and 56-Kd LYN proteins were unaltered by IL-3 stimulation, as assessed by immunoblotting. Furthermore, the magnitude of these inducible increases in LYN kinase activity was dependent on the concentration of IL-3, and correlated with IL-3--induced proliferation. The IL-3--induced upregulation of LYN kinase activity may be mediated by the 120-Kd common subunit of the human IL-3 and GM-CSF receptors, because GM-CSF also stimulated marked increases in the activity of the LYN kinase, whereas granulocyte-CSF (G-CSF) did not, despite inducing cellular proliferation. These observations provide the first example of an IL-3--regulable PTK, and strongly suggest that the p53/56-LYN kinase participates in early IL-3--initiated signalling events, at least in some human leukemic cell lines.

Cell Line↗
PubMedDate not supplied

A Drosophila genomic sequence with homology to human epidermal growth factor receptor.

Vertebrate genomes contain an extensive family of genes possessing varying degrees of homology to the v-src oncogene. Most src-related proteins identified to date are intracellular and membrane-associated, although some are transmembrane proteins and function as receptors for peptide growth factors. Three Drosophila gene sequences related to the v-src gene have been identified, each exhibiting a high degree of homology to one or more of the src-family members encoding an intracellular protein. We have isolated a panel of cloned Drosophila sequences exhibiting weak v-src hybridization and were interested to determine whether any members of this group represented homologues of additional known src-family genes, especially those functioning as growth factor receptors. As we report here, four of these clones, representing overlapping portions of the same genomic segment, hybridized preferentially with the v-erb-B oncogene and were further characterized. The deduced amino-acid sequence from a portion of this Drosophila genomic segment is 77% homologous to the kinase domain of human epidermal growth factor (EGF) receptor, a substantially greater degree of homology than was observed with any other known src-family member. By hybridization with a human EGF receptor complementary DNA clone probe, we demonstrate that the same genomic segment showing homology with the kinase domain also contains sequences related to the extracellular domain of the EGF receptor gene.

Animals↗
PubMed1994

ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.

Protein tyrosine kinases (PTKs) play an integral role in T cell activation and differentiation. Defects in the Src-family PTKs in mice and in T cell lines have resulted in variable defects in thymic development and in T cell antigen receptor (TCR) signal transduction. Here, three siblings are described with an autosomal recessive form of severe combined immunodeficiency disease (SCID) in which ZAP-70, a non-Src PTK, is absent as a result of mutations in the ZAP-70 gene. This absence is associated with defects in TCR signal transduction, suggesting an important functional role for ZAP-70.

Amino Acid 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↗
PubMed1989

Characterization and functional allotment of proto-oncogenes belonging to the src family.

Eight proto-oncogenes encode cytoplasmic protein tyrosine kinases whose primary structures are closely related to that of p60proto-src. This group of related genes is called the src-family. Their products share common structures: namely, a glycine residue at position 2 from the N terminus, a unique domain, a modulatory domain (SH2, SH3), a kinase (catalytic) domain, and a regulatory domain. Oncogenic activation of these genes may occur by various mutations such as 1) deletion or substitution of the tyrosine at the C-terminal position, 2) mutation at a defined position in the kinase domain or 3) deletion or mutation in the modulatory domain. The expressions of the proto-oncogenes of this family are tissue specific and are unique for each gene, suggesting functional allotment of these gene products. In the hematopoietic system, the genes appear to be expressed in specific cell lineages. In this paper, the functional allotments of these genes in the hematopoietic system are considered with special reference to the functions of lck, lyn, and fyn in cells of T and B lymphocyte lineages.

Amino Acid Sequence↗
PubMed1993

Fc gamma RIIIA-mediated signaling involves src-family lck in human natural killer cells.

The cell surface receptor for IgG Fc domain (Fc gamma RIIIA, CD16) is present as a multimeric complex on human NK cells and is essential for antibody-dependent cellular cytotoxicity. Although CD16 engagement induces tyrosine phosphorylation of several substrates, the intracytoplasmic domains of the CD16 molecule lack intrinsic protein tyrosine kinase consensus sequences. We report here that, in human NK cells, engagement of the CD16 receptor induces tyrosine phosphorylation of p56lck tyrosine kinase. CD16 triggering through anti-receptor antibody enhances in vitro kinase activity of phosphotyrosyl proteins and autophosphorylation activity of anti-lck immunoprecipitates. Similar results were obtained after stimulation with IL-12. In vivo labeling experiments showed that CD16 cross-linking primarily induces in vivo phosphorylation of p56lck at the tyr-394 autophosphorylation site. Using in vitro kinase assay of CD16 immunoprecipitates from lysates of human NK cells, we demonstrated that a phosphorylated protein of 62 to 65 kDa was co-precipitated with CD16. Reprecipitation experiments also suggested that a small amount of p56lck may interact with this complex. These results indicate that CD16 triggering involves p56lck and that this kinase is therefore involved in the signaling pathways regulating NK cell cytolytic functions. However, additional kinases not yet identified are also likely to play a role in the early signaling events through CD16.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1993

The interleukin-2 receptor complex and signal transduction: role of the beta-chain.

Proliferation of lymphocytes is regulated by a variety of cytokines, among which interleukin-2 (IL-2) is well characterized for its potent ability to promote cell growth. The IL-2 signal(s) is transmitted to the cell interior via its homologous receptor (IL-2R). The functional high affinity IL-2R is a multichain complex consisting of at least three distinct components, IL-2R alpha, beta and gamma. None of these components possess an intrinsic protein tyrosine kinase (PTK) domain. cDNA expression studies, have revealed the critical role of IL-2R beta, but not IL-2R alpha, in the IL-2R-mediated intracellular signaling process. Studies utilizing mutants of IL-2R beta identified an essential cytoplasmic region, defined as the 'serine-rich' region, for IL-2-induced cell growth. With respect to the involvement of PTK(s) in IL-2R mediated signal transduction, it has been demonstrated that p56lck, a member of the src-family PTKs interacts with the IL-2R beta. In fact, IL-2 stimulation increases the PTK activity of p56lck. Another cytoplasmic region of the receptor, defined as the 'acidic' region has been found to be critical for the association of p56lck with the IL-2R beta. Interestingly the 'serine-rich' and 'acidic' regions of IL-2R beta are both required for the PTK activation of p56lck. Expression studies with mutant IL-2R beta cDNAs have revealed a bifurcation in the IL-2 signaling pathway. One pathway involves the src-family PTK activation which is linked to the activation of p21ras and the subsequent induction of c-fos/c-jun protooncogenes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The regulation of B- and T-lymphocyte activation by the transmembrane protein tyrosine phosphatase CD45.

The expression of transmembrane protein tyrosine phosphatase CD45 is required for antigen-induced activation of T and B lymphocytes. During the past year, advances have been made in our understanding of CD45 regulation and its role in regulating Src-family members. The importance of CD45 in thymocyte maturation has been demonstrated through the generation of CD45-knockout mice.

Animals↗
PubMed1994

Inhibition of phospholipase D of human platelets by protein tyrosine kinase inhibitors.

Protein tyrosine kinases (PTKs) of the src family are thought to play an important role in platelet signal transduction, but little is known about the targets of these enzymes in platelets. We determined that exposure of human platelets to pervanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in the activity of phospholipase D (PLD), an enzyme that might be involved in signaling events leading to aggregation or secretion. To further investigate whether tyrosine phosphorylation is a step in the pathway of activation of PLD in response to thrombin, we tested the effects of a series of PTK inhibitors on the activity of platelet PLD. PLD was activated in response to 0.3 U/ml thrombin, and this activation was reduced by several of the PTK inhibitors, especially genistein, methyl 2,5-dihydroxycinnamate (MDHC), ST271, and the tyrphostins A25 and A47. In saponin-permeabilized platelets, we observed a marked inhibition of GTP-gamma S-stimulated PLD by many of the PTK inhibitors, consistent with the possibility that PTKs are involved in the regulation of PLD activity by a G-protein or small GTP-binding protein. MDHC did not affect PLD activity in permeabilized cells, which suggests that this compound might inhibit PLD in intact platelets via another pathway. The inhibitors were also tested for their effects on the phosphorylation of a peptide substrate of src-family PTKs in a platelet membrane preparation and in permeabilized platelets. Several of the compounds partially inhibited peptide phosphorylation in the membrane preparation and in permeabilized platelets, most notably ST271, ST638, and tyrphostin A25.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Translational regulation of the lymphocyte-specific protein tyrosine kinase p56lck.

The lck gene encodes a lymphocyte-specific membrane-associated protein tyrosine kinase that is implicated in signal transduction processes that regulate T-cell growth. Previous studies demonstrate that lck transcripts, unlike most other vertebrate mRNAs, contain 5' proximal AUG sequences upstream relative to the major open reading frame (5' AUGs) that serve as satisfactory targets for recognition by 40S ribosomal subunits. There exist two distinct lck transcripts, generated through alternative promoter utilization, that differ only in their 5' untranslated region sequences. Both transcripts are present in murine and human lymphocytes, and the structures of the two distinct 5' untranslated regions have been closely conserved since the divergence of these species. Moreover, both transcripts contain 5'-AUG elements. Intriguingly, inspection of primary sequence data for other src-family transcripts reveals that virtually all contain 5'AUG triplets, and many contain a large number of these potential initiators. By analogy with the well-studied translational regulation of the yeast GCN4 gene, I propose that control of ribosomal reinitiation permits tight regulation of expression of the lck gene product and of other src-like protein tyrosine kinases.

Animals↗