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PubMed1990

Coordinate expression of src family protooncogenes in T cell activation and its modulation by cyclosporine.

Activation of T lymphocytes induces transcription of several important genes which encode lymphokines and lymphokine receptors as well as "proliferation complementary" protooncogenes like c-myc or c-fos. Recently, the expression of lck gene, one of the src family gene, has also been shown to be modulated during T cell activation. We, therefore, assessed the question of whether other src family genes are expressed during the activation of T cells and of whether cyclosporine, a potent immunosuppressive drug, affects expression of these genes. We examined the expression of four different src family genes (lck, c-src, fyn, and c-fgr) in addition to the expression of IL-2, c-fos, c-myc, and actin genes in murine T cells which were activated with PMA plus ionomycin or PMA plus anti-CD3 mAb. We found that T cell activation was associated with the up-regulation of these src family genes and that the expression of these genes was specifically blocked in the presence of cyclosporine indicating that these activation-related genes were coordinately regulated.

Blotting, Northern↗
PubMed1990

Expression of cellular-yes protein in mammalian tissues.

Expression of the c-yes proto-oncogene in normal mammalian tissues was studied by immunohistochemistry and by immunoprecipitation assays using affinity purified anti-yes IgG. The antibody was raised against the amino-terminal domain of the human c-yes protein expressed in bacteria as a trpE-yes fusion protein. We detected p62c-yes in a variety of rat and human tissues but its relative level of expression varied significantly among different cell types. Certain epithelial cells, platelets and spermatid acrosomes showed the highest levels of p62c-yes. Intracellular localization of p62c-yes in epithelial cells differed among organs performing different physiological functions. The overall pattern of expression suggests that p62c-yes may be involved in numerous cellular pathways.

Acrosome↗
PubMed1989

The sequence of chicken c-yes and p61c-yes.

We have deduced the sequence of the protein encoded by the chicken c-yes gene from overlapping cDNA clones. The predicted protein, p61c-yes, contains 541 amino acids and has a molecular weight of 60,911 with the amino terminal methionine residue. Chicken p61c-yes differs from Y73 virus p90gag/v-yes in three respects. First, the carboxy-terminal eight amino acids of p61c-yes are replaced by three amino acids in p90gag/v-yes, which are encoded by the avian leukemia virus env gene. This alteration changes the position and context of a tyrosine residue in p61c-yes. Second, nucleotides which are present as 5' non-translated sequence in the p61c-yes mRNA, are translated in the p90gag/v-yes mRNA. Third, there are fourteen dispersed nucleotide differences in Y73 v-yes which result in six amino differences between the body of p90gag/v-yes and p61c-yes. Chicken p61c-yes differs from human p61c-yes at 43 residues, and from chicken pp60c-src at 122 residues.

Amino Acid Sequence↗
PubMed1989

The yes proto-oncogene is present in amphibians and contributes to the maternal RNA pool in the oocyte.

Low stringency screening of a cDNA library from the frog Xenopus laevis with a viral yes oncogene probe resulted in the isolation of clones with restriction maps which were distinct from previously identified X. laevis src cDNA clones. The polypeptide produced by in vitro translation of RNA synthesized from one of the clones was slightly larger in size than X. laevis pp60src, but gave protease cleavage patterns which were nearly identical to those yielded by pp60src. Determination of the DNA sequence of this clone revealed that it is derived from a X. laevis yes gene homologue. Transcripts from the yes gene were found in the ovary and consisted of a major 3.4 kb species and additional minor species. These findings indicate that the duplication event separating the src and yes genes occurred prior to the appearance of amphibians and that the yes gene product is likely to play an important role in oogenesis or early development.

Amino Acid Sequence↗
PubMed1989

Regulation of c-fgr proto-oncogene expression in Burkitt's lymphoma cells: effect of interferon treatment and relationship to EBV status and c-myc mRNA levels.

The proto-oncogene c-fgr is expressed in transformed human B lymphoid cell lines and has been reported to be induced in cells infected with the Epstein-Barr virus (EBV). We have compared the levels of c-fgr mRNA in several B cell lines and have examined the effects of interferon-induced changes in growth rate of Daudi cells on the concentration of this mRNA. High levels were found in exponentially growing EBV-positive Raji and Daudi cells but the amounts in B95-8 and P3HR-1 cells (also EBV-positive) were lower than in the EBV-negative cell line Ramos. Growth inhibition of Daudi cells by interferon-alpha is preceded by a reduction in c-fgr expression, with a 56% decrease observed within 6 h. The differences in the amounts of c-fgr mRNA in the various cell lines and in control versus interferon-treated cells are similar to the differences in the c-myc mRNA contents of these cells. These results indicate that c-fgr expression bears little relationship to the EBV status of B lymphoid cell lines but may play a role, in conjunction with c-myc expression, in growth regulation by interferons. Other conditions which influence Daudi cell proliferation, such as treatment with a phorbol ester or growth to high cell density, also inhibit c-fgr expression but to a lesser extent than interferon treatment.

Blotting, Northern↗
PubMed1990

Phosphorylation of src-phosphopeptides by casein kinases-1 and -2: favourable effect of phosphotyrosine.

The synthetic phosphotyrosyl tridecapeptide H-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-Gln-Gly-OH, reproducing a major phosphoacceptor site of protein tyrosine kinases of the src-family, can be phosphorylated at Thr-9 by both casein kinases -1 and -2. Its shorter derivative H-Asn-Glu-Tyr(P)-Thr-Ala-OH is not affected by casein kinase-1 while representing a substrate as good as the tridecapeptide for casein kinase-2. The unphosphorylated analogue H-Asn-Glu-Tyr-Thr-Ala-OH, however, is a much poorer substrate, and no significant phosphorylation could be observed of its O-methyl ether derivative H-Asn-Glu-Tyr(Me)-Thr-Ala-OMe. These data on one side corroborate the concept that casein kinase-1 recognizes residues located on the C-terminal edge of acidic stretches, providing, on the other, the evidence that phosphotyrosyl side chains can act as specificity determinants for casein kinase-2.

Amino Acid Sequence↗
PubMed1992

Interleukin 4 inhibits IL-2-induced proliferation of a human T-leukemia cell line without interfering with p56-LCK kinase activation.

Recently we described the establishment in culture and the immunophenotypic and functional characteristics of a human T-leukemia line TALL-103/2 derived from the T-cell receptor (TCR)-gamma/delta subset of T-lymphocytes. TALL-103/2 cells are absolutely dependent on interleukin 2 (IL-2) for their growth and survival in culture and thus provide a model cell line for studies of IL-2 signal transduction in a TCR-gamma/delta T-cell. In this report, we focus on the regulation of SRC-family protein tyrosine kinases (PTKs) by IL-2. TALL-103/2 cells were found to contain p56-LCK, p59-FYN, p62-YES and p53/56-LYN. Stimulation of growth factor-deprived TALL-103/2 cells with IL-2, however, induced increases in the relative activity only of the p56-LCK kinase. This IL-2-mediated increase in LCK kinase activity was manifested both by increased kinase autophosphorylation and by increased phosphorylation of the exogenous substrate enolase during in vitro kinase assays. Furthermore, immunoblot assays determined that the levels of p56-LCK protein were unaltered by IL-2-treatment, indicating that the measured elevations in LCK kinase activity reflected an increase in the specific activity of this PTK. In TALL-103/2 cells, IL-2 stimulated concentration-dependent increases in p56-LCK activity that displayed rapid and transient kinetics: detectable increases occurred within 1 minute after IL-2 stimulation, peaked at 10 minutes, and declined to baseline levels by 30 minutes. Treatment of TALL-103/2 cells with IL-4 abrogated IL-2-initiated proliferation, but did not inhibit IL-2-mediated activation of p56-LCK.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-Response Relationship, Drug↗
PubMed1989

Transformation of NIH 3T3 fibroblasts by an activated form of p59hck.

Phosphorylation of a tyrosine residue near the carboxy terminus of src-family protein tyrosine kinases is believed to regulate the biological activity of these gene products. Conversion of this tyrosine in p59hck (Tyr-501) to a phenylalanine residue by using oligonucleotide-directed mutagenesis yielded a product (p59hckF501) with very potent transforming activity. Quantitative analysis by a soft-agar cloning assay revealed that p59hckF501 was more than 100-fold more effective than a closely related transforming element, p56lckF505, in colony formation. Cells bearing p59hckF501 had increased levels of protein phosphotyrosine. The ability of p59hckF501 to transform NIH 3T3 cells was abolished by a second mutation believed to destroy the ATP-binding domain.

Amino Acid Sequence↗
PubMed1989

A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity.

A fourth type of rat phosphoinositide-specific phospholipase C (PLC IV) has been cloned for cDNA and sequenced. PLC IV is distinct from the other three types of rat PLC (PLC I, II, and III) with respect to primary structure and tissue distribution of its mRNAs. PLC IV contains two homologous regions included commonly in PLC I, II, and III and is most similar to PLC II (identity: 50.2%). PLC IV, in common with PLC II, has a sequence homologous to the N-terminal regulatory domains of nonreceptor tyrosine kinases of the src-family of oncogenes. Using an Escherichia coli expression system, we succeeded in producing active PLC IV in E. coli crude extracts. Various truncation experiments of the PLC IV cDNA revealed that the src-related domain is not necessary for catalytic activity while both domains homologous among PLC I-IV are essential. PLC IV is expressed in various rat tissues and abundant in spleen, suggesting that PLC IV plays a fundamental role in cellular functions such as growth and secretion.

Amino Acid Sequence↗
PubMed1991

Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn, and Yes protein-tyrosine kinases in human platelets.

Activation of platelets with thrombin and other agonists causes a rapid increase in the phosphorylation of multiple proteins on tyrosine. To identify candidate protein-tyrosine kinases (PTKs; EC 2.7.1.112) that may be responsible for these phosphorylation events, we analyzed the expression of seven Src-family PTKs and examined the association of these kinases with known platelet membrane glycoproteins. Five Src-related PTKs were detected in platelets: pp60SRC, pp60FYN, pp62YES, pp61HCK, and two LYN products of Mr 54,000 and 58,000. The Fgr and Lck PTKs were not detected. Although strict comparative quantification of protein levels was not possible, pp60SRC was detected at higher levels than any of the other kinases. In addition, glycoprotein IV (GPIV, CD36), one of the major platelet membrane glycoproteins, was associated in a complex with the Fyn, Yes, and Lyn proteins in platelet lysates. Similar complexes were also found in two GPIV-expressing cell lines, C32 melanoma cells and HEL cells. Since PTKs appear to be involved in stimulus-response coupling at the plasma membrane, these results suggest that ligand interaction with GPIV may activate signaling pathways that are triggered by tyrosine phosphorylation.

Antigens, CD↗
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↗
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↗
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↗
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↗
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↗