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.

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.

tkl is the avian homolog of the mammalian lck tyrosine protein kinase gene.

We have tested the possibility that tkl, a partially characterized avian tyrosine protein kinase gene, is the chicken homolog of lck, a lymphocyte-specific mammalian gene. Using polymerase chain reactions, we have cloned sequences encoding the previously unidentified amino terminus of the tkl gene product. The newly defined unique domain of Tkl displayed significant identity (68%) to the equivalent region of the mammalian lck gene product, p56lck. This identity included a glycine residue at position 2 (present in all Scr-related tyrosine protein kinases) and a cysteine motif at positions 20 and 23, which allows binding of p56lck to CD4 and CD8 in mammalian T lymphocytes. A specific RNase protection assay revealed that, in contrast to a previous report (K. Strebhardt, J. I. Mullins, C. Bruck, and H. Rübsamen-Waigmann, Proc. Natl. Acad. Sci. USA 84:8778-8782, 1987), tkl expression is restricted to the lymphoid tissues thymus and spleen. Moreover, the absence of tkl transcripts in the bursa of Fabricius suggested that this gene is expressed in avian T lymphocytes but not in B lymphocytes. A polyclonal rabbit antiserum raised against the unique domain of Tkl recognized a 56-kDa polypeptide with associated protein kinase activity from avian thymus-derived cells. Additional studies showed that p56tkl is structurally similar to mammalian p56lck and that it is physically associated with the avian CD4 and CD8 T-cell surface antigens. It was also determined that tkl transcripts have one major type of 5' untranslated region (UTR), which differs greatly from the two known 5' UTRs of mammalian lck mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Cloning and characterization of a novel Krüppel-like zinc finger gene, ZNF268, expressed in early human embryo.

With the aim of identifying genes involved in early human embryonic development, we have isolated a cDNA clone representing a novel human zinc finger gene ZNF268 from 3 week old human embryo cDNA library using a differential hybridization strategy. The complete cDNA sequence of ZNF268 contained an open reading frame of 2841 nucleotides that encodes a 947 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and 24 C-terminal zinc fingers. Northern blot analysis showed that ZNF268 mRNA is mainly expressed in 3-5 week old human embryos suggesting it could play certain roles in the embryogenesis. The gene consists of six exons spanning about 22 kb of genomic DNA. According to the genomic sequence from the HTGS database, the ZNF268 gene is assigned to human chromosome 5.

Construction and characterization of a cDNA library from 4-week-old human embryo.

Development and differentiation studies of early human embryos have been severely impeded by general difficulties in obtaining suitable samples. In order to isolate and identify new genes expressed during early human development, we constructed and characterized a PCR-based cDNA library using a 4-week-old chorion-free human embryo. The constructed cDNA library contained 6.3 x 10(6) directional recombinants, and its insert size ranged from 0.4 to 1.8 kb. The cDNA library proportionally represents the mRNA population, containing beta-actin, tPA and LINE1 repetitive sequences at the expected frequencies as in other conventionally constructed and PCR-based cDNA libraries. PCR analyses of the library for specific genes have also revealed the presence of cDNAs for developmentally important genes such as CD59, MCP, Quox-1 and ZNF268. Among the 70 randomly selected cDNA clones, 53% encoded previously known genes, 26% matched with anonymous sequences, and 17% showed no sequence similarity and were designated as human early embryo-specific ESTs. These results demonstrate the sequence complexity and relatively low redundancy of our cDNA library. Furthermore, approximately 40% of those randomly analyzed clones contained full-length encoding regions. To our knowledge, this is the first description of the PCR-based cDNA library from a 4-week-old chorion-free human embryo, and the presence of novel sequences within this library makes it a valuable and unique resource for studying gene expression and regulatory mechanisms that underlie the early process of human embryogenesis.

Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii.

The goal of this work was to investigate the mechanism of drug resistance in Leishmania enriettii as a model system for drug resistance both in human leishmaniasis and on other parasitic diseases. Parasites were selected in increasing concentrations of vinblastine, an inhibitor of microtubule assembly, and resistant clones were isolated which grew in concentrations 5-30 times the IC50 (30 micrograms ml-1) of parental cells. The vinblastine-resistant parasites were also resistant to puromycin, an unrelated drug which inhibits protein synthesis. This cross-resistance to unrelated drugs had previously been observed in mammalian cells and recently in L. donovani. The proposed mechanism for this cross-resistance is drug efflux mediated by increased expression of a P-glycoprotein molecule encoded by a multidrug resistance (mdr) gene. Here we report the identification, cloning and sequencing of an mdr-like gene from L. enriettii, lemdr1, and demonstrate that this gene is amplified on an extrachromosomal circle of 35-40 kb in vinblastine-resistant L. enriettii. The longest open reading frame in the cloned gene is 1280 amino acids with a predicted protein of 140 kDa. The predicted protein has a structure similar to that for all other reported P-glycoproteins namely 12 transmembrane domains and 2 ATP binding sites, arranged in 2 similar half-molecules. Comparison of the primary amino acid sequence with other known mdr gene products demonstrates a significant homology with 37% amino acid identity with human mdr1 and 83% identity with the L. donovani ldmdr1 gene. The lemdr1 gene was cloned in the expression vector pALTNEO and transfected into wild-type L. enriettii and the resulting transfected cells were resistant to vinblastine but at lower levels than in the selected mutant cells.

A homologous recombination strategy to analyze the vinblastine resistance property of the V-circle in Leishmania.

The generation of extrachromosomal DNA elements in Leishmania sp. can occur naturally or during in vitro selection with drugs. Previously, we had established a strong association between V-circle amplification and drug resistance in L. enriettii stepwise selected with increasing concentrations of vinblastine. Further, we demonstrated the presence of the lemdr1 gene in the amplified V-circle and subsequent functional analysis by transfection of this gene alone confirmed its role in conferring a drug-resistant phenotype, but the level of resistance was significantly lower than in cell lines obtained from stepwise drug selection. The aim of this work was to determine if other genes either on the V-circle or elsewhere in the genome were necessary for expression of vinblastine resistance. We report here the development of a homologous recombination method to convert the entire V-circle from the LeV160 cell line into a shuttle vector and further the targeted disruption of specific sites within the V-circle. Our results clearly demonstrate that the V-circle alone is sufficient to confer full vinblastine resistance and the disruption of the lemdr1 locus destroys the ability of the V-circle to confer vinblastine resistance.

Establishment and characterization of a new xenograft-derived human esophageal squamous cell carcinoma cell line SLMT-1 of Chinese origin.

A new human esophageal cancer cell line, named SLMT-1, was established from a nude-mouse xenograft of a well-differentiated esophageal squamous cell carcinoma (ESCC) of the lower esophagus from a male Hong Kong Chinese patient. SLMT-1, passaged over 34 times and with a doubling time of 31 hours, has the microscopic features of epithelial cells with adherent growth as a monolayer. The general biologic properties of SLMT-1 cells were characterized by (1) a positive test of tumorigenicity obtained by injecting cells subcutaneously into athymic nude mice and observing their development into well-differentiated squamous cell carcinoma; (2) immunohistochemical staining using antibodies (AE1/AE3, CAM5.2 and MAK 6) which show the presence of cytokeratin intermediate filaments; and (3) electron microscopy demonstrating the morphologic features of epithelial cells with the presence of desmosomes. The cytogenetic abnormalities found in both the primary culture and SLMT-1 included der(1;14)(q10;q10), add(1)(p1?), +1, +2, del(3)(q11), +6, +7, i(8)(q10), +8, +10, +11, -13, -15, +16, +17, -18, -19, -Y and marker chromosomes. Additional changes observed in the 34th passage included gains as well as losses of both numerical and structural abnormalities. Comparative genomic hybridization (CGH) indicated copy number gains on chromosomal regions 3q32-qter, 5p, 8p12-p11.2, 11q13-q22 and 13q22-qter, and loss of the Y. The gains of 8p12-p11.2 in SLMT-1 cells are novel to ESCC. Based on its distinct and common characteristics, the SLMT-1 cell line serves as a useful tool for studying the molecular and genetic basis of the pathogenesis of ESCC.

Functional reconstitution of purified chloroquine resistance membrane transporter expressed in yeast.

Malaria is one of the major parasitic diseases. Current treatment of malaria is seriously hampered by the emergence of drug resistant cases. A once-effective drug chloroquine (CQ) has been rendered almost useless. The mechanism of CQ resistance is complicated and largely unknown. Recently, a novel transmembrane protein, Plasmodium falciparum chloroquine resistance transporter (PfCRT), has fulfilled all the requirements of being the CQ resistance gene. In order to elucidate the mechanism how PfCRT mediates CQ resistance, we have cloned the cDNA from a CQ sensitive parasite (3D7) and tried to express it in Pichia pastoris (P. pastoris) but with unsuccessful results due to AT-rich sequences in the malaria genome. We have therefore, based on the codon usage in P. pastoris, chemically synthesized a codon-modified pfcrt with an overall 55% AT content. This codon-modified pfcrt has now been successfully expressed in P. pastoris. The expressed PfCRT has been purified with immuno metal affinity chromatography (IMAC) and then reconstituted into proteoliposome. It was found that proteoliposomes have a saturable, concentration and time-dependent CQ transport activity. In addition, we found that proteoliposomes with resistant PfCRT(r) (K76T or K76I) showed an increased CQ transport activity compared to liposomes with lipid alone, or proteoliposomes reconstituted with sensitive PfCRT(s) (K76) protein. This activity could be inhibited by nigericin and decreased with the removal of Cl(-). This work suggests that PfCRT is mediating CQR in P. falciparum by virtue of its changes in CQ transport activity depending on pH gradient and chloride ion in the food vacuole.

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.

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.

Differential intrinsic enzymatic activity of Syk and Zap-70 protein-tyrosine kinases.

Syk and Zap-70 are related protein-tyrosine kinases implicated in antigen and Fc receptor signaling. While Zap-70 is restricted to T-cells and natural killer cells, Syk accumulates in B-cells, mast cells, platelets, and immature T-cells. In addition, we found that an isoform of Syk (SykB), which carries a 23-amino acid deletion in the "linker" region, is prominently expressed in bone marrow. To better understand the relative impact of Syk, SykB, and Zap-70 on signal transduction, we compared their intrinsic enzymatic properties in transiently transfected COS-1 cells and in hemopoietic cells. Using modified versions of these enzymes bearing a common Myc epitope at the amino terminus, we determined that the ability of Syk and SykB to undergo autophosphorylation and to phosphorylate erythrocyte band 3 in immune complex kinase reactions was at least 100-fold greater than that of Zap-70. Similarly, Syk and SykB, but not Zap-70, caused prominent tyrosine phosphorylation of p120(c-)cbl in COS-1 cells. A similar pattern of activity was also noted for endogenous Syk and Zap-70 from hemopoietic cells. To understand the structural basis for these characteristics, we also created and analyzed a series of chimeras between Syk and Zap-70. These studies indicated that the catalytic domain of Syk and Zap-70, but not their SH2 domains, linker region or carboxyl-terminal tail, was responsible for their respective activity. Taken together, these data demonstrated that the intrinsic enzymatic activity of Syk and SykB is superior to that of Zap-70 and that such a distinction relates to structural variations in the catalytic domain.

Chk, a Csk family tyrosine protein kinase, exhibits Csk-like activity in fibroblasts, but not in an antigen-specific T-cell line.

The Csk family of tyrosine protein kinases comprises two members named Csk and Chk. These enzymes phosphorylate the carboxyl-terminal tyrosine of Src-related kinases in vitro, thereby repressing their activity. Csk has been found to be necessary for normal embryonic development, and to be a potent negative regulator of antigen receptor signaling in T-lymphocytes. As the functions of Chk in mammalian cells are not known, we examined its ability to carry out Csk-like functions in vivo. Like p50csk, Chk reduced the elevated phosphotyrosine levels and the augmented activity of Src family kinases in Csk-deficient fibroblasts. Contrary to Csk, however, Chk was inefficient at repressing antigen receptor-induced signals in a T-cell line (BI-141). We also noted that Chk, but not Csk, failed to stably associate with cellular membranes following addition of a membrane targeting signal to its amino terminus. This observation suggested that Chk may contain dominant targeting sequences disallowing its recruitment to cellular membranes. Hence, these data demonstrate that Chk can mediate some, but not all, Csk-related functions in vivo. Moreover, they suggest that the "restricted" function of Chk may relate at least in part to its inability to be recruited to certain cellular locales.

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.

Trimethylaminuria is caused by mutations of the FMO3 gene in a North American cohort.

Trimethylaminuria (TMAuria) (McKusick 602079) first described in 1970 is an autosomal recessive condition caused by a partial or total incapacity to catalyze the N-oxygenation of the odorous compound trimethylamine (TMA). The result is a severe body odor and associated psychosocial conditions. This inborn error of metabolism, previously thought to be rare, is now being increasingly detected in severe and milder presentations. Mutations of a phase 1 detoxicating gene, flavin-containing monooxygenase 3 (FMO3), have been shown to cause TMAuria. Herein we describe a cohort of individuals ascertained in North America with severe TMAuria, defined by a reduction of TMA oxidation below 50% of normal with genotype-phenotype correlations. We detected four new FMO3 mutations; two were missense (A52T and R387L), one was nonsense (E314X). The fourth allele is apparently composed of two relatively common polymorphisms (K158-G308) found in the general population. On the basis of this study we conclude that one common mutation and an increasing number of private mutations in individuals of different ethnic origins cause TMAuria in this cohort.

Mutations of the flavin-containing monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication.

Individuals with the recessive condition trimethylaminuria exhibit variation in metabolic detoxication of xenobiotics by hepatic flavin-containing monooxygenases. We show here that mutations in the human flavin-containing monooxygenase isoform 3 gene ( FMO3 ) impair N -oxygenation of xenobiotics and are responsible for the trimethylaminuria phenotype. Three disease-causing mutations in nine Australian-born probands have been identified which share a particular polymorphic haplotype. Nonsense and missense mutations are associated with a severe phenotype and are also implicated in impaired metabolism of other nitrogen- and sulfur-containing substrates including biogenic amines, both clinically and when mutated proteins expressed from cDNA are studied in vitro . These findings illustrate the critical role played by human FMO3 in the metabolism of xenobiotic substrates and endogenous amines.

Antiproliferative activity of the extract of Gleditsia sinensis fruit on human solid tumour cell lines.

BACKGROUND: The fruit extract of Gleditsia sinensis Lam. (GSE) is a traditional herbal medicine that is saponin-rich. However, its activity on solid tumour cell lines has never been demonstrated. METHODS: The activity of GSE was demonstrated in four cancer cell lines (breast cancer MCF-7, MDA-MB231, hepatoblastoma HepG2 and oesophageal squamous carcinoma cell line SLMT-1) using MTT assay, anchorage-independent clonogenicity assay, DNA laddering and in situ cell death detection. RESULTS: The mean MTT(50) (the mean concentration of GSE to reduce MTT activity by 50%) ranged from 16 to 20 microg/ml of GSE. An anchorage-independent clonogenicity assay showed that all of the four solid tumour cell lines gradually lost their regeneration potential after treatment with GSE, DNA fragmentation and TUNEL analysis demonstrated that the action of GSE is both dose- and time course-dependent. CONCLUSIONS: Our results suggest that GSE has a cytotoxic activity and can induce apoptosis in human solid tumour cell lines.