Betulinic acid prevents liver fibrosis by binding Lck and suppressing Lck in HSC activation and proliferation.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum japonicum Thunb. ex Murray (Hypericaceae), named 'Tianjihuang' is a traditional Chinese medicine with hepatoprotective, antibacterial, and antitumour effects. Betulinic acid (BA) is its active constituent and has been found to have a number of biological effects, including antiviral, anti-inflammatory, and anti-malarial therapeutic properties. Non-alcoholic fatty liver disease and acute alcoholic liver injury have both been proven to benefit from BA. BA's effects and mechanism on liver fibrosis are still unknown. AIM OF THE STUDY: The purpose of this study was to explore the influence of BA on lymphocyte-specific protein tyrosine kinase (Lck), a non-receptor Src family kinase, that reduces liver fibrosis by inhibiting the phosphorylation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways through the interaction of Lck and SOCS1. MATERIALS AND METHODS: A liver fibrosis model was established in vivo with CCl4 using haematoxylin and eosin (HE) staining, Masson staining, immunohistochemical staining, and immunofluorescence staining. Hepatic stellate cells were induced with transforming growth factor (TGF)-ß1 in vitro, using Western blotting, immunofluorescence staining, and a cell scratch assay. RESULTS: In a CCl4-induced mouse hepatic fibrosis model and in TGF-ß1-activated HSC-T6 cells, BA markedly reduced fibrosis, as demonstrated by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in vivo and in vitro. BA significantly suppressed the activity and expression of Lck in vitro. Overexpression of Lck may diminish the effect of BA on liver fibrosis. In vitro, BA also greatly increased the expression of suppressor of cytokine signalling 1 (SOCS1) while it considerably inhibited the expression of p-JAK and p-STAT1. CONCLUSIONS: These findings suggest that BA promotes the expression of SOCS1 by the inhibiting the interaction between Lck and SOCS1, followed by the inhibition of JAK/STAT phosphorylation to prevent the progression of liver fibrosis. Therefore, BA could be used as a promising natural supplement for the treatment of liver fibrosis.

[Mutation of palmitoylation site of linker for activation of T cells inhibits signal transduction mediated by glycosyl phosphatidyl inositol-anchored CD59 in T cells].

OBJECTIVE: To construct the lentivirus carrying the mutated palmitoylation site of the linker for activation of T cells (LAT) and infect Jurkat cells with it to establish stable cell line, and to investigate the effect of LAT palmitoylation mutation on T cell signaling induced by CD59. METHODS: Negative control (neg-EGFP) and LAT-M-EGFP fusion protein gene vectors were respectively constructed and then packaged using lentivirus. Subsequently, Jurkat cells were infected with them to establish stable cell lines. Confocal laser scanning microscopy was used to observe the infection efficiency and the distribution of fusion proteins in Jurkat cells. CCK-8 assay was used to detect the change of cell proliferation activity after CD59 mAb supplementation. Flow cytometry was used to determine the apoptosis rate. Western blotting was used to examine the levels of phospholipase C-γ1 (PLC-γ1) and lymphocyte-specific protein tyrosine kinase (LCK). RESULTS: Confocal laser scanning microscopy revealed that LAT molecules of LAT-M group scattered on cell membrane, and there was no obvious clustered region after cross linkage with CD59 mAb. Compared with the negative control group, the cell proliferation activity of LAT-M group significantly decreased, and the quantity of middle-late apoptotic cells significantly increased; Western blotting showed that the expression levels of PLC-γ1 and LCK in LAT-M group was roughly the same with those in negative control group, and after CD59 mAb stimulation, there was no obvious change in LAT-M group, while the levels in negative control group were reduced. CONCLUSION: LAT-M-EGFP fusion protein could not locate on lipid rafts of Jurkat cells infected with LAT palmitoylation mutation. In addition, the growth of the cells carrying the LAT-M-EGFP was inhibited. The palmitoylation mutation of LAT attenuated the signal transduction induced by glycosylphosphatidylinositol-anchored CD59 in T cells.

N-Octanoyl dopamine transiently inhibits T cell proliferation via G1 cell-cycle arrest and inhibition of redox-dependent transcription factors.

Recently, we developed a nonhemodynamic dopamine derivative, NOD, which has profound anti-inflammatory effects in vitro. As NOD also protects rats from ischemic AKI, the present study tested whether NOD is able to modulate cellular immunity for potential use as a T cell-suppressive agent. To this end, T cells were stimulated by anti-CD3/CD28 or PMA/ionomycin in the presence or absence of different concentrations of NOD. T cell proliferation, activation markers, intracellular cytokine expression, and activation of transcription factors were assessed. Whereas T cell proliferation was inhibited significantly by NOD at Day 3, proliferation was restored at Day 7 or later depending on the NOD concentration used. Inhibition of proliferation was reflected by a diminished CD25 expression and switch from naive to memory T cells. Early TCR activation events were unaffected, yet NF-κB and AP-1 were strongly inhibited by NOD. The inhibitory effect of NOD seemed to be dependent on its redox activity, as NOT, a redox-inactive NOD derivate, did not influence proliferation. NOD displayed synergistic effects with CNIs on T cell proliferation. Our data demonstrate that NOD displays T cell-suppressive activity. In keeping with its anti-inflammatory action and its beneficial effect on ischemia-induced AKI, NOD may be an interesting drug candidate to prevent CNI-related side-effects.

Dietary zinc deficiency in rodents: effects on T-cell development, maturation and phenotypes.

Zinc deficiency is one of the leading risk factors for developing disease and yet we do not have a clear understanding of the mechanisms behind the increased susceptibility to infection. This review will examine the interrelationships among the hypothalamus-pituitary-adrenal stress axis, p56(lck), and T-cell maturation in both zinc deficiency and responses during zinc repletion. We will highlight differences between the adult mouse model (wasting malnutrition) and growing rat model (stunting malnutrition) of dietary zinc deficiency and discuss the use of various controls to separate out the effects of zinc deficiency from the associated malnutrition. Elevated serum corticosterone in both zinc deficient and pair-fed rats does not support the hypothesis that zinc deficiency per se leads to corticosterone-induced apoptosis and lymphopenia. In fact, the zinc deficient rat does not have lymphopenia. Thymocytes from zinc deficient mice and rats have elevated levels of p56(lck), a signalling protein with a zinc clasp structure, but this does not appear to affect thymocyte maturation. However, post-thymic T-cell maturation appears to be altered based on the lower proportion of splenic late thymic emigrants in zinc deficient rats. Fewer new T-cells in the periphery could adversely affect the T-cell repertoire and contribute to immunodeficiency in zinc deficiency.

Tebrophen--an old polyphenol drug with anticancer potential.

In vitro high-throughput screening was carried out in order to detect new activities for old drugs and to select compounds for the drug development process comprising new indications. Tebrophen, a known antiviral drug, was found to inhibit activities on inflammation and cancer related targets. In primary screening this semisynthetic halogenated polyphenol was identified to inhibit the activities of kinases ZAP-70 and Lck (IC50 0.34 µM and 16 µM, respectively), as well as hydrolase DPPIV (at 80 µM 41% inhibition). Next, it showed no cytotoxic effects on standard cell lines within 24 h. However, tebrophen slowed propagation of breast cancer (MDA-MB-231), osteosarcoma (U2OS) and cervical carcinoma (HeLa), through at least 35 population doublings in a dose-dependent manner. It completely stopped the division of the prostate cancer (PC3) cell line at 50 µM concentration and the cells entered massive cell death in less than 20 days. On the other hand, tebrophen did not influence the growth of normal fibroblasts. According to the measured oxidative burst and estimated in silico parameters its direct antioxidative ability is limited. The obtained results indicate that tebrophen can be considered a promising lead molecule for generating more soluble derivatives with specific anticancer efficacy.

TCR-induced T cell activation leads to simultaneous phosphorylation at Y505 and Y394 of p56(lck) residues.

Biochemical studies have demonstrated that phosphorylation of lymphocyte cell kinase (p56(lck) ) is crucial for activation of signaling cascades following T cell receptor (TCR) stimulation. However, whether phosphorylation/dephosphorylation of the activating or inhibitory tyrosine residues occurs upon activation is controversial. Recent advances in intracellular staining of phospho-epitopes and cytometric analysis, requiring few cells, have opened up novel avenues for the field of immunological signaling. Here, we assessed p56(lck) phosphorylation, using a multiparameter flow-cytometric based detection method following T cell stimulation. Fixation and permeabilization in conjunction with zenon labeling technology and/or fluorescently labeled antibodies against total p56(lck) or cognate phospho-tyrosine (pY) residues or surface receptors were used for detection purposes. Our observations showed that activation of Jurkat or primary human T cells using H(2) O(2) or TCR-induced stimulation led to simultaneous phosphorylation of the activating tyrosine residue, Y394 and the inhibitory tyrosine residue, Y505 of p56(lck) . This was followed by downstream calcium flux and expression of T cell activation markers; CD69 and CD40 ligand (CD40L). However, the extent of measurable activation readouts depended on the optimal stimulatory conditions (temperature and/or stimuli combinations). Treatment of cells with a p56(lck) -specific inhibitor, PP2, abolished phosphorylation at either residue in a dose-dependent manner. Taken together, these observations show that TCR-induced stimulation of T cells led to simultaneous phosphorylation of p56(lck) residues. This implies that dephosphorylation of Y505 is not crucial for p56(lck) activity. Also, it is clear that cytometric analysis provides for a rapid, sensitive, and quantitative method to supplement biochemical studies on p56(lck) signaling pathways in T cells at single cell level.

Vitamin E reverses impaired linker for activation of T cells activation in T cells from aged C57BL/6 mice.

Supplemental vitamin E alleviates age-related defects in interleukin (IL)-2 production, T cell proliferation, and immune synapse formation. Here, we evaluated the effect of in vitro supplementation with 46 mumol/L of vitamin E on T cell receptor-proximal signaling events of CD4(+) T cells from young (4-6 mo) and old (22-26 mo) C57BL mice. Aged murine CD4(+) T cells stimulated via CD3 and CD28, tyrosine 191 of the adaptor protein Linker for Activation of T cells (LAT), was hypo-phosphorylated. Supplementation with vitamin E eliminated this difference in the tyrosine phosphorylation of LAT. By using a flow cytometric assay, the age-related differences in the activation-induced phosphorylation of LAT were observed in both naïve and memory T cell subsets. In addition, supplementation with vitamin E eliminates the age-related differences in LAT phosphorylation in both T cell subsets. Neither age nor vitamin E supplementation altered the fraction of LAT entering the membrane compartment. Furthermore, neither age nor vitamin E influenced the phosphorylation of Lck and Zap70, indicating that associated changes in LAT phosphorylation were not caused by alterations in activation states of the upstream kinases Lck and Zap70.

Electrostatic evaluation of isosteric analogues.

A method is presented for enumerating a large number of isosteric analogues of a ligand from a known protein-ligand complex structure and then rapidly calculating an estimate of their binding energies. This approach takes full advantage of the observed crystal structure, by reusing the atomic co-ordinates determined experimentally for one ligand, to approximate those of similar compounds that have approximately the same shape. By assuming that compounds with similar shapes adopt similar binding poses, and that entropic and protein flexibility effects are approximately constant across such an isosteric series ("the frozen ligand approximation"), it is possible to order their binding affinities relatively accurately. Additionally, the constraint that the atomic coordinates are invariant allows for a dramatic simplification in the Poisson-Boltzmann method used to calculation the electrostatic component of the binding energy. This algorithmic improvement allows for the calculation of tens of thousands of binding energies per second for drug-like molecules, enabling this technique to be used in screening large virtual libraries of isosteric analogues. Most significantly, this procedure is shown to be able to reproduce SAR effects of subtle medicinal chemistry substitutions. Finally, this paper reports the results of the proposed methodology on seven model systems; dihydrofolate reductase, Lck kinase, ribosome inactivating protein, L: -arabinose binding protein, neuraminidase, HIV-1 reverse transcriptase and COX-2.

Identification of substrates of human protein-tyrosine phosphatase PTPN22.

Stimulation of mature T cells activates a downstream signaling cascade involving temporally and spatially regulated phosphorylation and dephosphorylation events mediated by protein-tyrosine kinases and phosphatases, respectively. PTPN22 (Lyp), a non-receptor protein-tyrosine phosphatase, is expressed exclusively in cells of hematopoietic origin, notably in T cells where it represses signaling through the T cell receptor. We used substrate trapping coupled with mass spectrometry-based peptide identification in an unbiased approach to identify physiological substrates of PTPN22. Several potential substrates were identified in lysates from pervanadate-stimulated Jurkat cells using PTPN22-D195A/C227S, an optimized substrate trap mutant of PTPN22. These included three novel PTPN22 substrates (Vav, CD3epsilon, and valosin containing protein) and two known substrates of PEP, the mouse homolog of PTPN22 (Lck and Zap70). T cell antigen receptor (TCR) zeta was also identified as a potential substrate in Jurkat lysates by direct immunoblotting. In vitro experiments with purified recombinant proteins demonstrated that PTPN22-D195A/C227S interacted directly with activated Lck, Zap70, and TCRzeta, confirming the initial substrate trap results. Native PTPN22 dephosphorylated Lck and Zap70 at their activating tyrosine residues Tyr-394 and Tyr-493, respectively, but not at the regulatory tyrosines Tyr-505 (Lck) or Tyr-319 (Zap70). Native PTPN22 also dephosphorylated TCRzeta in vitro and in cells, and its substrate trap variant co-immunoprecipitated with TCRzeta when both were coexpressed in 293T cells, establishing TCRzeta as a direct substrate of PTPN22.

Dexamethasone induces rapid tyrosine-phosphorylation of ZAP-70 in Jurkat cells.

Steroid hormones are known to mediate rapid non-genomic effects occurring within minutes, besides the classical genomic actions mediated by the nuclear translocation of the cytoplasmic glucocorticoid receptor (GR). The glucocorticoid hormone (GC) has significant role in the regulation of T-cell activation; however, the cross-talk between the GC and T-cell receptor (TcR) signal transducing pathways are still to be elucidated. We examined the rapid effects of GC exposure on in vitro cultured human T-cells. Our results showed that Dexamethasone (DX), a GC analogue, when applied at high dose (10 microM), induced rapid (within 5 min) tyrosine-phosphorylation events in Jurkat cells. Short DX pre-treatment strongly inhibited the tyrosine-phosphorylation stimulated by CD3 cross-linking. Furthermore, we also investigated the phosphorylation status of ZAP-70, an important member of tyrosine kinase mediated signalling pathway of TcR-elicited T-cell activation. Here, we demonstrate that high dose DX induced a rapid ZAP-70 tyrosine-phosphorylation in Jurkat T-cells. DX-induced ZAP-70 phosphorylation could be inhibited by RU486 (GR antagonist), suggesting that this process was GR mediated. DX-induced ZAP-70 phosphorylation did not occur in the absence of active p56-lck as examined in the p56-lck kinase-deficient Jurkat cell line JCaM1.6. Our results show that DX, at a high dose, can rapidly influence the initial tyrosine-phosphorylation events of the CD3 signalling pathway in Jurkat cells, thereby modifying TcR-derived signals. Lck and ZAP-70 represent an important molecular link between the TcR and GC signalling pathways.

A rare mRNA variant of the human lymphocyte-specific protein tyrosine kinase LCK gene with intron B retention and exon 7 skipping encodes a putative protein with altered SH3-dependent molecular interactions.

A rare mRNA variant of the human lymphocyte-specific protein tyrosine kinase LCK gene that retains intron B and excludes exon 7 (B+7-) due to alternative splicing of the canonical LCK transcripts was identified and characterized. LCK B+7- mRNA is detected in all tested peripheral blood T lymphocytes total RNA samples but is apparently sequestered in the nucleus. The presence of intron B sequence does not disrupt the reading frame and results in the insertion of 58 aminoacids, containing a proline-rich region just upstream of p56lck SH3 domain. This putative isoform encodes an unstable 516 aminoacids protein (LckB+7-) which can be expressed in transfected COS-7 cells. Furthermore in Jurkat T cell extracts, a recombinant intron B plus SH3 p56lck domain fails to interact with some TCR-induced tyrosine phosphorylated polypeptides and known p56lck partners such as Sam68 and c-Cbl. The biological function of this rare messenger remains to be elucidated.

Tyrosine kinase, p56lck-induced cell motility, and urokinase-type plasminogen activator secretion involve activation of epidermal growth factor receptor/extracellular signal regulated kinase pathways.

We have recently reported that tyrosine kinase, p56(lck) regulates cell motility and nuclear factor kappaB-mediated secretion of urokinase-type plasminogen activator (uPA) through tyrosine phosphorylation of IkappaBalpha following hypoxia/reoxygenation (Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 52598-52612). However, the role of hypoxia/reoxygenation (H/R) on ERK1/2-mediated uPA secretion and cell motility and the involvement of p56(lck) and EGF receptor in these processes in breast cancer cells is not well defined. We provide here evidence that H/R induces Lck kinase activity and Lck-dependent tyrosine phosphorylation of EGF receptor in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. H/R also stimulates MEK-1 and ERK1/2 phosphorylations, and H/R-induced phosphorylations were suppressed by the dominant negative form of Lck (DN Lck, K273R) as well as pharmacological inhibitors of EGF receptor and Lck indicating that EGF receptors and Lck are involved in these processes. Transfection of these cells with wild type Lck or Lck F505 (Y505F) but not with Lck F394 (Y394F) induced phosphorylations of EGF receptor followed by MEK-1 and ERK1/2, suggesting that Lck is upstream of EGF receptor and Tyr-394 of Lck is crucial for these processes. H/R also induced uPA secretion and cell motility in these cells. DN Lck and inhibitors of Lck, EGF receptor, and MEK-1 suppressed H/R-induced uPA secretion and cell motility. To our knowledge, this is the first report that p56(lck) in presence of H/R regulates MEK-1-dependent ERK1/2 phosphorylation and uPA secretion through tyrosine phosphorylation of EGF receptor, and it further demonstrates that all of these signaling molecules ultimately control the motility of breast cancer cells.

CD38 signaling in T cells is initiated within a subset of membrane rafts containing Lck and the CD3-zeta subunit of the T cell antigen receptor.

In this study we present data supporting that most CD38 is pre-assembled in a subset of Brij 98-resistant raft vesicles, which were stable at 37 degrees C, and have relatively high levels of Lck and the CD3-zeta subunit of T cell antigen receptor-CD3 complex in contrast with a Brij 98-soluble pool, where CD38 is associated with CD3-zeta, and Lck is not detected. Our data further indicate that following CD38 engagement, LAT and Lck are tyrosine phosphorylated exclusively in Brij 98-resistant rafts, and some key signaling components translocate into rafts (i.e. Sos and p85-phosphatidylinositol 3-kinase). Moreover, N-Ras results activated within rafts immediately upon CD38 ligation, whereas activated Erk was mainly found in soluble fractions with delayed kinetics respective to Ras activation. Furthermore, full phosphorylation of CD3-zeta and CD3-epsilon only occurs in rafts, whereas partial CD3-zeta tyrosine phosphorylation occurs exclusively in the soluble pool, which correlated with increased levels of c-Cbl tyrosine phosphorylation in the non-raft fractions. Taken together, these results suggest that, unlike the non-raft pool, CD38 in rafts is able to initiate and propagate several activating signaling pathways, possibly by facilitating critical associations within other raft subsets, for example, LAT rafts via its capacity to interact with Lck and CD3-zeta. Overall, these findings provide the first evidence that CD38 operates in two functionally distinct microdomains of the plasma membrane.

Tyrosine kinase p56lck regulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through tyrosine phosphorylation of IkappaBalpha following hypoxia/reoxygenation.

Nuclear factor kappaB (NFkappaB) plays major role in regulating cellular responses as a result of environmental injuries. The molecular mechanism(s) by which hypoxia/reoxygenation (H/R) regulates p56lck-dependent activation of NFkappaB through tyrosine phosphorylation of IkappaBalpha and modulates the expression of downstream genes that are involved in cell migration in human breast cancer cells are not well defined. In this paper, we investigated the involvement of protein-tyrosine kinase p56lck in the redox-regulated activation of NFkappaB following H/R in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. We demonstrated that H/R induces tyrosine phosphorylation of p56lck, nuclear translocation of NFkappaB, NFkappaB-DNA binding, and transactivation of NFkappaB through tyrosine phosphorylation of IkappaBalpha. Transfection of these cells with wild type Lck but not with mutant Lck F394 followed by H/R induces the tyrosine phosphorylation of inhibitor of nuclear factor kappaB (IkappaBalpha) and transcriptional activation of NFkappaB, and these are inhibited by Lck inhibitors. In vitro kinase assay demonstrated that immunoprecipitated p56lck but not Lyn or Fyn directly phosphorylate IkappaBalpha in presence of H/R. Pervanadate, H2O2, and H/R induce the interaction between Lck and tyrosine-phosphorylated IkappaBalpha, and this interaction is inhibited by Src homology 2 domain inhibitory peptide, suggesting that tyrosine-phosphorylated IkappaBalpha interacts with Src homology 2 domain of Lck. Luciferase reporter gene assay indicated that Lck induces NFkappaB-dependent urokinase type plasminogen activator (uPA) promoter activity in presence of H/R. Furthermore, H/R stimulates the cell motility through secretion of uPA. To our knowledge, this is the first report that p56lck in presence of H/R regulates NFkappaB activation, uPA secretion, and cell motility through tyrosine phosphorylation of IkappaBalpha and further demonstrates an important redox-regulated pathway for NFkappaB activation following H/R injury that is independent of IkappaB kinase/IkappaBalpha-mediated signaling pathways.

Inhibitory effects of rosmarinic acid on Lck SH2 domain binding to a synthetic phosphopeptide.

In the course of screening inhibitors from the methanol (MeOH) extracts of 168 medicinal plants against lymphocyte cell-specific kinase (Lck) Src -homology 2 (SH2) binding to a synthetic phosphotyrosine-containing peptide (phosphopeptide), we isolated rosmarinic acid from the MeOH extract of Prunella vulgaris, which showed specific inhibitory activity. The IC 50 value for Lck SH2 binding to phosphopeptide (SGSGEEPQpYEEIPI) of hamster polyomavirus middle-sized tumor (hmT pY324) was 7 microM. However, even at concentrations of 0.1 to 1000 microM, no significant inhibitions were observed against other SH2 domains binding such as the growth factor receptor binding protein 2 (Grb2) SH2 domain to phosphopeptide of Shc and phospholipase Cgamma1 (PLCgamma1) SH2 domain to translational elongation factor 1alpha (EF1alpha) C-terminal. Rosmarinic acid inhibited interleukin-2 (IL-2) gene expression by 50 % at a concentration of 8 microM in Jurkat cells stimulated with anti-CD3 and anti-CD4 antibodies. FK506 and cyclosporin A (CsA) employed as positive controls showed less than 30 % inhibition at the same concentration. In addition, rosmarinic acid inhibited the intracellular [Ca 2+] i increase in Jurkat cells after T cell activation in a dose-dependent manner at concentrations of 1.4 to 140 microM of rosmarinic acid, which is one of the earliest responses of antigen-specific T cell receptor (TCR) and of the upstream pathway of IL-2 expression. Taken together, these results suggest that rosmarinic acid has the potential to specifically inhibit Lck SH2 domain binding to its cognate ligand, including ZAP-70, Cbl, HS-1, and PLCgamma1, and Lck-dependent Ca 2+ signaling pathway of its downstream effector and finally to modulate IL-2 gene expression after T cell activation.

Regulation of Lck and Fyn tyrosine kinase activities by transmembrane protein tyrosine phosphatase leukocyte common antigen-related molecule.

Leukocyte common antigen-related molecule (LAR) is a receptor-like protein tyrosine phosphatase (PTPase) with two PTPase domains. In the present study, we detected the expression of LAR in the brain, kidney, and thymus of mice using anti-LAR PTPase domain subunit monoclonal antibody (mAb) YU1. In the thymus, LAR was expressed on CD4(-)CD8(-) and CD4(-)CD8(low) thymocytes. The development of thymocytes in CD45 knockout mice is blocked partially in the maturation of CD4(-)CD8(-) to CD4(+)CD8(+). We postulated that LAR regulates Lck and Fyn in the immature thymocytes. Transfection of wild-type LAR activated extracellular signal-regulated kinase signal transduction pathway in CD45-deficient Jurkat cells stimulated with anti-CD3 mAb. LAR mutants, with Cys to Ser mutation in the catalytic center of PTPase D1, bound to tyrosine-phosphorylated Lck and Fyn, and LAR PTPase domain 2 was tyrosine phosphorylated by Fyn tyrosine kinase. The phosphorylated LAR was associated with Fyn Src homology 2 domain. Moreover, LAR dephosphorylated phosphorylated tyrosine residues in both the COOH terminus and kinase domain of Fyn in vitro. Our results indicate that Lck and Fyn would be substrates of LAR in immature thymocytes and that each LAR PTPase domain plays distinct functional roles in phosphorylation and dephosphorylation.

Dynamic association of CD45 with detergent-insoluble microdomains in T lymphocytes.

The receptor-like protein tyrosine phosphatase CD45 is essential for TCR signal transduction. Substrates of CD45 include the protein tyrosine kinases p56(lck) and p59(fyn), both of which have been shown to be enriched in detergent-insoluble microdomains. Here we find that there is a cholesterol-dependent association between CD45 and the raft-associated protein linker for activation of T cells, suggesting that CD45 and linker for activation of T cells may colocalize in lipid rafts. Consistent with this observation, we find that approximately 5% of total CD45 can be detected in Triton X-100-insoluble buoyant fractions of sucrose gradients, demonstrating that CD45 is not excluded from lipid rafts. Upon stimulation of T cells with anti-CD3, there is a reduction in the amount of CD45 found associating with lipid rafts. Our data suggest that CD45 is present in lipid rafts in T cells before activation, perhaps to activate raft-associated p56(lck), allowing membrane-proximal signaling events to proceed. Furthermore, the reduction in CD45 content of lipid rafts after CD3 stimulation may serve to limit the amounts of activated p56(lck) in rafts and thus possibly the duration of T cell responses.

Pycnogenol efficacy in the treatment of systemic lupus erythematosus patients.

A pilot study was performed to evaluate the efficacy of Pycnogenol treatment in systemic lupus erythematosus (SLE) patients. Eleven SLE patients were treated with first line medication according to disease activity and in addition, six of them received Pycnogenol and five a placebo. The SLE disease activity index (SLEDAI), serum anti-dsDNA antibodies, fibrinogen, C-reactive protein levels, erythrocyte sedimentation rate, production of reactive oxygen species (ROS) by neutrophils, spontaneous apoptosis and p56(lck) specific activity in peripheral blood lymphocytes were evaluated. Pycnogenol treatment determined a significant reduction of ROS production, apoptosis, p56(lck) specific activity and erythrocyte sedimentation rate. In addition, the decrease of SLEDAI was significant in the Pycnogenol treated group compared with the placebo group (p = 0.018). The results obtained suggest that Pycnogenol could be useful for second line therapy to reduce the inflammatory feature of SLE.

Interleukin-2 carbohydrate recognition modulates CTLL-2 cell proliferation.

Interleukin-2 (IL-2) specifically recognizes high-mannose type glycans with five or six mannosyl residues. To determine whether the carbohydrate recognition activity of IL-2 contributes to its physiological activity, the inhibitory effects of high-mannose type glycans on IL-2-dependent CTLL-2 cell proliferation were investigated. Man(5)GlcNAc(2)Asn added to CTLL-2 cell cultures inhibited not only phosphorylation of tyrosine kinases but also IL-2-dependent cell proliferation. We found that a complex of IL-2, IL-2 receptor alpha, beta, gamma subunits, and tyrosine kinases was formed in rhIL-2-stimulated CTLL-2 cells. Among the components of this complex, only the IL-2 receptor alpha subunit was stained with Galanthus nivalis agglutinin which specifically recognizes high-mannose type glycans. This staining was diminished after digestion of the glycans with endo-beta-N-acetylglucosaminidase H or D, suggesting that at least a N-glycan containing Man(5)GlcNAc(2) is linked to the extracellular portion of the IL-2 receptor alpha subunit. Our findings indicate that IL-2 binds the IL-2 receptor alpha subunit through Man(5)GlcNAc(2) and a specific peptide sequence on the surface of CTLL-2 cells. When IL-2 binds to the IL-2Ralpha subunit, this may trigger formation of the high affinity complex of IL-2-IL-2Ralpha, -beta, and -gamma subunits, leading to cellular signaling.

Detection of p56(lck) kinase activity using scintillation proximity assay in 384-well format and imaging proximity assay in 384- and 1536-well format.

p56(lck) is a lymphocyte-specific tyrosine kinase that plays an important role in both T-cell maturation and activation. We have developed a homogeneous assay in which p56(lck) catalyzes the transfer of the gamma-phosphate group from [gamma-(33)P]ATP to a biotinylated peptide substrate. The labeled peptide is then captured on a streptavidin-coated scintillation proximity assay (SPA) bead or imaging proximity bead. The SPA is counted in a microplate scintillation counter and the imaging proximity assay is counted in a charge-coupled device-based imaging system called LEADseekertrade mark, recently launched as a homogeneous imaging system by Amersham Pharmacia Biotech. We show, via time-dependence assays and inhibitor studies, that this assay can be performed in 1536-well microplate format using imaging proximity as the method of detection. The results compare favorably with the same assay performed in 384-well microplate format using both SPA and imaging proximity as the detection methods. From this study, we conclude that a kinase assay can be performed in 384- and 1536-well format using imaging as the detection method, with significant time savings over standard scintillation counting. In addition, we show cost saving advantages of 1536- over 384-well format in terms of reagent usage, higher throughput, and waste disposal.