Activity Of P56lck Research Articles

The Activity and Stability of p56Lck and TCR Signaling Do Not Depend on the Co-Chaperone Cdc37.

Lymphocyte-specific protein tyrosine kinase (Lck) is a pivotal tyrosine kinase involved in T cell receptor (TCR) signaling. Because of its importance, the activity of Lck is regulated at different levels including phosphorylation of tyrosine residues, protein–protein interactions, and localization. It has been proposed that the co-chaperone Cdc37, which assists the chaperone heat shock protein 90 (Hsp90) in the folding of client proteins, is also involved in the regulation of the activity/stability of Lck. Nevertheless, the available experimental data do not clearly support this conclusion. Thus, we assessed whether or not Cdc37 regulates Lck. We performed experiments in which the expression of Cdc37 was either augmented or suppressed in Jurkat T cells. The results of our experiments indicated that neither the overexpression nor the suppression of Cdc37 affected Lck stability and activity. Moreover, TCR signaling proceeded normally in T cells in which Cdc37 expression was either augmented or suppressed. Finally, we demonstrated that also under stress conditions Cdc37 was dispensable for the regulation of Lck activity/stability. In conclusion, our data do not support the idea that Lck is a Cdc37 client.

Src-type tyrosine kinase p56lck is critical for thymic stromal lymphopoietin-induced allergic rhinitis.

Thymic stromal lymphopoietin (TSLP) is a regulator of mast cell-mediated allergic inflammatory reactions, but the manner in which TSLP contributes to allergic rhinitis (AR) remains unclear. Here, we sought to determine that TSLP plays a crucial role in AR by interacting with Src-type tyrosine kinase p56lck and STAT6 and promoting mast cells degranulation. The effects of TSLP on mast cell degranulation and AR were analysed in human mast cell line (HMC-1 cells), ovalbumin (OVA)-induced AR animal model, and human subjects. Small interfering RNA experiments were performed in HMC-1 cells and OVA-induced AR model. Immune responses were analysed by enzyme-linked immunosorbent assay, Western blotting, immunoprecipitation, and histological studies. Thymic stromal lymphopoietin levels and mast cell-derived p56lck activation were elevated in human subjects with AR, and in AR mice, exogenous TSLP accelerated TH2-allergic inflammatory reactions by up-regulating p56lck and STAT6. On the other hand, depletion of TSLP, p56lck, and STAT6 ameliorated clinical symptoms in AR mice. The selective inhibitor of p56lck, damnacanthal, inhibits AR reactions. Collectively, these observations suggest a role for TSLP/p56lck/STAT6 in AR and offer insight into potential therapeutic strategies.

The SRC Kinase Family Member Lymphocyte Specific Kinase (p56Lck) Regulates Endothelial Cell Proliferation and Survival

Background and Objective: The Src family consists of Src, Lck (p56Lck), Fyn, Hck, Blk, Lyn, Fgr, Yes, and Yrk protein tyrosine kinases, which display cell and tissue specific expression. These non-receptor protein tyrosine kinases regulate many cellular processes including cell growth, differentiation, shape, migration, and survival. p56Lck is primarily expressed in T lymphocytes where it stimulates T cell receptor-mediated signaling to regulate thymocyte development. Similar to other SRC family members, p56Lck activity is negatively regulated by phosphorylation of Tyr505 and undergoes autophosphorylation on Tyr394 in the active state. Our previous studies showed that p56Lck activation occurred in proliferating endothelial cells exposed to cleaved high molecular weight kininogen (HKa), which induced endothelial cell apoptosis.The purpose of this study is to define the role of Lck in regulation of endothelial cell survival and angiogenesis.Methods: Primary cultures of endothelial cells were treated with Lck siRNA or with mammalian expression vectors or lentiviral constructs containing Lck cDNA. The proliferative capacity, viability, and tube-forming ability of Lck and control endothelial cells were determined.Results: Transfection of endothelial cells with Lck siRNA markedly changed their morphology and significantly enhanced their ability to proliferate in response to bFGF. In contrast, endothelial cells transfected with a Lck expression vector or transduced with a Lck-lentivirus not only failed to proliferate in response to growth factors, but also underwent apoptosis on several different extracellular matrix proteins. Finally, overexpression of Lck in endothelial cells led to impaired tube formation in a matrigel-based, in vitro angiogenesis assay; apoptosis of endothelial cells in this system was suggested by increased staining with Annexin V.Conclusion: Taken together, these results suggest that regulation of Lck activation provides a key node in determination of endothelial cell proliferation and survival that is likely to be relevant to the regulation of angiogenesis. Moreover, they suggest that specific endothelial cell Src family kinase members may play contrasting roles in regulation of these processes. Activation of Lck may represent a potential mechanism for controlling aberrant angiogenesis in pathological conditions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Type II PtdIns 4-kinase β associates with CD4-p56lck complex and is involved in CD4 receptor signaling.

Type II phosphatidylinositol (PtdIns) 4-kinases are involved in the synthesis of PtdIns 4-phosphates and modulate various cell functions like, intracellular signaling, cytoskeletal rearrangements, vesicular trafficking, and pathogen invasion. In CD3 receptor activated T cells, a type II PtdIns 4-kinase β is recruited to CD3 receptor zeta and plays a role in intracellular calcium release and probably in actin cytoskeleton reorganization. T cell receptor mediated activation is supported by CD4 receptor. The role of type II PtdIns 4-kinase β in CD4 receptor-mediated signaling was addressed in the present manuscript. Crosslinking of CD4 receptors with monoclonal antibodies showed an increase in CD4-associated PtdIns 4-kinase activity and requires p56(lck) activity. Biochemical characterization suggests that it belongs to type II PtdIns 4-kinase family. shRNA mediated knockdown of type II PtdIns 4-kinase β showed abrogation of CD4 receptor induced intracellular calcium release. These results suggest that type II PtdIns 4-kinase β plays an integral part in CD4 receptor-mediated signaling.

TCR‐induced T cell activation leads to simultaneous phosphorylation at Y505 and Y394 of p56lck 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.

ChemInform Abstract: Synthesis and Protein-Tyrosine Kinase Inhibitory Activities of Flavonoid Analogues.

Treatment of o-hydroxyacetophenones 2a-e with excess lithium bis(trimethylsilyl)amide followed by dialkyl carbonates gave alkyl 3-(2-hydroxyaryl)-3-oxopropanoates 3a-e. The latter substances were transformed through the reaction of their magnesium chelates with benzoyl chlorides into a series of 3-(alkoxycarbonyl)-2-arylflavones, which were subsequently elaborated into a variety of flavonoids. These compounds were tested for their abilities to inhibit the in vitro protein-tyrosine kinase activity of p56lck, an enzyme which is thought to play a key role in mediating signal transduction from the CD4 receptor during lymphocyte activation. All of the active compounds had either an amino or a hydroxyl substituent at the 4'-position of the 2-aryl ring. The most active substance prepared in this study is compound 17c, which is approximately 1 order of magnitude more potent than the natural product quercetin (1). Compound 17c was a competitive inhibitor of p56lck with respect to ATP and was highly selective for the inhibition of protein-tyrosine over protein-serine/threonine kinases.

T‐cell receptor proximal signaling via the Src‐family kinases, Lck and Fyn, influences T‐cell activation, differentiation, and tolerance

T-cell development in the thymus and activation of mature T cells in secondary lymphoid organs requires the ability of cells to respond appropriately to environmental signals at multiple stages of their development. The process of thymocyte selection insures a functional T-cell repertoire, while activation of naive peripheral T cells induces proliferation, gain of effector function, and, ultimately, long-lived T-cell memory. The T-cell immune response is initiated upon engagement of the T-cell receptor (TCR) and coreceptor, CD4 or CD8, by cognate antigen/major histocompatibility complexes presented by antigen-presenting cells. TCR/coreceptor engagement induces the activation of biochemical signaling pathways that, in combination with signals from costimulator molecules and cytokine receptors, direct the outcome of the response. Activation of the src-family kinases p56(lck) (Lck) and p59(fyn) (Fyn) is central to the initiation of TCR signaling pathways. This review focuses on our current understanding of the mechanisms by which these two proteins orchestrate T-cell function.

P56Lck Tyrosine Kinase Enhances the Assembly of Death-inducing Signaling Complex during Fas-mediated Apoptosis

Although the death-inducing signaling complex (DISC) is rapidly assembled, several lines of evidence suggest that formation of this complex is not the first consequence of cell surface CD95 (Fas) stimulation but rather a later step in this process. Activation of Fas triggers a cascade of signaling events that culminate in cellular apoptosis. Tyrosine kinases are critical effectors in T cell activation. However, their functional involvement in death receptor-mediated apoptosis is unknown. Here, we used p56(Lck)-deficient cells to show that CD95-induced cell death is highly dependent on p56(Lck) activity and its localization within plasma membrane. We found that p56(Lck) acts upstream of the mitochondria; in the absence of p56(Lck), Bid cleavage and the release of cytochrome c were severely impaired. Moreover, p56(Lck)-deficient cells or cells expressing an inactive form of p56(Lck) displayed defective formation of the DISC post CD95 stimulation. In vivo reconstitution of thymocytes from p56(lck)-deficient mice, which are resistant to apoptosis, with p56(Lck) restored Fas-mediated cell death. Our results support a novel model whereby sensitivity to apoptosis is regulated through quantitative changes in the stoichiometry of DISC components triggered by p56(Lck) activation and localization.

Suppression of Proximal T Cell Receptor Signaling and Lytic Function in CD8+ Tumor-Infiltrating T Cells

CD8(+) tumor-infiltrating lymphocytes (TIL) lack in vivo and in vitro lytic function due to a signaling deficit characterized by failure to flux calcium or activate tyrosine kinase activity upon contact with cognate tumor cells. Although CD3 zeta is phosphorylated by conjugation in vitro with cognate tumor cells, showing that TIL are triggered, PLC gamma-1, LAT, and ZAP70 are not activated and LFA-1 is not affinity-matured, and because p56(lck) is required for LFA-1 activation, this implies that the signaling blockade is very proximal. Here, we show that TIL signaling defects are transient, being reversed upon purification and brief culture in vitro, implying a fast-acting "switch". Biochemical analysis of purified nonlytic TIL shows that contact with tumor cells causes transient activation of p56(lck) ( approximately 10 s) which is rapidly inactivated. In contrast, tumor-induced activation of p56(lck) in lytic TIL is sustained coincident with downstream TCR signaling and lytic function. Shp-1 is robustly active in nonlytic TIL compared with lytic TIL, colocalizes with p56(lck) in nonlytic TIL, and inhibition of Shp-1 activity in lytic TIL in vitro blocks tumor-induced defective TIL cytolysis. Collectively, our data support the notion that contact of nonlytic TIL with tumor cells, and not with tumor-infiltrating myeloid-derived suppressor cells, causes activation of Shp-1 that rapidly dephosphorylates the p56(lck) activation motif (Y394), thus inhibiting effector phase functions.

Defective Adhesion in Tumor Infiltrating CD8+ T Cells

CD8(+) tumor-infiltrating lymphocytes (TIL) are defective in cytolysis due to tumor-induced inhibition of proximal TCR-mediated signaling, a defect that is relieved upon purification and brief culture. We show in this study that frequency of conjugation in vitro of nonlytic TIL with tumor cells is low in comparison with their lytic counterparts, and the strength of interaction and duration of conjugation are also reduced. Previous reports show that p56(lck) activation is required for TCR-initiated LFA-1 avidity up-regulation, raising the question: is low LFA-1 avidity the basis of reduced TIL conjugation frequency? When stimulated with phorbol ester, nonlytic TIL bind purified ICAM-1 equivalently as lytic TIL, suggesting that LFA-1 can be activated if proximal TCR signaling is bypassed. However, when treated with phorbol ester, the conjugation frequency of nonlytic TIL does not increase. CD2 and CD8 also mediate T cell adhesion to cognate target cells and are both expressed at lower levels in nonlytic TIL in addition to being excluded from the immune synapse formed upon conjugation. Collectively, these results imply that adhesion defects in nonlytic TIL result from a combination of decreased cell surface levels of adhesion molecules, deficient LFA-1 activation, and the failure to recruit essential adhesion receptors to the membrane contact site formed with cognate target cells.

Hypoxia Regulates Cross-talk between Syk and Lck Leading to Breast Cancer Progression and Angiogenesis

Hypoxia is a key parameter that controls tumor angiogenesis and malignant progression by regulating the expression of several oncogenic molecules. The nonreceptor protein-tyrosine kinases Syk and Lck play crucial roles in the signaling mechanism of various cellular processes. The enhanced expression of Syk in normal breast tissue but not in malignant breast carcinoma has prompted us to investigate its potential role in mammary carcinogenesis. Accordingly, we hypothesized that hypoxia/reoxygenation (H/R) may play an important role in regulating Syk activation, and Lck may be involved in this process. In this study, we have demonstrated that H/R differentially regulates Syk phosphorylation and its subsequent interaction and cross-talk with Lck in MCF-7 cells. Moreover, Syk and Lck play differential roles in regulating Sp1 activation and expressions of melanoma cell adhesion molecule (MelCAM), urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) in response to H/R. Overexpression of wild type Syk inhibited the H/R-induced uPA, MMP-9, and VEGF expression but up-regulated MelCAM expression. Our data also indicated that MelCAM acts as a tumor suppressor by negatively regulating H/R-induced uPA secretion and MMP-9 activation. The mice xenograft study showed the cross-talk between Syk and Lck regulated H/R-induced breast tumor progression and further correlated with the expressions of MelCAM, uPA, MMP-9, and VEGF. Human clinical specimen analysis supported the in vitro and in vivo findings. To our knowledge, this is first report that the cross-talk between Syk and Lck regulates H/R-induced breast cancer progression and further suggests that Syk may act as potential therapeutic target for the treatment of breast cancer.

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 μM), 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.

Complement regulatory protein Crry/p65-mediated signaling in T lymphocytes: role of its cytoplasmic domain and partitioning into lipid rafts

Crry/p65 is a type I glycoprotein, which protects mouse T cells from complement attack. We have previously shown that complement receptor I-related protein Crry/p65 (Crry) ligation has a costimulatory effect on mouse CD4+ T cell activation. Here, we have examined the mechanisms responsible for Crry costimulation, addressing the question of whether Crry potentiates signal transduction starting at the T cell receptor (TCR)/CD3 complex or promotes distinct costimulatory signals. We show that Crry increases early TCR-dependent activation signals, including p56lck-, zeta-associated protein-70 (ZAP-70), Vav-1, Akt, and extracellular signal-regulated kinase (ERK) phosphorylation but also costimulation-dependent mitogen-activated protein kinases (MAPK), such as the stress-activated c-Jun N-terminal kinase (JNK). It is intriguing that Crry costimulus enhanced p38 MAPK activation in T helper cell type 1 (Th1) but not in Th2 cells. A fraction of Crry is found consistently in the detergent-insoluble membrane fraction of Th1 or Th2 cells or CD4+ lymphoblasts. Crry costimulation induced clustering of lipid rafts, increasing their content in Crry, CD3epsilon, and p59-60 forms of p56lck, and caused actin polymerization close to the site of activation in Th2 cells. Such events were inhibited by wortmannin, suggesting a role for phosphatidylinositol-3 kinase in these effects. The Crry cytoplasmic domain was required for JNK activation and interleukin-4 secretion but not for the presence of Crry in rafts or activation of p56lck, ZAP-70, Akt, Vav-1, or ERK. This suggests that Crry costimulation involves two different but not mutually exclusive signal transduction modules. The dual function of Crry as a complement regulatory protein and as a T cell costimulator illustrates the importance of complement regulatory proteins as links between innate and adaptive immunity.

Differential role of lipid rafts in the functions of CD4 + and CD8 + human T lymphocytes with aging

Lipid rafts are critical to the assembly of the T-cell receptor (TCR) signaling machinery. It is not known whether lipid raft properties differ in CD4 + and CD8 + T cells and whether there are age-related differences that may account in part for immune senescence. Data presented here showed that time-dependent interleukin-2 (IL-2) production was different between CD4 + and CD8 + T cells. The defect in IL-2 production by CD4 + T cells was not due to lower levels of expression of the TCR or CD28. There was a direct correlation between the activation of p56 Lck and LAT and their association/recruitment with the lipid raft fractions of CD4 + and CD8 + T cells. p56 Lck, LAT and Akt/PKB were weakly phosphorylated in lipid rafts of stimulated CD4 + T cells of elderly as compared to young donors. Lipid rafts undergo changes in their lipid composition (ganglioside M1, cholesterol) in CD4 + and CD8 + T cells of elderly individuals. This study emphasizes the differential role of lipid rafts in CD4 + and CD8 + T-cell activation in aging and suggests that the differential localization of CD28 may explain disparities in response to stimulation in human aging.

Epitope‐specific crosslinking of CD45 down‐regulates membrane‐associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells

CD45 engagement by monoclonal antibodies on human activated T cells triggers tumour necrosis factor-alpha (TNF-alpha) gene transcription in an epitope-specific manner. To dissect the early signalling events leading to TNF-alpha gene expression, we established that CD45 crosslinking resulted in tyrosine phosphorylation of p56lck, ZAP-70, CD3-zeta, LAT and Vav. This was accompanied by down-regulation of membrane-associated protein tyrosine phosphatase activity in the absence of demonstration of enhanced p56lck, p72syk and ZAP-70 kinase activity, which remained constitutive. These early events eventually triggered an intracellular Ca(2+) rise and phosphoinositide turnover. We conclude that down-regulation of membrane-associated tyrosine phosphatase activity by CD45 extracytoplasmic domain multimerization led, in an epitope-specific fashion, to unopposed tyrosine kinase activity and to the activation of the T-cell receptor/CD3 complex signalling cascade, resulting in TNF-alpha gene expression. This model strongly suggests that CD45 extracytoplasmic tail multimerization may contribute to the modulation T-cell functions.

Effects of methyl-β-cyclodextrin on T lymphocytes lipid rafts with aging

Aging is associated with a decline in immune functions. Among them, T-cell activation is altered at several points of the signaling cascade following TCR engagement. Recent findings suggest that lipid rafts act as a platform in the initiation of T-cell activation. We have previously demonstrated that cholesterol content in T-cells increased with aging, especially in lipid rafts. Cholesterol, which is a major component of lipid rafts, serves to stabilize their structure. We hypothesized that restoring T-cell cholesterol content and membrane fluidity would restore, at least in part, T-cell function via lipid rafts. We measured the lipid rafts coalescence, the p56 Lck and linker of activated T-cell (LAT) signaling molecules recruitment and activation, the cholesterol content and fluidity in T-cell membrane after different methyl-β-cyclodextrin (MBCD) treatments. Our results show that high concentration of MBCD (10 mM) completely disorganized the lipid rafts in T-cell membranes of young and elderly donors, however, T-cells from elderly donors were less sensitive than T-cells of young donors to low concentration of MBCD (0.5 mM). p56 Lck and LAT recruitment and activation were affected in T-cells of both aged groups. MBCD treatment did not affect the cholesterol content and fluidity of T-cell membranes of young donors, while the cholesterol content was decreased and fluidity increased in lipid rafts of elderly donors. These results suggested that cholesterol extraction by MBCD increased the fluidity and disrupted lipid rafts organization. The increase in cholesterol content in lipid rafts with aging and its decrease by biochemical extraction were able to affect early signaling molecules activation. Restoring cholesterol content and fluidity may have beneficial effects, however, MBCD disorganized the membrane and this might not completely restore the T-cell activation via lipid rafts with aging. Altogether these results suggest that defects in cholesterol cellular homeostasis may be part of T-cell immunosenescence via lipid rafts dysfunction.

Tyrosine phosphorylation-dependent activation of NFκB is compromised in T cells from the elderly

NFκB induction and gene regulation are compromised in T lymphocytes during aging. This has been attributed to altered proteasomal function resulting in decreased ubiquitin-mediated degradation of IκBα. However, little is known about the impact of aging on the mechanisms that lead to the release of active NFκB employing pro-oxidant pathways. Oxidant-mediated activation of NFκB has been previously shown to involve proteasome independent mechanisms and hence may be an important alternate conduit to the induction of this central transcription factor in aging. Employing H 2O 2 and pervanadate we not only demonstrate lowered tyrosine phosphorylation of IκBα, but also compromised induction of nuclear NFκB in T cells from the elderly. Lowered tyrosine phosphorylation of IκBα may be due to a decrease in activity of p56 lck and ZAP-70, since treatment with piceatannol, an inhibitor of syk and src family kinases, mimics age associated decline in tyrosine phosphorylation of IκBα in T cells from young donors. Thus, alternate pathways of NFκB induction are also impaired in T cells from the elderly and may underlie immune-deficit accompanying aging.

Lysophosphatidylcholine is a regulator of tyrosine kinase activity and intracellular Ca 2+ level in Jurkat T cell line

Lysophospholipids, particularly lysophosphatidylcholine (lyso-PC), have been implicated in modulating T cell functions at the sites of inflammation and atherosclerosis. Although the chemotactic and immunomodulatory effects are well documented, the exact signaling pathway of lyso-PC action is poorly defined. In this work, we studied the earliest biochemical events in T cells triggered by lyso-PC. A marked and immediate tyrosine phosphorylation was induced in the leukemic T cell line, Jurkat. Phosphorylation of cellular substrates included src family kinase, p56 lck and syk family kinase, ZAP70. The lyso-PC induced tyrosine phosphorylation was largely dependent on the presence of functional p56 lck. Tyrosine phosphorylation was followed by the elevation of intracellular Ca 2+ concentration. The magnitude of the mobilization of the intracellular Ca 2+ was similar in the absence of the p56 lck activity in JCaM1.6 cells as in Jurkat cells, however, it was slightly but reproducibly delayed compared to that in the wild type cells. Inhibition of the Ser/Thr kinases and tyrosine kinases with staurosporine and genistein, respectively, decreased the rise in the intracellular Ca 2+ content. Moreover, pertussis toxin completely blocked the Ca 2+ signal supporting the role of the G-protein coupled LPC receptor in this event.

Role of tumour necrosis factor-alpha (TNF-alpha) in the induction of HIV-1 gp120-mediated CD4+ T cell anergy.

The HIV-1 envelope glycoprotein (gp 120) is known to induce antigen-specific and non-specific CD4+ T cell anergy. We found that early T cell activation, as indicated by HLA-DP expression in the early G1 (G1A) phase of the cell cycle, and the inhibition of mitogen-mediated IL-2 production induced by gp120, required TNF-alpha produced by gp120-stimulated macrophages. In the presence of an antibody to TNF-alpha, these changes induced by gp120 were inhibited, while recombinant TNF-alpha induced similar abnormalities of CD4+ T cells, even in the absence of gp120. On the other hand, inhibition of the mixed lymphocyte reaction (MLR) in CD4+ T cells by gp120, which may be related to gp120-mediated down-regulation of CD4 expression on T cells and activation of protein tyrosine kinase p56(lck) in CD4+ T cells, was observed even in the absence of macrophage-derived TNF-alpha induced by gp120. These observations indicate that both TNF-alpha-dependent and independent events contribute to gp120-mediated CD4+ T cell anergy, and TNF-alpha appears to play an important role in inducing CD4+ T cell anergy in HIV-1 infection.

Cutting Edge: A Chemical Genetic System for the Analysis of Kinases Regulating T Cell Development

To understand the regulatory activities of kinases in vivo requires their study across a biologically relevant window of activity. To this end, ATP analog-sensitive kinase alleles (ASKAs) specifically sensitive to a competitive inhibitor have been developed. This article tests whether ASKA technology can be applied to complex immunological systems, such as lymphoid development. The results show that when applied to reaggregate thymic organ culture, novel p56(Lck) ASKAs readily expose a dose-dependent correlation of thymocyte development with a range of p56(Lck) activity. By regulating kinase activity, rather than amounts of RNA or protein, ASKA technology offers a general means for assessing the quantitative contributions to immunology of numerous kinases emerging from genomics analyses. It can obviate the generation of multiple lines of mice expressing different levels of kinase transgenes and should permit specific biological effects to be associated with defined biochemical activities.