Cell-specific Protein-tyrosine Kinase Research Articles

Unveiling the Innate and Adaptive Immunity Interplay: Global Transcriptomic Profiling of the Host Immune Response in Candida albicans Endophthalmitis in a Murine Model.

Intraocular fungal infection poses a significant clinical challenge characterized by chronic inflammation along with vision impairment. Understanding the host defense pathways involved in fungal endophthalmitis will play a pivotal role in identifying adjuvant immunotherapy. Clinical isolates of Candida albicans (15,000 CFU/μL) were intravitreally injected in C57BL/6 mice followed by enucleation at 24 and 72 h postinfection. Histopathological analysis was performed to evaluate the retinal changes and the disease severity. RNA-seq analysis was conducted on homogenized eyeballs to assess the relevant gene profiles and their differentially expressed genes (DEGs). Pathway enrichment analysis was performed to further annotate the functions of the DEGs. Histopathological analysis demonstrated a higher disease severity with increased inflammatory cells at 72 hpi and transcriptome analysis revealed 27,717 DEGs, of which 1493 were significant (adj p value ≤0.05, FC ≥ 1.5). Among these, 924 were upregulated, and 569 were downregulated. Majority of the upregulated genes were associated with the inflammatory/host immune response and signal transduction and enriched in the T-cell signaling pathway, natural killer cell-mediated cytotoxicity, C-type receptor signaling pathway, and NOD-like receptor signaling pathway. Furthermore, inflammation-associated genes such as T-cell surface glycoprotein CD3, cathelicidin antimicrobial peptide, and lymphocyte cell-specific protein tyrosine kinase were enriched, while pathways such as MAPK, cAMP, and metabolic pathways were downregulated. Regulating the T-cell influx could be a potential strategy to modulate excessive inflammation in the retina and could potentially aid in better vision recovery in fungal endophthalmitis.

C-CBL/LCK/c-JUN/ETS1/CD28 axis restrains childhood asthma by suppressing Th2 differentiation

BackgroundAsthma is a common immune disease with high morbidity in children. Type 2 inflammation is the center of asthma development, and mainly mediated by a subset of CD4 + T cells, T helper 2 (Th2) cells. Excess Th2 differentiation was generally associated with asthmatic attack. Casitas B-lineage lymphoma (c-CBL) was reported to involved in T cell development and databank showed its decreased expression in CD4 + T cells from peripheral blood of asthmatic children. This study aims to investigate the role of c-CBL in childhood asthma and Th2 differentiation, and explore the underlying mechanism.MethodsWe collected peripheral blood samples from clinical childhood asthma cases and healthy controls, and determined c-CBL expression in CD4 + T cells. Asthma was induced in neonatal mice by ovalbumin (OVA) intraperitoneal injection and aerosol inhalation, and c-CBL expression in CD4 + T cells from peripheral blood and spleen was measured. Gain-of-function experiments was performed to confirm the effects of c-CBL on Th2 differentiation in vitro. Finally, c-CBL was delivered into asthmatic mice via lentivirus infection to verify its effects on experimental asthma.Resultsc-CBL was lowly expressed in CD4 + T cells from asthmatic children than those of healthy controls. Similarly, it was downregulated in CD4 + T cells from peripheral blood and spleen of asthma mice. Overexpression of c-CBL restrained lung pathological injury and type 2 inflammation in experimental asthmatic mice. Gain-of-function experiments demonstrated that c-CBL inhibited Th2 differentiation of CD4 + T cells from healthy children, and mediated the ubiquitination of lymphocyte cell-specific protein-tyrosine kinase (LCK). LCK acted as a kinase to phosphorylate and activate c-JUN, which was predicted to bind promoter sequence of CD28 by bioinformatic analysis. Dual-luciferase reporter assay verified that c-JUN and ETS1 synergically enhanced transcription of CD28, and this transcription activation was aggravated by LCK overexpression.Conclusionc-CBL alleviated asthma and suppressed Th2 differentiation by facilitating LCK ubiquitination, interrupting c-JUN activation and CD28 expression in vivo and in vitro. c-CBL/LCK/c-JUN/ETS1/CD28 axis was partially involved in childhood asthma, and may provide novel insights for clinical treatment for asthma.

A multimodal radiomic machine learning approach to predict the LCK expression and clinical prognosis in high-grade serous ovarian cancer

We developed and validated a multimodal radiomic machine learning approach to noninvasively predict the expression of lymphocyte cell-specific protein-tyrosine kinase (LCK) expression and clinical prognosis of patients with high-grade serous ovarian cancer (HGSOC). We analyzed gene enrichment using 343 HGSOC cases extracted from The Cancer Genome Atlas. The corresponding biomedical computed tomography images accessed from The Cancer Imaging Archive were used to construct the radiomic signature (Radscore). A radiomic nomogram was built by combining the Radscore and clinical and genetic information based on multimodal analysis. We compared the model performances and clinical practicability via area under the curve (AUC), Kaplan–Meier survival, and decision curve analyses. LCK mRNA expression was associated with the prognosis of HGSOC patients, serving as a significant prognostic marker of the immune response and immune cells infiltration. Six radiomic characteristics were chosen to predict the expression of LCK and overall survival (OS) in HGSOC patients. The logistic regression (LR) radiomic model exhibited slightly better predictive abilities than the support vector machine model, as assessed by comparing combined results. The performance of the LR radiomic model for predicting the level of LCK expression with five-fold cross-validation achieved AUCs of 0.879 and 0.834, respectively, in the training and validation sets. Decision curve analysis at 60 months demonstrated the high clinical utility of our model within thresholds of 0.25 and 0.7. The radiomic nomograms were robust and displayed effective calibration. Abnormally high expression of LCK in HGSOC patients is significantly correlated with the tumor immune microenvironment and can be used as an essential indicator for predicting the prognosis of HGSOC. The multimodal radiomic machine learning approach can capture the heterogeneity of HGSOC, noninvasively predict the expression of LCK, and replace LCK for predictive analysis, providing a new idea for predicting the clinical prognosis of HGSOC and formulating a personalized treatment plan.

Investigating small molecule compounds targeting psoriasis based on cMAP database and molecular dynamics simulation.

Psoriasis (PSO) is a chronic inflammatory skin disease that severely affects the physical and mental health of patients. Drug resistance has been developed upon current drug treatments, and there is no specific therapy. The aim of this study was to screen promising novel drug candidates for PSO using molecular dynamics (MD) simulations. The data of PSO were downloaded from gene expression omnibus (GEO) database and subjected to variance analysis. Target proteins and small molecule compounds targeting PSO were predicted in the connective map (cMAP) database. Molecular docking, MD simulation, and trajectory analysis were conducted to predict the binding of target proteins to compounds. 1999 differentially expressed genes in PSO were obtained by differential analysis. Through cMAP database prediction, a low Score value of -45.69 for lymphocyte cell-specific protein-tyrosine kinase (LCK) was revealed, and aminogenistein was identified as the compound targeting LCK, and LCK was notably highly expressed in the PSO samples. The drugScore of the binding pocket P_0 was 0.814656, which was docked with aminogenistein. The results showed that there were more than one binding site between LCK and aminogenistein with binding energy less than -7.0kJ/mol, and the docking was relatively stable. The results of root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), Gyrate, number of hydrogen bonds and total free binding energy in MD simulations showed that the binding of aminogenistein to LCK was relatively solid. Aminogenistein has good protein-ligand interaction and stability with LCK, a target of PSO, and is a novel drug candidate for PSO.

"Drying effect" of fructus aurantii components and the mechanism of action based on network pharmacology and in vitro pharmacodynamic validation.

Background: Fructus aurantii (FA) is the dried, unripe fruit of the plant Citrus aurantium L. and its cultivated varieties. We investigated the drying effect of FA components and how this drying affect is achieved. Methods: We employed systems pharmacology to predict the components and targets of FA that produce its drying effect. These predictions were verified by computer simulation and animal experiments. In the latter, we measured the bodyweight, water consumption, urine output, fecal water content, rate of salivary secretion, and cross-sectional area of the long axis of the submandibular gland of mice. Immunohistochemistry was used to measure expression of aquaporin (AQP)5 in the submandibular gland, AQP2 in the kidney, and AQP3 in the colon. ELISA kits were used to measure the horizontal variation of cyclic adenosine monsophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and interferon-γ. Results: Sixty-seven potentially active components of FA were screened out. FA could produce a drying effect after regulating 214 targets through 66 active components. A total of 870 gene ontology (GO) terms and 153 signaling pathways were identified. The hypoxia inducible factor-1 signaling pathway, phosphoinositide 3-kinase-protein kinase B (PI3K-AKT) signaling pathway, calcium signaling pathway, and Ras signaling pathway may have important roles in the drying effect of FA. Four components of FA were identified: sinensetin, tangeretin, 5-demethylnobiletin and chrysin. These four components could increase the serum level of interferon-γ and ratio of cyclic adenosine monophosphate:cyclic guanosine monophosphate in mice, and affect their water consumption, urine output, fecal water content and rate of salivary secretion. Conclusion: Four components of FA (tangeretin, sinensetin, chrysin, 5-Demethylmobiletin) were closely related to the Janus kinase-signal transducer and activator of transcription-3 (JAK-STAT3), PI3K-AKT, and the other signaling pathways. They can regulate the protein expression of JAK2, STAT3, PI3K, lymphocyte cell-specific protein-tyrosine kinase, vascular endothelial growth factor A, and protein kinase B1, affect water metabolism in the body and, finally, result in a drying effect.

Oncogenic deubiquitination controls tyrosine kinase signaling and therapy response in acute lymphoblastic leukemia.

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.

Application of an antibody microarray for serum protein profiling of coronary artery stenosis

Protein expression profiling in the serum is used to identify novel biomarkers and investigate the signaling pathways in various diseases. The aim of the present study was to evaluate serum biomarkers associated with coronary artery stenosis resulting from atherosclerosis.The study included 4 groups of subjects: group A and B with and without coronary lesions, respectively, were selected from a previously reported cohort study on coronary atherosclerosis, control group C comprised of asymptomatic subjects and group D was used for independent validation of the microarray data by ELISA. Labeled serum proteins were profiled by an Explorer antibody array, which included 656 specific antibodies in two replicates (FullMoon Biosystems, USA). Cadherin-P, interleukin-5, glutathione S-transferase Mu, and neuronal nitric oxide synthase were sex-independently increased in Group A compared with those in group B. The microarray data on cadherin-P were externally validated in an independent group D using ELISA. Fibroblast growth factor-1, FGF-2, collagen II, granulocyte-macrophage colony-stimulating factor, IL-1 alpha, angiopoietin-2, granulocyte colony-stimulating factor, lymphocyte cell-specific protein tyrosine kinase, and IkappaB kinase b were increase in men in group A compared with group B. Cyclin-dependent kinase 1, DNA fragmentation factor subunit alpha DFF45/ICAD, adenovirus type 2 E1A, calponin, ADP-ribosylation factor-6, muscle-specific actin, thyroid hormone receptor alpha, and alpha-methylacyl-CoA racemase were specifically increased in women in Group A compared with group B. Alterations in the levels of specific proteins may point to the signaling pathways contributing to coronary atherosclerosis, and these proteins will be useful biomarkers for the progression of cardiovascular diseases.

S109: ONCOGENIC DEUBIQUITINATION CONTROLS TYROSINE KINASE SIGNALING AND THERAPY RESPONSE IN ACUTE LYMPHOBLASTIC LEUKEMIA

Background: Dysregulation of kinase signaling pathways via mutations favors tumor cell survival and resistance to therapy and it is common in cancer. Our data unveil how dysregulated deubiquitination controls signaling pathways, leading to cancer cell survival and drug non-response, and suggest novel therapeutic combinations towards targeting T-cell acute lymphoblastic leukemia (T-ALL). Here, we reveal a novel mechanism of post-translational regulation of kinase signaling and nuclear receptor activity via deubiquitination in acute leukemia. Aims: This study aims at 1) characterizing the function of an oncogenic complex composed by two deubiquitinating enzymes in in vitro and in vivo leukemia systems and 2) testing the association of deubiquitinase activity with resistance to therapy in acute lymphoblastic leukemia. Methods: We use genetic mouse and human:mouse xenograft models of T-cell leukemia, biochemical studies (quantitative global proteomics, phosphoproteomics and ubiquitination analysis) and high-throughput molecular biology (chromatin conformation capture (HiC), chromatin accessibility (ATAC-Seq) and gene expression (RNA-Seq)) analyses. Results: We observed that the ubiquitin specific protease 11 (USP11) is highly expressed in lymphoblastic leukemia and associates with poor prognosis in this disease. USP11 ablation inhibits leukemia growth in vitro and in vivo, sparing normal hematopoiesis and thymus development, suggesting that USP11 could be a therapeutic target in leukemia. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK). Deubiquitination of LCK controls its activity, thereby altering T cell receptor signaling. Impairment of LCK activity leads to increased expression of the glucocorticoid receptor transcript, culminating into transcriptional activation of pro-apoptotic target genes, and sensitizes cells to glucocorticoids in T cell leukemia patient samples. The transcriptional activation of pro-apoptotic target genes, such as BCL2L11, is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Pharmacological inhibition of USP7 or genetic knockout of USP7 in combination treatment of glucocorticoid displayed improved anti-T-ALL efficacy in vivo. Image:Summary/Conclusion: Our data unveil how dysregulated deubiquitination controls signaling pathways, leading to cancer cell survival and drug non-response, and suggest novel therapeutic combinations towards targeting T-cell leukemia.

Identification of lymphocyte cell-specific protein-tyrosine kinase (LCK) as a driver for invasion and migration of oral cancer by tumor heterogeneity exploitation

BackgroundCancer metastases are the main cause of lethality. The five-year survival rate for patients diagnosed with advanced stage oral cancer is 30%. Hence, the identification of novel therapeutic targets is an urgent need. However, tumors are comprised of a heterogeneous collection of cells with distinct genetic and molecular profiles that can differentially promote metastasis making therapy development a challenging task. Here, we leveraged intratumoral heterogeneity in order to identify drivers of cancer cell motility that might be druggable targets for anti-metastasis therapy.MethodsWe used 2D migration and 3D matrigel-based invasion assays to characterize the invasive heterogeneity among and within four human oral cancer cell lines in vitro. Subsequently, we applied mRNA-sequencing to map the transcriptomes of poorly and strongly invasive subclones as well as primary tumors and matched metastasis.ResultsWe identified SAS cells as a highly invasive oral cancer cell line. Clonal analysis of SAS yielded a panel of 20 subclones with different invasive capacities. Integrative gene expression analysis identified the Lymphocyte cell-specific protein-tyrosine kinase (LCK) as a druggable target gene associated with cancer cell invasion and metastasis. Inhibition of LCK using A-770041 or dasatinib blocked invasion of highly aggressive SAS cells. Interestingly, reduction of LCK activity increased the formation of adherens junctions and induced cell differentiation.ConclusionAnalysis of invasive heterogeneity led to the discovery of LCK as an important regulator of motility in oral cancer cells. Hence, small molecule mediated inhibition of LCK could be a promising anti-metastasis therapy option for oral cancer patients.

Pretreatment with LCK inhibitors chemosensitizes cisplatin\u2010resistant endometrioid ovarian tumors

BackgroundOvarian cancer is the most fatal gynecologic malignancy in the United States. While chemotherapy is effective in the vast majority of ovarian cancer patients, recurrence and resistance to standard systemic therapy is nearly inevitable. We discovered that activation of the non-receptor tyrosine kinase Lymphocyte Cell-Specific Protein-Tyrosine Kinase (LCK) promoted cisplatin resistance. Here, we hypothesized that treating high grade, platinum resistant endometrioid cancer cells with an LCK inhibitor (LCKi) followed by co-treatment with cisplatin would lead to increased cisplatin efficacy. Our objective was to assess clinical outcomes associated with increased LCK expression, test our hypothesis of utilizing LCKi as pre-treatment followed by co-treatment with cisplatin in platinum resistant ovarian cancer in vitro, and evaluate our findings in vivo to assess LCKi applicability as a therapeutic agent.ResultsKaplan-Meier (KM) plotter data indicated LCK expression is associated with significantly worse median progression-free survival (HR 3.19, p = 0.02), and a trend toward decreased overall survival in endometrioid ovarian tumors with elevated LCK expression (HR 2.45, p = 0.41). In vitro, cisplatin resistant ovarian endometrioid cells treated first with LCKi followed by combination LCKi-cisplatin treatment showed decreased cell viability and increased apoptosis. Immunoblot studies revealed LCKi led to increased expression of phosphorylated H2A histone family X (gamma-H2AX), a marker for DNA damage. In vivo results demonstrate treatment with LCKi followed by LCKi-cisplatin led to significantly slowed tumor growth.ConclusionsWe identified a strategy to therapeutically target cisplatin resistant endometrioid ovarian cancer leading to chemosensitization to platinum chemotherapy via treatment with LCKi followed by co-treatment with LCKi-cisplatin.

Polypharmacological study of Ceritinib using a structure based in silico approach

Drug repurposing has gained mass recognition over the past few years as it has paved new therapeutic applications for already approved FDA drugs. It focuses on finding new molecular targets of drugs for medical uses different than the one originally proposed. Ceritinib, an Anaplastic Lymphoma Kinase (ALK) inhibitor is given orally in the treatment of non-small cell lung cancer (NSCLC). This treatment has been reported to be associated with a number of side effects such as hyperglycemia, convulsion, pneumonitis etc. The side effects are usually due to the unintended interaction of the drug with other protein targets. In silico polypharmacological studies of Ceritinib suggests that it binds to multiple targets other than the intended one which may largely be due to different proteins possessing similar binding sites. ProBis server was used to retrieve probable off-targets of Ceritinib based on presence of structurally similar protein binding sites as that of ALK. Ceritinib was found to bind effectively to three proteins namely Lymphocyte Cell-Specific Protein-Tyrosine Kinase, Tropomyosin receptor kinase B and Aurora kinase B having favorable binding energies and inhibition constants, with no reported side-effects as compared to their marketed drugs. Therefore, it is concluded from the present study that Ceritinib may act as an effective therapeutic target against its polypharmacological targets.

Glucocorticoid resistance is reverted by LCK inhibition in pediatric T-cell acute lymphoblastic leukemia

AbstractPediatric T-acute lymphoblastic leukemia (T-ALL) patients often display resistance to glucocorticoid (GC) treatment. These patients, classified as prednisone poor responders (PPR), have poorer outcome than do the other pediatric T-ALL patients receiving a high-risk adapted therapy. Because glucocorticoids are administered to ALL patients during all the different phases of therapy, GC resistance represents an important challenge to improving the outcome for these patients. Mechanisms underlying resistance are not yet fully unraveled; thus our research focused on the identification of deregulated signaling pathways to point out new targeted approaches. We first identified, by reverse-phase protein arrays, the lymphocyte cell-specific protein-tyrosine kinase (LCK) as aberrantly activated in PPR patients. We showed that LCK inhibitors, such as dasatinib, bosutinib, nintedanib, and WH-4-023, are able to induce cell death in GC-resistant T-ALL cells, and remarkably, cotreatment with dexamethasone is able to reverse GC resistance, even at therapeutic drug concentrations. This was confirmed by specific LCK gene silencing and ex vivo combined treatment of cells from PPR patient-derived xenografts. Moreover, we observed that LCK hyperactivation in PPR patients upregulates the calcineurin/nuclear factor of activated T cells signaling triggering to interleukin-4 (IL-4) overexpression. GC-sensitive cells cultured with IL-4 display an increased resistance to dexamethasone, whereas the inhibition of IL-4 signaling could increase GC-induced apoptosis in resistant cells. Treatment with dexamethasone and dasatinib also impaired engraftment of leukemia cells in vivo. Our results suggest a quickly actionable approach to supporting conventional therapies and overcoming GC resistance in pediatric T-ALL patients.

NO2 inhalation enhances asthma susceptibility in a rat model.

Nitrogen dioxide (NO2) is a major air pollutant. Epidemiologic studies have found that NO2 exposure is associated with an increased risk of asthma. Nevertheless, the potential molecular mechanisms remain unclear. In this study, we investigated the effect of NO2 inhalation on the occurrence of allergic airway inflammation and its underlying mechanisms. Firstly, male Wistar rats were exposed to 2 and 5mg/m3 NO2 (28days, 5h/day). The results showed that NO2 exposure could induce pulmonary inflammatory response, mucus formation, and Th1/Th2 imbalance in the lung of normal rats, resulting in allergic asthma-like features. Secondly, male Wistar rats were exposed to 5mg/m3 NO2 (42days, 5h/day), sensitized with ovalbumin (OVA), challenged with aerosolized OVA, and characterized in asthma models. Results showed that NO2 exposure aggravated lung inflammation in the OVA-sensitized rats, accompanied by the increase in inflammatory cell infiltration, mucus hypersecretion, and collagen deposition. Furthermore, NO2 exposure promoted the increase in the expression of mucin gene (MUC5AC) and pro-inflammatory factors [interleukin (IL)-1β, intercellular adhesion molecule-1 (ICAM-1), and IL-6] as well as serum OVA-specific immunoglobulin E (IgE) production. Taken together, we established that NO2 exposure promotes allergic airway inflammation and increases the asthma susceptibility. The underlying mechanisms involve the promotion of activation of interleukin-4/signal transducer and activator of transcription-6 (IL-4/STAT6) pathway [IL-4 receptor (IL-4R) α, janus kinase (JAK) 1, JAK 3, and STAT6] and related transcription factor [T cell-specific protein-tyrosine kinase (Lck), extracellular-regulated kinase (ERK)1/2, and nuclear factor-κB (NF-κB)]. In particular, the imbalance of Th1/Th2 cell differentiation [IL-4, interferon (IFN)-γ, GATA-binding protein-3 (GATA-3), and T-box expressed in T cells (T-bet)] plays a pivotal role in NO2-induced inflammatory responses. These findings may provide a better understanding of mechanism of NO2-associated respiratory diseases.

Guanine exchange-dependent and -independent effects of Vav1 on integrin-induced T cell spreading.

Vav1 is a 95-kDa member of the Dbl family of guanine exchange factors and a prominent hemopoietic cell-specific protein tyrosine kinase substrate, the involvement of which in cytoskeletal rearrangements has been linked to its ability to activate Rho family small GTPases. Beta1 integrin ligation by fibronectin induced Vav1 phosphorylation in peripheral blood lymphocytes and in two different T cell lines. Vav1 overexpression led to massive T cell spreading on beta1 integrin ligands, and, conversely, two dominant negative mutants blocked integrin-induced spreading. Vav1 and beta1 integrin ligation synergistically activated Pak, but not Rac, Cdc42, or c-Jun N-terminal kinase. In addition, Vav1 cooperated with constitutively active V12Rac mutant, but not with V12Cdc42, to induce T cell spreading after integrin occupancy. More importantly, a Vav1 mutant that lacked guanine exchange factor activity still cooperated with V12Rac. In contrast, a point mutation in the SH2 domain of Vav1 abolished this synergistic effect. Therefore, our results suggest a new regulatory effect of Vav1 in T cell spreading, which is independent of its guanine exchange factor activity.

Localization of Zap70, the gene for a T cell-specific protein tyrosine kinase, to mouse and rat chromosomes by fluorescence in situ hybridization and molecular genetic linkage analyses.

Localization of Zap70, the gene for a T cell-specific protein tyrosine kinase, to mouse and rat chromosomes by fluorescence in situ hybridization and molecular genetic linkage analyses.

Tyrosine phosphorylation and recruitment of ZAP-70 to the CD3-TCR complex are defective after CD2 stimulation.

CD2 has been described as an alternative transduction pathway sharing the same biochemical cascade as CD3. The T cell-specific protein tyrosine kinase (PTK) ZAP-70 is believed to play a key role in early tyrosine phosphorylations of cellular proteins induced by CD3 stimulation. We show in the present report that ZAP-70 is insignificantly tyrosine phosphorylated and recruited to CD3 after CD2 stimulation in Jurkat T cells. The same result is obtained for p72syk, a PTK structurally and functionally related to ZAP-70. Several studies have suggested a model in which p56lck would be responsible after CD3 triggering for the phosphorylation of particular tyrosine residues in the immunoreceptor tyrosine-based activation motifs of the CD3 chains, inducing the recruitment and the tyrosine phosphorylation of ZAP-70. In Jurkat cells, p56lck is required for CD3- or CD2-induced tyrosine phosphorylations and clearly activated after CD2 cross-linking. However, we find that the CD3 complex, and especially its zeta-chain, are faintly tyrosine phosphorylated after CD2 triggering. By contrast, CD2 induces PLCgamma-1 tyrosine phosphorylation as efficiently as CD3, with a correlated inositol phosphate production and intracellular calcium increase, and even a higher production of IL-2. Interestingly, the SH2 domains of PLCgamma-1 associate with ZAP-70 upon CD3 stimulation while they bind, in CD2-activated cells, to a heavily tyrosine-phosphorylated 62-kDa protein. Altogether, these findings suggest that CD2 could bypass the PTK ZAP-70 for PLCgamma-1 activation and involves a preferential cascade comprising p56lck and a p62 protein, possibly acting as an anchor molecule.

P56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation.

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70.

We have developed a rapid and sensitive two capillary-column chromatography and mass spectrometry-based method for the determination of protein phosphorylation sites following recovery of individual phosphopeptides from two-dimensional phosphopeptide maps. With a standard phosphopeptide, we demonstrate detection sensitivity of at least 250 fmol for this system. We applied this technique to the analysis of in vitro sites of tyrosine phosphorylation induced on the T cell-specific protein tyrosine kinase ZAP-70 in the absence and presence of p56lck. We show that ZAP-70 has a primary autophosphorylation site at Tyr-292, with a secondary site at Tyr-126. We also show additional phosphorylation at Tyr-69, Tyr-178, Tyr-492, and Tyr-493 upon the addition of the protein tyrosine kinase, p56lck. By comparative two-dimensional phosphopeptide mapping, we show that ZAP-70 isolated from Jurkat T cells also autophosphorylates at Tyr-292 and Tyr-126. Similar analysis of 32P-labeled Jurkat cells stimulated with anti-T cell receptor antibodies reveals Tyr-492 and Tyr-493 as the principal sites of T cell antigen receptor-induced tyrosine phosphorylation, with additional phosphorylation at the Tyr-292, but not the Tyr-126 autophosphorylation site. The high degree of sensitivity achieved with this technology should greatly facilitate the direct biochemical determination of inducible protein phosphorylation events, an experimental strategy that until now has been both time consuming and difficult.

Functional analysis of T-cell antigen 4-1BB in activated intestinal intra-epithelial T lymphocytes

The 4-1BB antigen has been recently demonstrated to be a 30 kDa inducible T-cell antigen and is expressed on the cell surface of activated splenic T cells and thymocytes. This novel T-cell antigen also associates physically with the T cell-specific protein tyrosine kinase, p56 lck. We show here that the inducible T-cell antigen 4-1BB is expressed on activated intestinal intra-epithelial T lymphocytes (IEL). After activation, or in the presence of IL-2, the activated IELs showed higher levels of 4-1BB. Functional studies revealed that the activated IELs triggered with anti-4-1BB monoclonal antibody could enhance the level of IEL cytotoxicity against anti-CD3-secreting hybridoma cells. Cross-linking of anti-4-1BB antibody also enhanced the proliferation of IELs. These results suggest that the inducible antigen 4-1BB has broad biological functions which not only play a role in activated splenic T cells and thymocytes but also in the mucosal immune system.

Peripheral T cells in mice lacking p56lck do not express significant antiviral effector functions.

Mutant mice lacking p56lck, the T cell-specific protein tyrosine kinase, have a profound thymic atrophy and possess only an immature thymocyte population. Only 5 to 10% of normal levels of mature T cells (TCR-alpha beta+, CD4+, or CD8+) are present in these mice. These T cells, but also B cells of mutant mice, can be activated by mitogens, and T cells proliferate upon stimulation with IL-2 or mAb against the TCR. Thus, it appears that selected T cells can mature without lck and that they may have circumvented the need for this enzyme. This study evaluated the capacity of these mice to generate antiviral immune responses: 1) mutant mice failed to generate virus-specific CTL after acute infection with lymphocytic choriomeningitis virus (LCMV) or vaccinia virus. 2) LCMV was not cleared from the spleen on day 10 after infection. 3) Infection of LCMV into footpads of mutant mice did not induce a T cell-dependent swelling reaction. 4) Intracerebral inoculation with LCMV did not induce in mutant mice any clinical signs of sickness. 5) Vesicular stomatitis virus (VSV)-primed mutant mice could not eliminate nucleoprotein of VSV recombinant vaccinia virus upon intracerebral infection. 6) VSV infection of mutant mice elicited normal, specific T cell-independent IgM responses, but no significant switch to IgG. These results show that, despite the presence of in vitro activatable CD8+ and CD4+ T cells in the mutant mice, no biologically relevant functions can be detected. Therefore, these data indicate that p56lck is directly or indirectly crucially involved in in vivo Ag-induced T cell responses.