Opposing Effects of Chelidonine on Tyrosine and Serine Phosphorylation of STAT3 in Human Uveal Melanoma Cells.

STAT3 is a transcription factor that regulates various cellular processes with oncogenic potential, thereby promoting tumorigenesis when activated uncontrolled. STAT3 activation is mediated by its tyrosine phosphorylation, triggering dimerization and nuclear translocation. STAT3 also contains a serine phosphorylation site, with a postulated regulatory role in STAT3 activation and G2/M transition. Interleukin-6, a major activator of STAT3, is present in elevated concentrations in uveal melanomas, suggesting contribution of dysregulated STAT3 activation to their pathogenesis. Here, we studied the impact of chelidonine on STAT3 signaling in human uveal melanoma cells. Chelidonine, an alkaloid isolated from Chelidonium majus, disrupts microtubules, causes mitotic arrest and provokes cell death in numerous tumor cells. According to our flow cytometry and confocal microscopy data, chelidonine abrogated IL-6-induced activation and nuclear translocation, but amplified constitutive serine phosphorylation of STAT3. Both effects were restricted to a fraction of cells only, in an all-or-none fashion. A partial overlap could be observed between the affected subpopulations; however, no direct connection could be proven. This study is the first proof on a cell-by-cell basis for the opposing effects of a microtubule-targeting agent on the two types of STAT3 phosphorylation.

IL-2 receptors preassemble and signal in the ER/Golgi causing resistance to antiproliferative anti-IL-2Rα therapies.

Interleukin-2 (IL-2) and IL-15 play pivotal roles in T cell activation, apoptosis, and survival, and are implicated in leukemias and autoimmune diseases. Their heterotrimeric receptors share their ß- and γc-chains, but have distinct α-chains. Anti-IL-2Rα (daclizumab) therapy targeting cell surface-expressed receptor subunits to inhibit T cell proliferation has only brought limited success in adult T cell leukemia/lymphoma (ATL) and in multiple sclerosis. We asked whether IL-2R subunits could already preassemble and signal efficiently in the endoplasmic reticulum (ER) and the Golgi. A combination of daclizumab and anti-IL-2 efficiently blocked IL-2-induced proliferation of IL-2-dependent wild-type (WT) ATL cells but not cells transfected with IL-2, suggesting that in IL-2-producing cells signaling may already take place before receptors reach the cell surface. In the Golgi fraction isolated from IL-2-producing ATL cells, we detected by Western blot phosphorylated Jak1, Jak3, and a phosphotyrosine signal attributed to the γc-chain, which occurred at much lower levels in the Golgi of WT ATL cells. We expressed EGFP- and mCherry-tagged receptor chains in HeLa cells to study their assembly along the secretory pathway. Confocal microscopy, Förster resonance energy transfer, and imaging fluorescence cross-correlation spectroscopy analysis revealed partial colocalization and molecular association of IL-2 (and IL-15) receptor chains in the ER/Golgi, which became more complete in the plasma membrane, further confirming our hypothesis. Our results define a paradigm of intracellular autocrine signaling and may explain resistance to antagonistic antibody therapies targeting receptors at the cell surface.

Minimum degree of overlap between IL-9R and IL-2R on human T lymphoma cells: A quantitative CLSM and FRET analysis.

The heterodimeric receptor complex of IL-9 consists of the cytokine-specific α-subunit and the common γc -chain shared with other cytokines, including IL-2, a central regulator of T cell function. We have shown previously the bipartite spatial relationship of IL-9 and IL-2 receptors at the surface of human T lymphoma cells: in addition to common clusters, expression of the two receptor kinds could also be observed in segregated membrane areas. Here we analyzed further the mutual cell surface organization of IL-9 and IL-2 receptors. Complementing Pearson correlation data with co-occurrence analysis of confocal microscopic images revealed that a minimum degree of IL-9R/IL-2R co-localization exists at the cell surface regardless of the overall spatial correlation of the two receptor kinds. Moreover, our FRET experiments demonstrated molecular scale assemblies of the elements of the IL-9/IL-2R system. Binding of IL-9 altered the structure and/or composition of these clusters. It is hypothesized, that by sequestering receptor subunits in common membrane areas, the overlapping domains of IL-9R and IL-2R provide a platform enabling both the formation of the appropriate receptor complex as well as subunit sharing between related cytokines. © 2018 International Society for Advancement of Cytometry.

Membrane Potential Distinctly Modulates Mobility and Signaling of IL-2 and IL-15 Receptors in T Cells.

The high electric field across the plasma membrane might influence the conformation and behavior of transmembrane proteins that have uneven charge distributions in or near their transmembrane regions. Membrane depolarization of T cells occurs in the tumor microenvironment and in inflamed tissues because of K+ release from necrotic cells and hypoxia affecting the expression of K+ channels. However, little attention has been given to the effect of membrane potential (MP) changes on membrane receptor function. Therefore, we studied the influence of membrane de- and hyperpolarization on the biophysical properties and signaling of interleukin-2 (IL-2) and interleukin-15 (IL-15) receptors, which play important roles in T cell function. We investigated the mobility, clustering, and signaling of these receptors and major histocompatibility complex (MHC) I/II glycoproteins forming coclusters in lipid rafts of T cells. Depolarization by high K+ buffer or K+ channel blockers resulted in a decrease in the mobility of IL-2Rα and MHC glycoproteins, as shown by fluorescence correlation spectroscopy, whereas hyperpolarization by the K+ ionophore valinomycin increased their mobility. Contrary to this, the mobility of IL-15Rα decreased upon both de- and hyperpolarization. These changes in protein mobility are not due to an alteration of membrane fluidity, as evidenced by fluorescence anisotropy measurements. Förster resonance energy transfer measurements showed that most homo- or heteroassociations of IL-2R, IL-15R, and MHC I did not change considerably, either. MP changes modulated signaling by the two cytokines in distinct ways: depolarization caused a significant increase in the IL-2-induced phosphorylation of signal transducer and activator of transcription 5, whereas hyperpolarization evoked a decrease only in the IL-15-induced signal. Our data imply that the MP may be an important modulator of interleukin receptor signaling and dynamics. Enhanced IL-2 signaling in depolarized Treg cells highly expressing IL-2R may contribute to suppression of antitumor immune surveillance.

Profiling DNA damage and repair capacity in sea urchin larvae and coelomocytes exposed to genotoxicants.

The ability to protect the genome from harmful DNA damage is critical for maintaining genome stability and protecting against disease, including cancer. Many echinoderms, including sea urchins, are noted for the lack of neoplastic disease, but there are few studies investigating susceptibility to DNA damage and capacity for DNA repair in these animals. In this study, DNA damage was induced in adult sea urchin coelomocytes and larvae by exposure to a variety of genotoxicants [UV-C (0-3000 J/m(2)), hydrogen peroxide (0-10mM), bleomycin (0-300 µM) and methylmethanesulfonate (MMS, 0-30 mM)] and the capacity for repair was measured over a 24-h period of recovery. Larvae were more sensitive than coelomocytes, with higher levels of initial DNA damage (fast micromethod) for all genotoxicants except MMS and increased levels of mortality 24h following treatment for all genotoxicants. The larvae that survived were able to efficiently repair damage within 24-h recovery. The ability to repair DNA damage differed depending on treatments, but both larvae and coelomocytes were able to most efficiently repair H2O2-induced damage. Time profiles of expression of a panel of DNA repair genes (ddb1, ercc1, xpc, xrcc1, pcna, ogg1, parp1, parp2, ape, brca1, rad51, xrcc2, xrcc3, xrcc4, xrcc5, xrcc6 and gadd45), throughout the period of recovery, showed greater gene induction in coelomocytes compared with larvae, with particularly high expression of xrcc1, ercc1, parp2 and pcna. The heterogeneous response of larvae to DNA damage may reflect a strategy whereby a subset of the population is equipped to withstand acute genotoxic stress, while the ability of coelomocytes to resist and repair DNA damage confirm their significant role in protection against disease. Consideration of DNA repair capacity is critical for understanding effects of genotoxicants on organisms, in addition to shedding light on life strategies and disease susceptibility.

Distinct spatial relationship of the interleukin-9 receptor with interleukin-2 receptor and major histocompatibility complex glycoproteins in human T lymphoma cells.

The interleukin-9 receptor (IL-9R) consists of an α subunit and a γ(c) chain that are shared with other cytokine receptors, including interleukin-2 receptor (IL-2R), an important regulator of T cells. We previously showed that IL-2R is expressed in common clusters with major histocompatibility complex (MHC) glycoproteins in lipid rafts of human T lymphoma cells, which raised the question about what the relationship between clusters of IL-2R/MHC and IL-9R is. Confocal microscopy colocalization and fluorescence resonance energy transfer experiments capable of detecting membrane protein organization at different size scales revealed nonrandom association of IL-9R with IL-2R/MHC clusters at the surface of human T lymphoma cells. Accommodation of IL-9Rα in membrane areas segregated from the IL-2R/MHC domains was also detected. The bipartite nature of IL-9R distribution was mirrored by signal transducer and activator of transcription (STAT) activation results. Our data indicate that co-compartmentalization with MHC glycoproteins is a general property of γ(c) receptors. Distribution of receptor chains between different membrane domains may regulate their function.

Comparative DNA damage and repair in echinoderm coelomocytes exposed to genotoxicants.

The capacity to withstand and repair DNA damage differs among species and plays a role in determining an organism's resistance to genotoxicity, life history, and susceptibility to disease. Environmental stressors that affect organisms at the genetic level are of particular concern in ecotoxicology due to the potential for chronic effects and trans-generational impacts on populations. Echinoderms are valuable organisms to study the relationship between DNA repair and resistance to genotoxic stress due to their history and use as ecotoxicological models, little evidence of senescence, and few reported cases of neoplasia. Coelomocytes (immune cells) have been proposed to serve as sensitive bioindicators of environmental stress and are often used to assess genotoxicity; however, little is known about how coelomocytes from different echinoderm species respond to genotoxic stress. In this study, DNA damage was assessed (by Fast Micromethod) in coelomocytes of four echinoderm species (sea urchins Lytechinus variegatus, Echinometra lucunter lucunter, and Tripneustes ventricosus, and a sea cucumber Isostichopus badionotus) after acute exposure to H2O2 (0-100 mM) and UV-C (0-9999 J/m2), and DNA repair was analyzed over a 24-hour period of recovery. Results show that coelomocytes from all four echinoderm species have the capacity to repair both UV-C and H2O2-induced DNA damage; however, there were differences in repair capacity between species. At 24 hours following exposure to the highest concentration of H2O2 (100 mM) and highest dose of UV-C (9999 J/m2) cell viability remained high (>94.6 ± 1.2%) but DNA repair ranged from 18.2 ± 9.2% to 70.8 ± 16.0% for H2O2 and 8.4 ± 3.2% to 79.8 ± 9.0% for UV-C exposure. Species-specific differences in genotoxic susceptibility and capacity for DNA repair are important to consider when evaluating ecogenotoxicological model organisms and assessing overall impacts of genotoxicants in the environment.

Sea urchin coelomocytes are resistant to a variety of DNA damaging agents.

Increasing anthropogenic activities are creating environmental pressures that threaten marine ecosystems. Effective environmental health assessment requires the development of rapid, sensitive, and cost-effective tools to predict negative impacts at the individual and ecosystem levels. To this end, a number of biological assays using a variety of cells and organisms measuring different end points have been developed for biomonitoring programs. The sea urchin fertilization/development test has been useful for evaluating environmental toxicology and it has been proposed that sea urchin coelomocytes represent a novel cellular biosensor of environmental stress. In this study we investigated the sensitivity of coelomocytes from the sea urchin Lytechinus variegatus to a variety of DNA-damaging agents including ultraviolet (UV) radiation, hydrogen peroxide (H(2)O(2)), methylmethane sulfonate (MMS) and benzo[a]pyrene (BaP). LD(50) values determined for coelomocytes after 24h of exposure to these DNA damaging agents indicated a high level of resistance to all treatments. Significant increases in the formation of apurinic/apyrimidinic (AP or abasic) sites in DNA were only detected using high doses of H(2)O(2), MMS and UV radiation. Comparison of sea urchin coelomocytes with hemocytes from the gastropod mollusk Aplysia dactylomela and the decapod crustacean Panulirus argus indicated that sensitivity to different DNA damaging agents varies between species. The high level of resistance to genotoxic agents suggests that DNA damage may not be an informative end point for environmental health assessment using sea urchin coelomocytes however, natural resistance to DNA damaging agents may have implications for the occurrence of neoplastic disease in these animals.

Non-random distribution of interleukin receptors on the cell surface.

Spatial organization of cell surface proteins plays a key role in the process of transmembrane signalling. Receptor clustering and changes in their cell surface distribution are often determining factors in the final outcome of ligand-receptor interactions. There are several techniques for assessing the distribution of protein molecules. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining distance relationships of cell surface molecules. However, it does not provide information on the distribution of molecular clusters. Different kinds of microscopies fill this gap. The evaluation of the images provided by the listed techniques is often questionable. Herein we show the applicability of Ripley's K(t) function as a tool for analyzing the cell surface receptor patterns (Y. Nakamura, et al., Nature 1994, 369, 330-333). We have implemented an effective image processing algorithm for fast localization of gold labels on biological samples. We investigated spatial organization of Interleukin-2R alpha and -15R alpha (IL-2R alpha and IL-15R alpha) on a human CD4+leukaemia T-cell line, Kit225 FT7.10 by using transmission electron microscopy (TEM). TEM analysis showed co-clustering of the two types of alpha-chains even on the few-hundred-nanometer scale. The analysis of our data may contribute to our understanding the action of the IL-2/IL-15 receptor system in T-cell function.

Nanometer-scale organization of the alpha subunits of the receptors for IL2 and IL15 in human T lymphoma cells.

Interleukin 2 and interleukin 15 (IL2 and IL15, respectively) provide quite distinct contributions to T-cell-mediated immunity, despite having similar receptor composition and signaling machinery. As most of the proposed mechanisms underlying this apparent paradox attribute key significance to the individual alpha-chains of IL2 and IL15 receptors, we investigated the spatial organization of the receptors IL2Ralpha and IL15Ralpha at the nanometer scale expressed on a human CD4+ leukemia T cell line using single-molecule-sensitive near-field scanning optical microscopy (NSOM). In agreement with previous findings, we here confirm clustering of IL2Ralpha and IL15Ralpha at the submicron scale. In addition to clustering, our single-molecule data reveal that a non-negligible percentage of the receptors are organized as monomers. Only a minor fraction of IL2Ralpha molecules reside outside the clustered domains, whereas approximately 30% of IL15Ralpha molecules organize as monomers or small clusters, excluded from the main domain regions. Interestingly, we also found that the packing densities per unit area of both IL2Ralpha and IL15Ralpha domains remained constant, suggesting a 'building block' type of assembly involving repeated structures and composition. Finally, dual-color NSOM demonstrated co-clustering of the two alpha-chains. Our results should aid understanding the action of the IL2R-IL15R system in T cell function and also might contribute to the more rationale design of IL2R- or IL15R-targeted immunotherapy agents for treating human leukemia.

Colorectal carcinoma rearranges cell surface protein topology and density in CD4+ T cells.

Previously, we described conserved protein clusters including MHC I and II glycoproteins, ICAM-1 adhesion molecules, and interleukin-2 and -15 receptors in lipid rafts of several human cell types. Differential protein-protein interactions can modulate function, thus influence cell fate. Therefore, we analyzed supramolecular clusters of CD4(+) T cells from draining lymph nodes and peripheral blood of colorectal carcinoma patients, and compared these to healthy controls. Superclusters of MHC I and II with IL-2/15 receptors were identified by confocal microscopy on all cell types. Flow-cytometric FRET revealed molecular associations of these proteins with each other and with ICAM-1 as well. In draining lymph nodes expression levels of all these proteins were lower, and interactions, particularly between IL-2/15 receptors and MHC molecules weakened or disappeared as compared to the control. Stimuli/local conditions can rearrange cell surface protein patterns on the same cell type in the same patient, having important implications on further function and cell fate.

Lack of age-associated telomere shortening in long- and short-lived species of sea urchins.

The red sea urchin, Strongylocentrotus franciscanus, can live in excess of 100 years while the sea urchin Lytechinus variegatus has an estimated lifespan of only 3-4 years. In an effort to understand the molecular mechanism underlying the difference in their longevity we characterized telomere biology in these species of sea urchins. Telomerase activity was found throughout early stages of development in L. variegatus and is maintained in adult tissues of L. variegatus and S. franciscanus. Terminal restriction fragment analysis indicated a lack of age-associated telomere shortening. These data suggest that long- and short-lived sea urchins do not utilize telomerase repression as a mechanism to suppress neoplastic transformation.

Apoptotic response of uveal melanoma cells upon treatment with chelidonine, sanguinarine and chelerythrine.

The benzophenanthridine alkaloids sanguinarine, chelerythrine and chelidonine were reported previously to provoke cell death in a variety of tumor cells suggesting their potential application as anticancer agents. Here we tested their effects on a primary human uveal melanoma cell line, OCM-1. Flow cytometric analysis of annexin V binding/PI exclusion and DNA fragmentation disclosed that all these alkaloids could induce apoptosis in OCM-1 cells. Moreover, necrotic cell death was also observed upon alkaloid treatment. As it was also evidenced by light microscopic inspection of cellular morphology, chelidonine primarily caused apoptosis, while sanguinarine and chelerythrine were effective via a so-termed bimodal cell death (apoptosis and primary necrosis). The relative efficiencies of the two modes depended on the applied dose. This study is the first implication for the possible use of these alkaloids in the therapy of uveal melanomas, for which no really efficient therapeutic regimen is available so far.

Computer program for analyzing donor photobleaching FRET image series.

BACKGROUND: The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel-by-pixel fashion from the image series recorded on the donor-only and donor and acceptor double-labeled samples. Because of the large number of pixels and the time-consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements. METHODS: New evaluation software was developed to analyze pbFRET data for Windows platform in National Instrument LabVIEW 6.1. This development environment contains a mathematical virtual instrument package, in which the Levenberg-Marquardt routine is also included. As a reference experiment, FRET efficiency between the two chains (beta2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC II molecules were determined in the plasma membrane of JY, human B lymphoma cells. RESULTS: The bleaching time constants calculated on pixel-by-pixel basis can be displayed as a color-coded map or as a histogram from raw image format. CONCLUSION: In this report we introduce a new version of pbFRET analysis and data processing software that is able to generate a full analysis pattern of donor photobleaching image series under various conditions. .

Membrane topography of HLA I, HLA II, and ICAM-1 is affected by IFN-gamma in lipid rafts of uveal melanomas.

The lateral distribution and colocalization of HLA I, HLA-DR, and ICAM-1 proteins was studied for the first time in the plasma membrane of two human uveal melanoma cell lines, OCM-1 and OCM-3. Our fluorescence resonance energy transfer and confocal laser scanning microscopic experiments revealed that these molecules are mostly confined to the same membrane regions, where they form similar protein patterns (homo- and hetero-associates) to those found previously on other cell types of lymphoid as well as colorectal carcinoma origin. Confocal microscopic colocalization experiments with GM(1) gangliosides and the GPI-anchored CD59 molecules showed enrichment of HLA I, HLA-DR, and ICAM-1 molecules in specific membrane domains (lipid rafts) excluding the transferrin receptor. IFN-gamma remarkably increased the expression levels of these molecules and rearranged their association patterns, which can affect the adoptive immune response of effector cells.

IL-2 and IL-15 receptor alpha-subunits are coexpressed in a supramolecular receptor cluster in lipid rafts of T cells.

The private alpha-chains of IL-2 and IL-15 receptors (IL-2R and IL-15R) share the signaling beta- and gamma(c)-subunits, resulting in both common and contrasting roles of IL-2 and IL-15 in T cell function. Knowledge of the cytokine-dependent subunit assembly is indispensable for understanding the paradox of distinct signaling capacities. By using fluorescence resonance energy transfer and confocal microscopy, we have shown that IL-2R alpha, IL-15R alpha, IL-2/15R beta and gamma(c)-subunits, as well as MHC class I and II glycoproteins formed supramolecular receptor clusters in lipid rafts of the T lymphoma line Kit 225 FT7.10. Fluorescence crosscorrelation microscopy demonstrated the comobility of IL-15R alpha with IL-2R alpha and MHC class I. A model was generated for subunit switching between IL-2R alpha and IL-15R alpha upon the binding of the appropriate cytokine resulting in the formation of high-affinity heterotrimeric receptors. This model suggests a direct role for the alpha-subunits, to which no definite function has been assigned so far, in tuning cellular responses to IL-2 or IL-15. In addition, both alpha-chains were at least partially homodimerized/oligomerized, which could be the basis of distinct signaling pathways by the two cytokines.

Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells.

Human embryonic stem (ES) cells are pluripotent cells with the potential to differentiate into a variety of cell types, which could be used for cell transplantation therapies as well as drug discovery studies. However, the large-scale culture of undifferentiated human ES cells is currently limited by their dependency on mouse embryonic fibroblast feeder layers. The proteomics approach was employed to characterize the environment that supports the growth of undifferentiated human ES cells and to identify factors critical for their independent growth. Conditioned medium from mouse embryonic fibroblast feeder layers, STO cell line, was concentrated and subjected to analyses by two-dimensional electrophoresis mass spectrometry. In total, 136 unique protein species were identified which included some that are known to participate in cell growth and differentiation, extracellular matrix formation and remodeling, in addition to the unexpected but interesting finding of many nominally intracellular proteins. This approach has thus revealed the complexity of the environment provided by the feeder cells and provides a useful starting point for future studies. Moreover, candidates from the initial list of identified proteins can be further investigated for their effects on the growth and differentiation of human ES cells in a defined culture environment.

GPI-microdomains (membrane rafts) and signaling of the multi-chain interleukin-2 receptor in human lymphoma/leukemia T cell lines.

Subunits (alpha, beta and gamma) of the interleukin-2 receptor complex (IL-2R) are involved in both proliferative and activation-induced cell death (AICD) signaling of T cells. In addition, the signaling beta and gamma chains are shared by other cytokines (e.g. IL-7, IL-9, IL-15). However, the molecular mechanisms responsible for recruiting/sorting the alpha chains to the signaling chains at the cell surface are not clear. Here we show, in four cell lines of human adult T cell lymphoma/leukemia origin, that the three IL-2R subunits are compartmented together with HLA glycoproteins and CD48 molecules in the plasma membrane, by means of fluorescence resonance energy transfer (FRET), confocal microscopy and immuno-biochemical techniques. In addition to the beta and gamma(c) chains constitutively expressed in detergent-resistant membrane fractions (DRMs) of T cells, IL-2Ralpha (CD25) was also found in DRMs, independently of its ligand-occupation. Association of CD25 with rafts was also confirmed by its colocalization with GM-1 ganglioside. Depletion of membrane cholesterol using methyl-beta-cyclodextrin substantially reduced co-clustering of CD25 with CD48 and HLA-DR, as well as the IL-2 stimulated tyrosine-phosphorylation of STATs (signal transducer and activator of transcription). These data indicate a GPI-microdomain (raft)-assisted recruitment of CD25 to the vicinity of the signaling beta and gamma(c) chains. Rafts may promote rapid formation of a high affinity IL-2R complex, even at low levels of IL-2 stimulus, and may also form a platform for the regulation of IL-2 induced signals by GPI-proteins (e.g. CD48). Based on these data, the integrity of these GPI-microdomains seems critical in signal transduction through the IL-2R complex.

Differential association of CD45 isoforms with CD4 and CD8 regulates the actions of specific pools of p56lck tyrosine kinase in T cell antigen receptor signal transduction.

An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4(+)CD8(+) HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was found as homodimers and also preferentially associated with CD4 and CD8 at the cell-surface. A comparison was therefore made of T cell antigen receptor signaling between sub-clones expressing either CD45R0 or CD45RBC. Under basal conditions CD4-associated p56(lck) tyrosine kinase activity and cellular protein tyrosine phosphorylation levels were higher in the CD45R0(+) than in the CD45RBC(+) sub-clones. Upon CD3-CD4 ligation, TCR-zeta phosphorylation, ZAP-70 recruitment to the p21/p23 TCR-zeta phosphoisomers, ZAP-70 phosphorylation, as well as p56(lck), c-Cbl and Slp-76 phosphorylation, were all markedly increased in CD45R0(+) compared with CD45RBC(+) cells. T cell antigen receptor (TCR) stimulation alone also promoted c-Cbl phosphorylation in CD45R0(+) but not in CD45RBC(+) cells. Our results are consistent with a model in which association of CD45R0 with CD4 generates a more active pool of CD4-associated p56(lck) kinase molecules. Upon CD3-CD4 co-ligation, the active p56(lck) increases the intensity of T cell antigen receptor signal transduction coupling by promoting TCR-zeta chain phosphorylation and ZAP-70 recruitment.