Stimulation of NK-like YT cells via leukocyte function-associated antigen (LFA)-1. Possible involvement of LFA-1-associated tyrosine kinase in signal transduction after recognition of NK target cells

The YTA-1 anti-LFA-1 alpha mAb activates protein tyrosine kinase (PTK), augments NK cytotoxicity, and induces proliferation of fresh CD3- large granular lymphocytes. We demonstrate here that LFA-1 is physically associated in the YT human NK-like cell line cells with a PTK(s) that is distinct from Src family PTKs such as Lck, Fyn, or Lyn. In vitro kinase assays revealed similar association of protein kinase activity with LFA-1 in normal CD3- large granular lymphocytes. Tyrosine phosphorylation of the proteins associated with LFA-1 drastically increased in YT cells after stimulation with NK-sensitive K562 cells but not with NK-resistant P815 cells. Furthermore, pretreatment of YT cells with TS1/22 anti-LFA-1 alpha and TS1/18 anti-LFA-1 beta mAbs abrogated not only the cytotoxicity against K562 cells but also an increase in tyrosine phosphorylation of LFA-1-associated molecules induced by K562 stimulation. These results provide biochemical evidence that the PTK(s) associated with LFA-1 is involved in the signal transduction that follows the recognition of NK target cells.     

Genetic evidence of a role for Lck in T-cell receptor function independent or downstream of ZAP-70/Syk protein tyrosine kinases

T-cell antigen receptor (TCR) engagement results in sequential activation of the Src protein tyrosine kinases (PTKs) Lck and Fyn and the Syk PTKs, ZAP-70 and Syk. While the Src PTKs mediate the phosphorylation of TCR-associated signaling subunits and the phosphorylation and activation of the Syk PTKs, the lack of a constitutively active Syk PTK has prohibited the analysis of Lck function downstream of these initiating signaling events. We describe here the generation of an activated Syk family PTK by substituting the kinase domain of Syk for the homologous region in ZAP-70 (designated as KS for kinase swap). Expression of the KS chimera resulted in its autophosphorylation, the phosphorylation of cellular proteins, the upregulation of T-cell activation markers, and the induction of interleukin-2 gene synthesis in a TCR-independent fashion. The KS chimera and downstream ZAP-70 or Syk substrates, such as SLP-76, were still phosphorylated when expressed in Lck-deficient JCaM1.6 T cells. However, expression of the KS chimera in JCaM1.6 cells failed to rescue downstream signaling events, demonstrating a functional role for Lck beyond the activation of the ZAP-70 and Syk PTKs. These results indicate that downstream TCR signaling pathways may be differentially regulated by ZAP-70 and Lck PTKs and provide a mechanism by which effector functions may be selectively activated in response to TCR stimulation.     

Uncoupling of nonreceptor tyrosine kinases from PLC-gamma1 in an SLP-76-deficient T cell

Activation of nonreceptor protein tyrosine kinases (PTKs) is essential for T cell receptor (TCR) responsiveness; however, the function of individual PTK substrates is often uncertain. A mutant T cell line was isolated that lacked expression of SLP-76 (SH2 domain-containing leukocyte protein of 76 kilodaltons), a hematopoietically expressed adaptor protein and PTK substrate. SLP-76 was not required for TCR-induced tyrosine phosphorylation of most proteins, but was required for optimal tyrosine phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1), as well as Ras pathway activation. TCR-inducible gene expression was dependent on SLP-76. Thus, coupling of TCR-regulated PTKs to downstream signaling pathways requires SLP-76.     

Interactions of vancomycin resistant enterococci with biomaterial surfaces

Many cytokines transmit signals to the cell interior through activation of receptor-associated, Janus family protein tyrosine kinases (Jak PTKs). The interleukin-2 receptor (IL-2R) is associated with the Jak1 and Jak3 PTKs, and ligand-induced activation of these PTKs is essential for lymphocyte proliferation. Here, the nonreceptor PTK, Pyk2, was found to be activated following IL-2 stimulation in a Jak-dependent manner. Furthermore, physical association was detected between endogenous Pyk2 and Jak3, and a dominant interfering mutant of Pyk2 inhibited IL-2-induced cell proliferation without affecting Stat5 activation. Collectively, these results suggest that Pyk2 is a newly identified component of the Jak-mediated IL-2 signaling pathway.     

CD50 monoclonal antibodies inhibit neutrophil activation

The constitutive high expression of CD50 (ICAM-3) on resting leukocytes, coupled with the observation that CD50 is the primary LFA-1 ligand on resting T cells, suggests that CD50 may be an important LFA-1 ligand in the initiation of the immune/inflammatory response. CD50 mAbs have been reported to increase homotypic adhesion of lymphocytes, and lymphocyte adhesion to HUVEC and extracellular matrix proteins. In this study, the effects of CD50 mAbs on neutrophil activation were examined. CD50 mAbs were found to inhibit neutrophil adhesion induced by FMLP and 12-O-tetradecanoyl-phorbol-13-acetate to resting and TNF-activated HUVEC. CD50 mAbs also inhibited neutrophil adhesion stimulated by CD66a, CD66b, CD66c, and CD66d mAbs to HUVEC. CD50 mAbs inhibited the up-regulation of CD11b/CD18 to the neutrophil surface, and the down-regulation of surface CD62L expression. The potential contribution of src family kinases to the previously described tyrosine kinase activity associated with CD50 in neutrophils was also examined. hck and lyn were found to account for much of the tyrosine kinase activity associated with CD50 in neutrophils. The data indicate that CD50 in neutrophils functions not only as a potential ligand for LFA-1, but also regulates the surface expression and activity of CD11b/CD18 and CD62L. In contrast to the effects in lymphocytes, CD50 appears to function as a negative regulator of neutrophil activation.     

Integrin-associated protein (CD47) is a comitogenic molecule on CD3-activated human T cells

IAP is a glycoprotein functionally and physically associated with some integrins, i.e., the leukocyte response integrin and the beta3 integrin chain on placenta, platelets, and polymorphonuclear cells. IAP may act as a transducer element in activation mediated via these integrins. Since IAP is present at high density on peripheral T lymphocytes we have investigated its involvement in T cell activation. We tested three mAbs against IAP, namely B6H12, BRIC126, and 2D3, which recognize two distinct epitopes. IAP cross-linking with B6H12 or BRIC126, but not 2D3, transduces costimulatory signals within highly purified CD3-activated T lymphocytes, i.e., enhancement of proliferation, CD25 expression, and IL-2 secretion, while no effect was observed upon CD2 stimulation. However, we could not observe any functional association between IAP and integrins on peripheral T cells. In an attempt to explore further the activation signal delivered by IAP, we show here that IAP cross-linking with the comitogenic B6H12 mAb induces the phosphorylation on tyrosine of several proteins, one of which is identified as p56(lck) protein tyrosine kinase. Moreover, we observed that IAP is associated with p56(lck) on PMA-activated, but not on resting, T cells. These data suggest that on T cells, IAP may be involved directly via a specific ligand in cell-matrix or cell-cell interactions. Such interactions could trigger protein tyrosine phosphorylation pathways, which play an important role in both maturation and activation of T cells.     

Tyrosine phosphorylation and association with phospholipase C gamma-1 of the GAP-associated 62-kD protein after CD2 stimulation of Jurkat T cell

Numerous substrates are tyrosine phosphorylated upon CD2 stimulation of human Jurkat T cells using a mitogenic pair of CD2 monoclonal antibodies, including the phospholipase C (PLC)gamma-1-p35/36 complex. Most of these substrates are identically tyrosine phosphorylated after CD3 ligation, suggesting that both stimuli share the same biochemical pathway. We show, however, in this report that a 63-kD protein is specifically phosphorylated on tyrosine residues after ligation of the CD2 molecule. The tyrosine phosphorylation of p63 can be induced independently of other substrates when using a single CD2 mAb recognizing the D66 epitope of the molecule. Importantly, this CD2-induced tyrosine phosphorylation of p63 can also occur in the absence of the CD3 zeta chain membrane expression, and is also distinct from the protein tyrosine kinases p56lck and p59fyn. We demonstrate, moreover, that p63 is physically linked with PLC gamma-1 and p35/36 upon CD2 stimulation. Finally, we also show that a 62-kD protein coimmunoprecipitating with the p21ras GTPase activating protein (GAP) is heavily tyrosine phosphorylated only after CD2 stimulation. This ultimately suggests that p63 may represent in fact the 62-kD protein that associates with GAP after tyrosine phosphorylation. Taken together, these results demonstrate the occurrence in Jurkat cells of a tyrosine kinase pathway specifically coupled to the CD2 molecule. They also suggest a function of the p62-GAP-associated protein as a link between PLC gamma-1 and p21ras activation pathways after CD2 activation.     

Multiple Grb2-mediated integrin-stimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.     

Coordinated regulation of the tyrosine phosphorylation of Cbl by Fyn and Syk tyrosine kinases

Cross-linking of the T cell antigen receptor (TCR)-CD3 complex induces rapid tyrosine phosphorylation and activation of Src (Lck and Fyn) and Syk (Syk and Zap-70) family protein tyrosine kinases (PTKs) which, in turn, phosphorylate multiple intracellular substrates. Cbl is a prominent PTK substrate suggesting a pivotal role for it in early signal transduction events. However, the regulation of Cbl function and tyrosine phosphorylation in T cells by upstream PTKs remains poorly understood. In the present study, we used genetic and biochemical approaches to demonstrate that Cbl directly interacts with Syk and Fyn via its N-terminal and C-terminal regions, respectively. Tyr-316 of Syk was required for the interaction with Cbl as well as for the maximal tyrosine phosphorylation of Cbl. However, both wild-type Syk and Y316F-mutated Syk phosphorylated equally well the C-terminal fragment of Cbl in vivo, suggesting the existence of an alternative, N terminus-independent mechanism for the Syk-induced tyrosine phosphorylation of Cbl. This mechanism appears to involve Fyn, since, in addition to its association with the C-terminal region of Cbl, Fyn also associated with Syk and enhanced the Syk-induced tyrosine phosphorylation of Cbl. These findings implicate Fyn as an adaptor protein that facilitates the interaction between Syk and Cbl, and suggest that Src and Syk family PTKs coordinately regulate the tyrosine phosphorylation of Cbl.     

Vascular density in melanoma xenografts correlates with vascular permeability factor expression but not with metastatic potential

Previous studies have shown that human airway epithelial cells (AEC) can stimulate allogeneic peripheral blood T-lymphocyte (PBT) proliferation. We now sought to determine which AEC surface molecule/T-cell coreceptors are involved in this process. AEC-induced PBT proliferation was inhibited by 25 microM genestein or herbamycin A (0.9 and 1.8 microM), both tyrosine kinase inhibitors. Anti-phosphotyrosine immunoblots performed on PBT lysates after coculture with AEC demonstrated phosphorylation of 56kD and 60kD bands. To determine whether CD3 associated p59fyn, or CD4 and CD8 associated p56lck phosphotyrosine kinases (PTK) were involved, we assayed kinase activity in lymphocyte lysates immunoprecipitated with anti-p56lck and p59fyn mAbs. PBT cells or murine T-cell line transfectants expressing human CD4 (3G4) or human CD8alpha (3G8) were cocultured with AEC or A549, an alveolar-like cell line lacking class II Ag expression. After A549 or AEC coculture, p56lck activity in PB T-cells peaked at 2 min whereas p59fyn kinase activity continued to rise at 8 min. AEC (expressing class II Ags) stimulate PTK activity in both 3G8 and 3G4 cells. A549 stimulated p56lck in 3G8, but not in 3G4 cells. This activation of p56lck was not blocked by preincubation of A549 with anti-class I or anti-CD1d mAbs. An antibody generated in our laboratory, which recognizes an epithelial specific surface molecule (mAb L12) and which blocks AEC driven PBT proliferation, was shown to block PTK activity of peripheral blood T-cell lysates, though not of 3G8 lysates. These studies suggest that AEC are capable of stimulating CD4 and CD8 associated lck and CD3 associated fyn kinases through class II dependent and independent pathways.     

Induction of late activation events by Igbeta signaling subunit of B-cell receptor in Jurkat T-cell without tyrosine phosphorylation

T-lymphocyte activation consists of multiple intracellular signaling events, eventually leading to cellular proliferation by the control of cytokine gene expression and the acquisition of diverse effector function. To investigate the functional specificity of ITAM (Immunoreceptor Tyrosine-based Activation Motif), chimeric molecules CD8-zeta, CD8-Igalpha, CD8-Igbeta, which contain the extracellular and transmembrane domains of the human CD8alpha molecule and the cytoplasmic tail of T-cell receptor (TcR) chain, Igalpha or Igbeta subunit of B-cell receptor, respectively, were stably expressed in a Jurkat cell line. Upon stimulation with anti-CD8 mAb OKT8, CD8-zeta and CD8-Igalpha chimeric proteins induced tyrosine phosphorylation of various cytoplasmic substrates as seen in TcR stimulation. They were also capable of stimulating IL-2 gene expression in a NF-AT dependent manner and inducing CD69 expression on the surface. However, stimulation of CD8-Igbeta can induce activation of CD69 surface expression and IL-2 gene expression equivalent to the level by CD8-Igalpha and CD8-zeta without induction of the tyrosine phosphorylation of intracellular signaling molecules. These results suggested that some of signaling chains containing ITAM may utilize a signal pathway without substrate tyrosine phosphorylation during T-cell activation leading to the IL-2 secretion.     

Stimulation through CD50 (ICAM-3) induces both activation and programmed cell death of human thymocytes

CD50 (ICAM-3) has been identified as the third CD11a/CD18 (LFA-1) counter receptor. We investigated the expression and possible role of this molecule in the induction of early and late activation events in human thymocytes. We observed that CD50 expression is acquired by early T cell progenitors (CD34+) and maintained during thymic development, reaching the highest levels in the most mature population of thymocytes (CD3high). Neither basal nor cytokine-induced expression of CD50 was observed on untransformed human thymic epithelial cell lines. Cross-linking of CD50 expressed on the surface of human thymocytes, by using mAbs recognizing epitopes not related to the CD11a binding site, transduced transmembrane signals leading to an increase of intracellular calcium concentration. This calcium mobilization was inhibited when CD50 was co-cross-linked with CD45, suggesting that tyrosine phosphorylation is also involved in CD50 signaling. The same anti-CD50 mAbs that were able to affect intracellular calcium levels were shown to induce CD69 but not CD25 expression on human thymocytes. This effect was preferentially observed on CD3low/CD3high thymocyte subpopulations. Cross-linking of CD50 also significantly increased activation-induced cell death of human thymocytes. These results support the idea that CD50 molecule can play a role in developing functionally mature T lymphocytes.     

Distribution and developmental regulation of metabotropic glutamate receptor 7a in rat brain

Ligation of the TCR-CD3 complex initiates a cascade of tyrosine phosphorylation that results in T cell activation. Initial activation of tyrosine kinases depends on the phosphorylation of activation motifs on CD3 chains. We previously found that a 90-kDa protein was tyrosine phosphorylated upon TCR cross-linking and the induction of the phosphorylation was dependent on the structure of the CD3 complex. In this study, we further characterized p90 phosphorylation. Phosphorylation of p90 was induced only by stimulation through the TCR-CD3 complex but not by other kinds of stimulation including CD28- or hydrogen peroxide-mediated activation and was dynamically regulated. Phosphorylated p90 was associated with the TCR-CD3 complex upon T cell activation. In a normal T cell population, thymocytes but not splenic T cells induced the tyrosine phosphorylation of p90 upon TCR cross-linking. These results suggest that p90 is a novel phosphoprotein associated with the TCR-CD3 complex and may play a role in TCR signaling during thymocyte differentiation.     

Update in geriatrics

We have recently observed an abnormal pattern of protein tyrosine phosphoryl-ation in resting T lymphocytes obtained from peripheral blood of patients with systemic lupus erythematosus (SLE). To examine whether these findings may be related to dysregulated protein tyrosine kinase (PTK) function, we tested the relative amount and enzyme activity of the main PTKs involved in the earliest signalling steps triggered via the CD3 pathway. Cell lysates from peripheral blood T cells in SLE patients showed lower amounts of p59(fyn) and p56(lck) as shown by immunoblot. In contrast, the amount of ZAP-70, a PTK of the syk family, was comparable in both groups. However, p59(fyn) immuno-precipitates obtained from unstimulated peripheral blood SLE T cells showed enhanced PTK activity as compared to controls, whereas the PTK activity of p56(lck) and ZAP-70 molecules was comparable in both groups. The unchecked activity of the TCR/CD3-associated src kinase p59(fyn) may alter the balance needed for regulated T cell responses in SLE patients.     

The Syk/ZAP-70 protein tyrosine kinase connection to antigen receptor signalling processes

The T- and B-cell receptor (TCR and BCR) signal transduction processes involve a coordinated interplay between two classes of non-receptor protein tyrosine kinases (PTKs), the Src-family and the Syk/ZAP-70 family of PTKs. Following antigen-receptor stimulation, the Src-family of PTKs mediate the phosphorylation of tyrosine residues contained in a signalling motif localized in the TCR and BCR subunits. The phosphorylation of this signalling motif recruits the Syk/ZAP-70 family of PTKs into the antigen receptor complex. This mechanism requires the tandem SH2 domains in ZAP-70 complexing to two critically spaced phosphotyrosine residues within the signalling motif. The clustering of Syk/ZAP-70 and cross-talk between this family and the Src-PTKs regulates subsequent signalling events that lead to a variety of cellular responses, such as antibody secretion, lymphokine production, cytolytic activity, proliferation, differentiation and cell survival.     

Pyk2 is differentially regulated by beta1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

Beta1 integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by beta1 integrin signaling in human T cells. Stimulation of Jurkat T cells with the alpha4beta1 integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and alpha4beta1 integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4+ T cells confirmed the ability of CD3-derived signals to synergize with beta1 integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell co-stimulation mediated specifically by beta1 integrins.     

Characterization of p59fyn-mediated signal transduction on T cell activation

Protein tyrosine kinase p59fyn is associated with the TCR-CD3 complex and is suggested to play a role in T cell activation. To determine the molecular mechanism of p59fyn-mediated signal transduction in T cell activation, we established murine T cell hybridoma lines that expressed an elevated amount of wild-type or mutant fyns. Clones that expressed high levels of normal p59fyn and active p59fyn, encoded by wild-type and f-14 mutant fyn respectively, showed enhanced IL-2 production upon stimulation by anti-CD3 antibodies or natural antigen. On the other hand, clones that expressed kinase negative p59fyn and p59fyn with an SH2 (Src-homology 2) deletion encoded by t-1 mutant fyn showed little induction of IL-2 production upon stimulation. These data suggest that p59fyn is important in T cell signaling and that the SH2 sequence plays a critical role in the reaction. Induction of tyrosine phosphorylation of multiple proteins upon antigenic stimulation was augmented similarly in the cells that respectively expressed wild-type and f-14 mutant fyns at elevated levels. The proteins that became highly tyrosine-phosphorylated included phospholipase C (PLC-gamma 1), p95vav, ZAP-70, the MAP kinase, CD3 zeta and unidentified proteins of 120, 100 and 80 kDa. Tyrosine phosphorylation of the 120, 95 and 68 kDa proteins associated with PLC-gamma 1 was also observed in these cells upon stimulation. In contrast, only the 100 kDa protein and the MAP kinase were increasingly tyrosine phosphorylated in the antigen-stimulated cells expressing t-1 fyn. These data suggest that PLC-gamma 1, PLC-gamma 1 associated molecules, p95vav, the 80 kDa protein, ZAP-70 and the CD3 zeta chain may be substrates of p59fyn or of other tyrosine kinases regulated by p59fyn and be important in T cell signaling.