High Mda-7 expression promotes malignant cell survival and p38 MAP kinase activation in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL)-B-cells are quiescent differentiated cells that produce interleukin (IL)-10 and accumulate due to resistance to apoptosis. The mechanisms underlying such resistance are poorly understood. Herein we show that all CLL B-cells tested (30/30) display high mRNA and protein expression of the tumor suppressor Mda-7/IL-24, an IL-10 family member, in comparison to normal B cells. A downstream Mda-7 signaling target, p38 mitogen-activated protein kinase (MAPK) was highly phosphorylated in all CLL cells but not in normal B-cells. Mda-7 expression and p38 MAPK phosphorylation diminished in culture and the latter could be reinduced by recombinant (r)-IL-24 or LPS and Mda-7 transfection. Mda-7/IL-24 siRNA specifically inhibited p38 MAPK phosphorylation in CLL without affecting p38 MAPK, bcl2, or Lyn expression, further demonstrating the direct role of Mda-7/IL-24 in p38 MAPK activation. Both pharmacological inhibition of p38 MAPK and Mda-7 silencing augmented spontaneous apoptosis by three-fold in CLL cells cultured in autologous serum, which was reversed by LPS and r-IL-24. We established the role of p38 MAPK in CLL cell survival and demonstrated a paradoxical effect, whereby Mda-7 and IL-24, inducers of apoptosis in diverse cancer cells, promote the survival of CLL B-cells through p38 MAPK activation.

A human TCR-Ig chimeric protein used to generate a TCR alpha chain variable region-specific mAb.

In a recent report, a construction containing the alpha chain-variable region (V alpha) coding sequence of a cDNA clone derived from a diphtheria toxoid-specific human T cell (P28), fused to a human immunoglobulin kappa light chain constant region (Ck), was used stably to transfect a murine myeloma cell. In the present study, these transfected cells were employed as an immunogen to raise a mAb, termed 1C5V alpha, specific both for the V alpha Ck chimeric protein secreted by the transfectant and the P28 T cell antigen receptor-V alpha region. mAb 1C5V alpha specifically immunoprecipitates the V alpha Ck protein as a family of 32-35 kDa bands present in the 35S-methionine-labeled culture supernatant from the transfected cells. It specifically binds clone P28. Surface molecules recognized by mAb 1C5V alpha are physically linked to the CD3 molecules since cell treatment with either 1C5V alpha or anti-CD3 mAbs caused the simultaneous down-regulation of the CD3/TCR molecular complex. This link is further supported by immunoprecipitation experiments. Thus, both the 1C5V alpha and the anti-CD3 mAbs precipitate the 16-28 kDa CD3 molecules and the disulfide-linked form of P28 TCR from 125I-labeled P28 T cells. Studies performed in order to define whether a stimulus directly acting on the TCR-V alpha region may trigger the intracellular events observed during human T cell activation showed that (a) mAb 1C5V alpha efficiently triggers the phospholipase C transduction pathway revealed by an accelerated phosphoinositides turn-over and an increased production of phosphorylated derivatives of inositol phosphates; (b) mAb 1C5V alpha induces an up-regulation of IL2R mRNA, accompanied by a slight increase of IL2 and IFN alpha mRNA transcripts evidently amplified in the presence of PMA; (c) soluble mAb 1C5V alpha is strongly mitogenic together with PMA. These results provide the first evidence for the structural authenticity of a secreted water-soluble chimeric form of the variable region of a human TCR alpha chain. They further demonstrate that such chimeric proteins may be valuable tool to further dissect the various functional structure of the human TCR.

Identification of cross-reactive T cell restriction epitopes located on the DR7 beta 1 and DR beta 4 molecules.

L cell fibroblasts transfected with HLA class II cDNA clones isolated from a cDNA library produced from a DR7 homozygous cell line were used as antigen-presenting cells (APC) for three HLA DR-restricted, diphtheria toxoid-specific T-cell clones in order to assess the antigen-presenting ability of the transfectants and to define the class II restriction of each clone. Class II-expressing transfectants are capable of presenting antigen to antigen-specific T-cell clones, although the transfectants are less efficient at antigen presentation than conventional APC. Paraformaldehyde fixation of transfectants prior to antigen pulsing abrogated antigen presentation, demonstrating that the transfectants require antigen processing. Antigen presentation by transfectants is completely inhibited by CD4-specific monoclonal antibodies (mAb) and one of four DR-specific mAb, whereas antigen presentation by conventional APC is only partially inhibited. Both the DR alpha:DR7 beta 1 and DR alpha:DR beta 4 (DR omega 53) molecules of the DR7 allotype serve as restriction elements for the diphtheria toxoid-specific T-cell clones. One clone is restricted by the DR7 beta 1 molecule, another clone by the DR beta 4(DR omega 53) molecule, and a third clone by a cross-reactive T cell epitope on DR7 beta 1 and DR beta 4(DR omega 53) molecules. The two DR beta 4(DR omega 53)-restricted clones react, however, differently with a panel of HLA-DR DR omega 53-positive human peripheral blood lymphocytes used as APC. Therefore the data presented here clearly document that the DR beta 4 (DR omega 53) chain may serve as restriction elements for DT-specific T-cell clones. They also provide the first evidence for functional cross-reactivity of the products of two different DR beta loci and in addition emphasize the high complexity of the supertypic HLA-DR omega 53 specificity.

PPAR-alpha is a therapeutic target for chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) cells with aggressive clinical properties express lipoprotein lipase (LPL), which generates activating ligands for the nuclear receptor peroxisome proliferator activated receptor (PPAR)α and allows fatty acids to be used as fuel. However, the role of PPARα in CLL is unclear. PPARα was found to be expressed by circulating CLL cells and highly associated with advanced stage disease. Consistent with this observation, palmitate oxidation rates in circulating CLL cells were similar to more conventional fat-burning cells such as muscle. Transgenic expression of PPARα in CD5(+) Daudi cells increased both their expression of immunosuppressive factors (that is, interleukin (IL)10 and phospho-STAT3) and resistance to metabolic and cytotoxic stressors. In contrast, marked downregulation of PPARα expression accompanied immunogenic death of proliferating CLL cells. The PPARα antagonist MK886 killed circulating CLL cells directly, caused proliferating CLL cells to enter an immunogenic death pathway and cleared CLL xenografts from immunodeficient mice. These results suggest that PPARα is a biological mediator of CLL and MK886 is a clinically relevant agent with activity against CLL.

Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like/progenitor cells.

Malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew; these cells are known as cancer stem-like cells (CSCs) or tumor-initiating cells. Primitive mammary CSCs/progenitor cells can be propagated in culture as floating spherical colonies termed 'mammospheres'. We show here that the expression of the autophagy protein Beclin 1 is higher in mammospheres established from human breast cancers or breast cancer cell lines (MCF-7 and BT474) than in the parental adherent cells. As a result, autophagic flux is more robust in mammospheres. We observed that basal and starvation-induced autophagy flux is also higher in aldehyde dehydrogenase 1-positive (ALDH1(+)) population derived from mammospheres than in the bulk population. Beclin 1 is critical for CSC maintenance and tumor development in nude mice, whereas its expression limits the development of tumors not enriched with breast CSCs/progenitor cells. We found that decreased survival in autophagy-deficient cells (MCF-7 Atg7 knockdown cells) during detachment does not contribute to an ultimate deficiency in mammosphere formation. This study demonstrates that a prosurvival autophagic pathway is critical for CSC maintenance, and that Beclin 1 plays a dual role in tumor development.

Triggering of a sustained calcium response through a p56lck-dependent pathway by exogenous ganglioside GM1 in human T lymphocytes.

Various biologic effects induced by free external gangliosides, including cell-signaling events, have been described in several cell systems. We show in this report that free monosialoganglioside GM1, following its rapid and saturable binding to the cell membrane of human Jurkat T cells, triggers in a few seconds a sustained elevation of the intracellular free calcium concentration. It also induces in parallel the early tyrosine phosphorylation of numerous proteins, including phospholipase C gamma-1. Parallel experiments performed with asialo-GM1 or the ceramide part of the molecule do not reproduce these effects, demonstrating the prominent role played by the sialylated part of the ganglioside. A marked conversion of the T cell-specific tyrosine kinase p56lck to a slow migrating 60-kDa form is also found following GM1 addition. It is accompanied in the same time by an increased kinase activity in p56lck immunoprecipitates. Finally, the marked calcium response and tyrosine phosphorylations triggered by GM1 cannot be observed in a p56lck-negative T cell variant. Together these results demonstrate that the monosialoganglioside GM1 can behave as an authentic activation molecule on human T lymphocytes, likely through a p56lck tyrosine kinase-dependent pathway.

The proto-oncogene Vav product is constitutively tyrosine-phosphorylated in normal human immature T cells.

The proto-oncogene Vav product is markedly tyrosine-phosphorylated after the recruitment of various receptors of cells of hematopoietic origin, including mature T cells. Recent studies on Vav-deficient mice have clearly implicated the product of the proto-oncogene Vav in intrathymic T cell development. Vav tyrosine phosphorylation is probably crucial to connect early tyrosine kinase(s) to downstream molecular events leading to cell division and maturation that occur in the thymus. We investigated the tyrosine phosphorylation of Vav in human thymocytes. Immunoblotting experiments demonstrate that, as in mature T cells, tyrosine phosphorylation of Vav is induced following thymocyte stimulation through the T cell receptor. The main observation, however, is that an important fraction of cellular Vav is constitutively tyrosine-phosphorylated in freshly isolated cells. This phenomenon takes place apparently both in the CD4+CD8+ and the more mature CD4+CD8- and CD4-CD8+ thymocyte cell subsets. Co-immunoprecipitation experiments showed, moreover, that a small amount of Vav is engaged in the multimolecular complex that includes elements of the T cell receptor and the T cell specific ZAP-70 tyrosine kinase. Altogether, these data suggest that a critical pathway for T cell development in the human thymus likely involves the permanent activation of Vav in vivo.

Cell calcium signaling via GM1 cell surface gangliosides in the human Jurkat T cell line.

The cell surface ganglioside GM1 is the specific receptor for the B subunit of cholera toxin. We show here that in the human Jurkat T cell line an increase in intracellular free Ca2+ concentration can be elicited by using B subunits to ligate GM1 molecules. This Ca2+ signaling effect is clearly mediated through GM1 because it can be observed after direct insertion of exogenous GM1 in a Jurkat cell variant deficient in GM1 expression. The observed Ca2+ response clearly involves both the release of Ca2+ from intracellular stores and a Ca2+ influx from extracellular spaces. It is sustained in the presence of 1 mM extracellular Ca2+, whereas it becomes transient in Ca(2+)-free medium. We show that the GM1-mediated stimulation partially empties the CD3-dependent and inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ pool suggesting a dependence of the Ca2+ response from activation of phospholipase C (PLC) metabolism. Accordingly, tyrosine phosphorylation of PLC gamma-1 can be evidenced but only in Jurkat cells highly expressing GM1. GM1 stimulation results in an IL-2 production comparable to that obtained after CD3 activation. Finally, the GM1-linked cell Ca2+ activation pathway is also observed in a Jurkat cell clone lacking Ag-specific receptor expression suggesting that the presence of functional CD3/TCR molecules is not essential for GM1-induced cell Ca2+ response. Altogether, these data show that cell surface gangliosides GM1 may act as a signaling molecule in Jurkat T cells possibly by a new pathway, a finding of importance when considering a possible function for ubiquitous membrane carbohydrate structures in T cell recognition systems.

Cyclic AMP- and inositol phosphate-independent inhibition of Ca2+ influx by cholera toxin in CD3-stimulated Jurkat T cells. A study with a cholera toxin-resistant cell variant and the Ca2+ endoplasmic reticulum-ATPase inhibitor thapsigargin.

In this report, we describe a Jurkat cell variant, termed JCT8, the selection of which is based upon its resistance to cell-growth inhibition mediated by the holotoxin of Vibrio cholerae, cholera toxin (CT). JCT8 cells exhibit normal cAMP production in response to various cAMP inducers, including CT, together with conserved ADP ribosylation in vitro of G-protein Gs alpha by the A subunit of the toxin. However, after a 4-h pretreatment with CT, JCT8 cells have a conserved expression of cell-surface CD3 molecules. These effects are in contrast to those elicited by the toxin in long term PGE2-desensitized Jurkat cells, which remain as sensitive as the wild type to the inhibitory action of CT on cell growth and CD3 cell-surface expression, despite poor responsiveness to CT with regard to cAMP production. In JCT8 cells, Ca2+ mobilization induced via the CD3/TCR is maintained after CT treatment contrasting with its complete suppression in the wild-type and in the PGE2-desensitized cells. However, as in the other cell types, CT still suppresses Ca2+ influx in JCT8 cells. Increase in inositol phosphates by CD3 stimulation of JCT8 cells, including of inositol 1,4,5-triphosphate (I(1,4,5)P3), is only partially antagonized by CT. This suggests either that in JCT8 cells there is a different susceptibility of Ca2+ mobilization and influx to partial inhibition by CT of CD3-triggered phospholipase C (PLC)-induced phosphoinositide hydrolysis or that an additional and PLC-independent suppressive effect of the toxin on Ca2+ influx may exist. To investigate this particular point further, we use Thapsigargin, a Ca(2+)-endoplasmic reticulum ATPase inhibitor that can mobilize in human T lymphocytes I(1,4,5)P3-dependent intracellular Ca2+ pools by a PLC-independent pathway. We demonstrate that the Ca2+ influx triggered in the wild-type Jurkat cells or in JCT8 cells by Thapsigargin is antagonized by CT. The present data are therefore consistent with the idea that CT specifically impairs in the Jurkat T cell model the entry of Ca2+ from extracellular spaces by a mechanism independent not only from cAMP but also in part from inhibition by the toxin of phosphoinositide hydrolysis.

NF-kappa B activation by tumor necrosis factor alpha in the Jurkat T cell line is independent of protein kinase A, protein kinase C, and Ca(2+)-regulated kinases.

NF-kappa B is a DNA-binding regulatory factor able to control transcription of a number of genes, including human immunodeficiency virus (HIV) genes. In T cells, NF-kappa B is activated upon cellular treatment by phorbol esters and the cytokine tumor necrosis factor alpha (TNF alpha). In the present work, we investigated the molecular events leading to NF-kappa B activation by TNF alpha in a human T cell line (Jurkat) and its subclone JCT6, which presents a deficiency in the PKA transduction pathway. We found that in both cell lines, both phorbol ester and TNF alpha were able to activate NF-kappa B. Phorbol activation was positively modulated by Ca2+ influx while TNF alpha activation was not. Furthermore, while PMA activation was inhibited by the PKC inhibitor staurosporin, the TNF alpha effect was unchanged. TNF alpha did not activate cAMP production and its signal was not modulated by cAMP activators. Moreover, cAMP activators did not activate NF-kappa B in Jurkat cells. Thus, TNF alpha-induced NF-kappa B activation was found to be mediated by none of the major signal-mediating kinases such as protein kinase C (PKC), protein kinase A, or Ca(2+)-regulated kinases. Furthermore, we found that cytoplasmic acidification facilitated NF-kappa B activation by both TNF alpha and PKC, by a mechanism that increases NF-kappa B/I kappa B dissociation without affecting the NF-kappa B translocation step.

Cyclic AMP-mediated alteration of the CD2 activation process in human T lymphocytes. Preferential inhibition of the phosphoinositide cycle-related transduction pathway.

Activation of human T lymphocytes via the CD2 molecule produces an enhanced turnover of phosphatidylinositol (PI) cycle-related phospholipids accompanied by the increased production of diacylglycerol (DG) and phosphorylated derivatives of inositol (IP). In this report we demonstrate that increased levels of intracellular cyclic AMP induced in human T lymphocytes by prostaglandin E2 or dibutyryl cAMP antagonize these early biochemical events of the CD2 activation process. Thus, a substantial inhibition of the CD2-induced increase in 32P-phosphatidic acid and 32P-PI values is observed. In parallel, both the DG production and the IP release triggered by the CD2 signal are strongly reduced contrasting with an almost conserved Ca2+ response. We also report here that cAMP does inhibit the CD2-induced proliferation in a dose-dependent manner while the proliferation generated independently of DG and IP production by a combination of Ca2+ ionophore A23187 and 12-O-tetradecanoylphorbol 13-acetate is not affected. These results therefore suggest that (a) intracellular cAMP levels may participate in the regulation of the PI cycle-related transduction pathway involved in the activation process of human T lymphocytes via the CD2 molecule; (b) the observed cAMP-mediated functional inhibitory effects are mainly related to an alteration of this cellular transduction signal; and (c) considering the putative critical second messenger role in the T cell proliferative response of DG and IP, respectively thought to activate the protein kinase C and to raise the intracellular free Ca2+, the lowering of DG production may be the key event responsible for this cAMP-mediated effect.

PGE2-induced cyclic AMP accumulation in human CD4+ T cells is strongly enhanced during the CD2-mediated activatory process.

The effect of a mitogenic combination of two different anti-CD2 monoclonal antibodies on the PGE2-stimulated and basal cAMP production in human CD4+ T cell clones was investigated. The anti-CD2 stimulation strongly potentiates the PGE2-induced cAMP production while both PMA and A23187 produced a less potent effect. On the opposite the anti-CD2 treatment is without any effect on the basal cAMP level contrasting with a marked increase of intracellular cAMP concentrations with A23187 or the combination of A23187 and PMA. These results suggest that activation of CD4+ human T cells via the CD2 molecule significantly influences the cAMP-related transduction pathway. Although PMA and A23187 also modulate the activity of this pathway, their effect in this model is more likely mediated through an amplification of basal cAMP production.

The lymphocyte surface antigen CD38 acts as a nicotinamide adenine dinucleotide glycohydrolase in human T lymphocytes.

The extracellular domain of the lymphocyte surface antigen CD38 has been recently shown to share a high sequence homology with a nicotinamide adenine dinucleotide (NAD+)-specific hydrolyzing enzyme cloned from the ovotestis of the gastropod Aplysia (E. States, D.J., Walseth, T.F., Lee, H. C., Trends Biochem. Sci. 1992. 17:495). In agreement with this finding, we present here evidence that CD38-overexpressing T cells, such as human thymocytes and cells from the human HPB-ALL T cell line, exhibit a NAD(+)-hydrolyzing enzymatic activity present on the outer surface of the cell membrane. In contrast, T lymphocytes with relatively low levels of CD38 marker, such as the human Jurkat cell line, display a lower activity. This suggests a relationship between ecto-NAD+ glycohydrolase activity and CD38 expression, as confirmed here when comparing wild-type Jurkat cells and a Jurkat cell variant overexpressing the CD38 molecule. Moreover, CD38 immunoprecipitates from thymocytes behave as an authentic NAD+ glycohydrolase enzyme: it transforms NAD+ stoichiometrically into nicotinamide plus adenosine 5'-diphosphoribose. Altogether these results strongly support the assumption that CD38 is actually a lymphocyte-specific NAD(+)-hydrolyzing enzyme, a finding that give new prospects to understand the in vivo function of this cell membrane protein.

Ca2+ influx in human T lymphocytes is induced independently of inositol phosphate production by mobilization of intracellular Ca2+ stores. A study with the Ca2+ endoplasmic reticulum-ATPase inhibitor thapsigargin.

Thapsigargin (TG), a sesquiterpene lactone and non-phorbol 12-myristate 13-acetate tumor promoter, stimulates a rapid increase in intracellular free Ca2+ [( Ca2+]i) in human T lymphocytes clone P28. The [Ca2+]i response to TG is sustained in the presence of 1 mM extracellular Ca2+, while it becomes transient in Ca2(+)-free medium suggesting that TG activates both the release of Ca2+ from intracellular stores and the entry of Ca2+ from extracellular spaces. TG-induced Ca2+ influx is completely abolished after cell depolarization caused by increased extracellular concentrations of K+. The rise in [Ca2+]i stimulated by TG occurs in the absence of detectable production of inositol phosphates. Moreover, TG does not alter the early biochemical events of T cell activation triggered through the CD2 or the CD3 T cell antigens. Indeed, both inositol phosphate production and intracellular pH increase induced by specific monoclonal antibodies (mAb) remain unchanged after TG treatment. These data suggest that in human T lymphocytes TG releases Ca2+ from an intracellular pool by a mechanism which is independent of the phospholipase C metabolic pathway. Preincubation with TG of T cell clone P28 empties both the CD2 and the CD3-sensitive intracellular Ca2+ pool(s). Conversely, prestimulation of T cell clone P28 by CD3 or CD2-specific mAb inhibits the Ca2(+)-mobilizing effect of TG. Thus it appears that TG and CD2- or CD3-specific mAb mobilize Ca2+ from common Ca2+ pool(s). Taken together, these results demonstrate that Ca2+ influx in human T cells may be linked to mobilization of intracellular Ca2+ pools and by a mechanism independent of phosphoinositide hydrolysis. They further indicate that the release of intracellular Ca2+ pool(s) may play a major role in the opening of cell membrane Ca2+ channels observed during the CD2- or CD3-induced stimulation of human T lymphocytes.

CD1 stimulation of human T cell lines induces a rapid increase in the intracellular free Ca2+ concentration and the production of IL-2.

The effect of a battery of CD1 mAb on intracellular free Ca2+ concentration and IL-2 production has been examined on different T cell lines in this study. Both 0249F and NU-T2 two CD1b specific mAb tested, induced a rapid increase in the intracellular Ca2+ concentration on HPBALL T cells whereas only one (L161) among three different CD1c mAb (L161, 10C3, and M241) produced a similar effect. In contrast the addition of four different CD1a mAb directed against two different epitopic groups of this molecule were uneffective in modifying the intracellular Ca2+. Both L161 and 0249F also induced a comparable increase in the intracellular Ca2+ concentration on MOLT 4 and JURKAT, two other T cell lines of similar phenotype. The effect of L161 mAb on the IL-2 production of the IL-2 producing T cell line JURKAT was also examined. The association of the latter with PMA strongly induced the production of IL-2 on this cell model while either L161 or PMA alone had no effect. Although the natural ligand and the function of CD1 molecules are still unknown, the accumulation of these data strongly suggest that CD1b and CD1c might represent two activatory pathways for immature T cells operating before the classical CD2 and CD3 activation pathways.

In vivo association of CD5 with tyrosine-phosphorylated ZAP-70 and p21 phospho-zeta molecules in human CD3+ thymocytes.

CD5 is a 67-kDa T cell surface Ag that can be found physically associated with the CD3-TCR molecular complex. In different experimental models it has been shown to act as a costimulatory receptor for T cell activation. Unexpectedly, studies in CD5-deficient mice suggested a negative role for the CD5 Ag in CD3-TCR signaling in the thymus. In this report we assessed the constitutive interactions of CD5 in freshly isolated human thymocytes with signaling elements of the CD3-TCR complex. We determined that the ZAP-70 protein tyrosine kinase was present in CD5 immunoprecipitates. The two molecules were constitutively tyrosine phosphorylated in a complex also associating with unphosphorylated as well as phosphorylated zeta-chains. Although both p21 and p23 tyrosine-phosphorylated forms of zeta as well as phospho-CD3 epsilon molecules were constitutively present in human thymocytes and could be immunoprecipitated with ZAP-70- or CD3 epsilon-specific Abs, the p21 species of zeta was predominant in CD5 immune complexes. The interaction between CD5 and ZAP-70 was not observed in CD3-negative thymocytes, where the constitutive tyrosine phosphorylation of ZAP-70 was very low. We conclude that CD5 may affect in vivo the signaling capacity of TCRs expressed by human thymocytes by altering the phosphorylation state of CD3 and/or by retaining ZAP-70 with the p21 species of zeta.

The pseudo-immunoreceptor tyrosine-based activation motif of CD5 mediates its inhibitory action on B-cell receptor signaling.

Genetic studies revealed that CD5 could be a negative regulator of the B-cell antigen receptor (BCR). We explore here the effect of human CD5 on BCR-triggered responses. B cells were obtained expressing a chimera composed of extracellular and transmembrane domains of Fcgamma type IIB receptor fused to CD5 cytoplasmic domain (CD5cyt). Coligation of the chimera with the BCR induces CD5cyt tyrosine phosphorylation. A rapid inhibition of BCR-induced calcium response is observed, as well as a partial but delayed inhibition of phospholipase Cgamma-1 phosphorylation. Activation of extracellular regulated kinase-2 is also severely impaired. Moreover, at the functional level, interleukin-2 production is abolished. Src homology 2 domain-bearing tyrosine phosphatase SHP-1 and Src homology 2 domain-bearing inositol 5'-phosphatase SHIP usually participate in negative regulation of the BCR. We show that they do not associate with the phosphorylated CD5 chimera. We finally demonstrate that the pseudo-immunoreceptor tyrosine based activation motif present in CD5cyt is involved because its deletion eliminates the inhibitory effect of the chimera, both at biochemical and functional levels. These results demonstrate the inhibitory role of CD5 pseudo-immunoreceptor tyrosine based activation motif tyrosine phosphorylation on BCR signaling. They further support the idea that CD5 uses mechanisms different from those already described to negatively regulate the BCR pathway.

A dual participation of ZAP-70 and scr protein tyrosine kinases is required for TCR-induced tyrosine phosphorylation of Sam68 in Jurkat T cells.

Sam68 has been initially described as a substrate of src kinases during mitosis in fibroblasts. Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction between Sam68 and the two src kinases involved in T cell activation, p59(fyn) and p56(lck), as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cgamma-1 and Grb2, has been shown. In this study we analyze the contribution of p56(lck), as well as the role of ZAP-70, the second class of protein tyrosine kinase involved in T cell activation, in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using the src inhibitor PP1 [4-amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo [3,4-d] pyrymidine] and cell variants with defective expression of p56(lck) or expressing a dominant negative form of ZAP-70, we demonstrate that, while both p56(lck) and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p56(lck) and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, we observe that p56(lck) activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that p59(fyn) and p56(lck) differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP-70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation.

Fyn membrane localization is necessary to induce the constitutive tyrosine phosphorylation of Sam68 in the nucleus of T lymphocytes.

A close relationship between Sam68, a tyrosine and proline-rich RNA-binding protein, and Src protein tyrosine kinases (PTK) has already been established, also in T lymphocytes. A constitutive phosphorylation of the molecule has also been documented in various transformed T cells, which probably reflects an increased expression of PTK of the Src family. Using the hybridoma T cell line, T8.1, or Jurkat T cells, we investigated the respective contribution of the two Src kinases Fyn and Lck, expressed in T cells, in this phenomenon. By overexpressing the two proteins, we show that the constitutive phosphorylation of Sam68 in vivo directly correlates with cellular Fyn levels, but not with Lck expression, despite the capacity of the PTK to strongly phosphorylate the molecule in vitro. Overexpressed Fyn is mainly localized at the cell membrane. We find that Sam68 phosphorylation, including in the nuclear fraction in which the molecule is predominantly expressed, is lost with a delocalized Fyn mutant deleted of its N-terminal membrane-anchoring domain. Finally, we demonstrate, using a construct encoding a Sam68 molecule without its nuclear localization signal, that nuclear expression of Sam68 is not required for phosphorylation. We conclude that the constitutive phosphorylation of Sam68 in T cells is a Fyn-dependent process occurring in a cell-membrane compartment from which phospho-Sam68 molecules can thereafter accumulate into the nucleus.

Coordinated regulation in human T cells of nucleotide-hydrolyzing ecto-enzymatic activities, including CD38 and PC-1. Possible role in the recycling of nicotinamide adenine dinucleotide metabolites.

The human leukocyte surface Ag CD38 was recently identified as a nicotinamide adenine dinucleotide (NAD)(+)-glycohydrolase ecto-enzyme, degrading NAD into nicotinamide and ADP-ribose. We show here that expression of CD38 is increased in the Jurkat T cell line after treatment with agents that augment intracellular cAMP, with the permeant cAMP analogue dibutyryl-cAMP (db-cAMP), and also with PMA, which activates protein kinase C. Treatment of human PBL T cells with db-cAMP or submitogenic doses of PMA also increased CD38 expression. Two other nucleotide-hydrolyzing activities were induced on the T cell surface concomitantly with CD38: the human PC-1 molecule, a nucleotide phosphodiesterase/pyrophosphatase that produces AMP from NAD or ADP-ribose, and a nucleotidase that produces adenosine from AMP, but which may be distinct from the CD73 5'-nucleotidase. All three enzymes were up-regulated after stimulation of human peripheral blood T cells with PHA. The coordinated regulation of these ecto-enzymes suggested that, besides a possible signaling function, they may recycle extracellular NAD by degrading it to adenosine and nicotinamide, which can be taken up by cells. In support of this hypothesis, db-cAMP-treated Jurkat cells could degrade extracellular NAD for de novo synthesis of purines, while untreated cells could not. Activated lymphocytes are often located in tissues in which cell death is common. It is suggested that the coordinated expression of these enzymes may allow activated T cells to re-use NAD and nucleotides from dead cells.