Altered glycosylation of leukosialin, CD43, in HIV-1-infected cells of the CEM line.

CD43 (leukosialin, gpL115, sialophorin) is a major sialoglycoprotein widely expressed on hematopoietic cells that is defective in the congenital immunodeficiency Wiskott-Aldrich syndrome. It is thought to play an important role in cell-cell interactions and to be a costimulatory molecule for T lymphocyte activation. Using a metabolic 35SO4(2-) radiolabeling assay or biotinylation of cell surface proteins, we describe here that CD43 are sulfated molecules the glycosylation of which is altered in human immunodeficiency virus type 1 (HIV-1)-infected leukemic T cells of the CEM line. Hyposialylation of O-glycans and changed substitution on N-acetylgalactosamine residues are observed. The glycosylation defect is associated with an impairment of CD43-mediated homotypic aggregation which can be restored by resialylation. The hyposialylation of CD43 on HIV-1+ cells may explain the high prevalence of autoantibodies directed against nonsialylated CD43 that have been detected in HIV-1-infected individuals. A defect in glycosylation of important molecules such as CD43 or, as we recently described, CD45 may explain alterations of T cell functions and viability in HIV-1-infected individuals. In addition, a possible implication of hyposialylation in the HIV-1-infected cells entrapment in lymph nodes could be envisioned.

Inhibition of the NF-kappaB survival pathway via caspase-dependent cleavage of the IKK complex scaffold protein and NF-kappaB essential modulator NEMO.

Apoptosis is mediated by cysteine-dependent, aspartate-directed proteases of the caspase family that proteolyse strategic intracellular substrates to induce cell suicide. We describe here that engagement of apoptotic processes by Fas triggering or by staurosporine stimulation leads to the caspase-dependent inactivation of the nuclear factor kappa B (NF-kappaB) pathway after cleavage of IKK1 (IkappaB kinase 1) and NEMO (NF-kappaB essential modulator), which are needed to transduce NF-kappaB activation signals. In this study, we have analyzed in more detail, the role of NEMO cleavage, as NEMO, but not IKK1, is important for the pro-survival actions of NF-kappaB. We demonstrate that NEMO is cleaved after Asp355 to remove the last 64 C-terminal amino acids. This short form was unable to rescue NF-kappaB activation by tumor necrosis factor-alpha (TNF-alpha) when transfected in NEMO-deficient cells. Consequently, inactivation of NEMO resulted in an inhibition of the expression of antiapoptotic NF-kappaB-target genes coding for caspase inhibitors (cIAP-1, cIAP-2) or adaptors of the TNF receptor family. NEMO-deficient Jurkat cells transiently expressing a non-cleavable mutant of NEMO were less sensitive to TNF-alpha-induced apoptosis. Therefore, downmodulation of NF-kappaB activation via the proteolytic cleavage of NEMO could represent an amplification loop for apoptosis.

Activation of nuclear factor kappaB and Bcl-x survival gene expression by nerve growth factor requires tyrosine phosphorylation of IkappaBalpha.

NGF has been shown to support neuron survival by activating the transcription factor nuclear factor-kappaB (NFkappaB). We investigated the effect of NGF on the expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein. Treatment of rat pheochromocytoma PC12 cells, human neuroblastoma SH-SY5Y cells, or primary rat hippocampal neurons with NGF (0.1-10 ng/ml) increased the expression of bcl-xL mRNA and protein. Reporter gene analysis revealed a significant increase in NFkappaB activity after treatment with NGF that was associated with increased nuclear translocation of the active NFkappaB p65 subunit. NGF-induced NFkappaB activity and Bcl-xL expression were inhibited in cells overexpressing the NFkappaB inhibitor, IkappaBalpha. Unlike tumor necrosis factor-alpha (TNF-alpha), however, NGF-induced NFkappaB activation occurred without significant degradation of IkappaBs determined by Western blot analysis and time-lapse imaging of neurons expressing green fluorescent protein-tagged IkappaBalpha. Moreover, in contrast to TNF-alpha, NGF failed to phosphorylate IkappaBalpha at serine residue 32, but instead caused significant tyrosine phosphorylation. Overexpression of a Y42F mutant of IkappaBalpha potently suppressed NFG-, but not TNF-alpha-induced NFkappaB activation. Conversely, overexpression of a dominant negative mutant of TNF receptor-associated factor-6 blocked TNF-alpha-, but not NGF-induced NFkappaB activation. We conclude that NGF and TNF-alpha induce different signaling pathways in neurons to activate NFkappaB and bcl-x gene expression.

Mast cells and cellularity of the colonic mucosa correlated with fatigue and depression in irritable bowel syndrome.

BACKGROUND: A subset of patients with irritable bowel syndrome (IBS) have an increased number of mast cells (MCs) in the colonic mucosa. Psychological factors are believed to contribute to the course of IBS. AIMS: To examine associations between fatigue, depression and MCs of the colonic mucosa in IBS. METHODS: Colonic biopsies were taken from 50 Rome II IBS patients, 21 healthy controls and 11 depressed/fatigued patients without IBS. The cellularity of the lamina propria was determined as the number of inflammatory cells per high power field (hpf) through a 400x microscope. The Fatigue Impact Scale (FIS) and the short form Beck Depression Inventory (BDI) evaluated the severity of fatigue and depression. RESULTS: IBS patients had a significant increase in the cellularity of the lamina propria compared with controls or with depressed patients (mean (SD) 94.5 (48-110) vs 68 (58-82) and 78 (87-90) cells per hpf, p = 0.005 and p = 0.05, respectively), in particular of MCs (9.3 (5.6-11.7) vs 4.0 (2.7-6.8) and 4.3 (2.8-7.8) cells per hpf, p = 0.001 and p = 0.005, respectively). Both the FIS and BDI scores were significantly higher in IBS or in depressed patients than in controls (p<0.001). In IBS, the FIS score correlated significantly with the cellularity of the lamina propria (r = 0.51, p<0.0001) and MCs (r = 0.64, p<0.0001). In IBS, the BDI score correlated significantly with MCs (r = 0.29, p = 0.03). CONCLUSIONS: Elevated MCs counts are a key feature of the low-grade inflammatory infiltrate in the caecal mucosa of IBS. Fatigue and depression are associated with mucosal cell counts, in particular MCs, suggesting that psychological factors are associated with the low-grade inflammatory infiltrate in IBS.

AS602868, a dual inhibitor of IKK2 and FLT3 to target AML cells.

Acute myeloid leukemia (AML) cells carry molecular defects that promote their leukemic proliferation, resistance to apoptosis and defect in differentiation. Pharmacological targeting of the nuclear factor kappaB (NF-kappaB) pathway has been shown to promote apoptosis of primary AML cells and to sensitize blasts to neoplastic drugs (Frelin, Blood 2005, 105, 804). The Fms-like tyrosine kinase 3 (FLT3), which sustains proliferation of normal hematopoietic progenitors is frequently overexpressed or mutated in AML patients. Using Ba/F3 murine pre-B cells transfected with various mutants of FLT3 (ITD, D835V, D835Y) and the MV4-11 human AML line, we show that normal or oncogenic stimulation of FLT3 led to activation of NF-kappaB. Pharmacological inhibition of either FLT3 with AG1296 or NF-kappaB with the small molecule inhibitor of IkappaB kinase-2 AS602868 reduced viability and triggered cell death. Moreover, AS602868 was also found to interfere directly with FLT3 kinase activation. AS602868 thus appears to target two different kinases that play a crucial role in the pathogenesis of AML, making it particularly attractive as a new therapeutical approach for AML.

Activation of nuclear factor kappaB through the IKK complex by the topoisomerase poisons SN38 and doxorubicin: a brake to apoptosis in HeLa human carcinoma cells.

The transcription factor nuclear factor (NF) kappaB is involved in the regulation of cell survival. NFkappaB is activated in many malignant tumors and seems to play a role in the resistance to cytostatic treatments and escape from apoptosis. We have studied the effects on NFkappaB activation of two topoisomerase poisons and DNA damaging agents that are used in chemotherapy: SN38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT11, and doxorubicin. In HeLa cells, both drugs activate NFkappaB using a preexisting pathway that requires a functional IkappaB-specific kinase complex, IkappaB-specific kinase activation, IkappaB-alpha phosphorylation, and degradation. Blocking NFkappaB activation by stable expression of a mutant super-repressor IkappaB-alpha molecule sensitized HeLa cells to the apoptotic actions of drugs and tumor necrosis factor. RNase protection assay analysis demonstrate that NFkappaB is involved in the regulation of a complex pattern of gene activation and repression during the cellular response of HeLa cells to topoisomerase poisons. The blockade of NF-kappaB activation seems to shift the death/survival balance toward apoptosis.

Cleavage and relocation of the tyrosine kinase P59FYN during Fas-mediated apoptosis in T lymphocytes.

Ligation of Fas with its natural ligand or with anti-Fas antibodies induces an apoptotic program in Fas sensitive cells. We report here the identification of the tyrosine kinase p59Fyn as a substrate for CPP32-like proteinases and more particularly caspase 3 during Fas-mediated apoptosis in Jurkat T cells. Inhibition of CPP32-like proteinases by Ac-Asp-Glu-Val-Asp-aldehyde but not by Ac-Tyr-Val-Ala-Asp-aldehyde prevents CPP32, PARP and p59Fyn cleavage indicating that CPP32 or CPP32-like proteinases are responsible for the cleavage of p59Fyn. Cleavage occurs in the N-terminal domain of p59Fyn between Asp19 and Gly20 and is accompanied by relocation of an active p57Fyn kinase to cytoplasm of Fas-stimulated Jurkat cells as judged by both biochemical and confocal microscopy experiments. Thus, p59Fyn relocation and activity may play an important role during Fas-mediated cell death in human T lymphocytes.

Tumor necrosis factor alpha-mediated inhibition of melanogenesis is dependent on nuclear factor kappa B activation.

Melanogenesis is a physiological process resulting in the synthesis of melanin pigments which play a crucial protective role against skin photocarcinogenesis. In vivo, solar ultraviolet light triggers the secretion of numerous keratinocyte-derived factors that are implicated in the regulation of melanogenesis. Among these, tumor necrosis factor alpha (TNFalpha), a cytokine implicated in the pro-inflammatory response, down-regulates pigment synthesis in vitro. In this report, we aimed to determine the molecular mechanisms by which this cytokine inhibits melanogenesis in B16 melanoma cells. First, we show that TNFalpha inhibits the activity and protein expression of tyrosinase which is the key enzyme of melanogenesis. Further, we demonstrate that this effect is subsequent to a down-regulation of the tyrosinase promoter activity in both basal and cAMP-induced melanogenesis. Finally, we present evidence indicating that the inhibitory effect of TNFalpha on melanogenesis is dependent on nuclear factor kappa B (NFkappaB) activation. Indeed, overexpression of this transcription factor in B16 cells is sufficient to inhibit tyrosinase promoter activity. Furthermore, a mutant of inhibitory kappa B (IkappaB), that prevents NFkappaB activation, is able to revert the effect of TNFalpha on the tyrosinase promoter activity. Taken together, our results clarify the mechanisms by which TNFalpha inhibits pigmentation and point out the key role of NFkappaB in the regulation of melanogenesis.

Activation of human T cells is associated with tyrosine phosphorylation of several cellular proteins.

Human T lymphocytes are activated to proliferate after triggering the T Cell Antigen Receptor Complex. CD3-Ti, with either antigen, mitogenic lectins or monoclonal antibodies against its different subunits. Stimulation of Jurkat leukemic human T cells with anti-CD3 or anti-Ti monoclonal antibodies was found to induce, within 1 min, an increase in the phosphorylation of a set of cellular proteins that can be precipitated with anti-phosphotyrosine antibodies. Seven phosphotyrosine-containing proteins were separated with respective mol. wt of 21, 25, 38, 55, 70, 80 and 110 kDa, among which the 38 kDa species is predominant. Moreover, incubation of Jurkat T cells with sodium orthovanadate, a potent inhibitor of phosphotyrosine protein-phosphatases, was found to potentiate the effects of anti-CD3 mAb on tyrosine phosphorylation. In addition vanadate also induced IL-2 secretion in Jurkat cells when associated with the phorbol ester TPA, further demonstrating the importance of these phosphorylation reactions in the process of T cell activation. Our results therefore allow us to identify several protein substrates of a tyrosine kinase activity, whose stimulation appears to be an early event in human T cell activation through the antigen receptor pathway.

Isolation and characterization of a T lymphocyte mutant defective in the protein kinase C signal transduction pathway.

The phorbol ester TPA is a potent protein kinase C (PKC) activator and a cofactor in the activation of the human Jurkat leukemic T cell line. We have studied the implication of the PKC signaling pathway in the process of T cell activation by generating TPA resistant mutants of Jurkat. These mutants were obtained by recovery of cells that survived a growth arrest induced by TPA. Several cellular phenomena dependent on TPA were dramatically altered in the mutated cells. The mutants were unable to form homoaggregates upon TPA stimulation. Moreover, they did not produce interleukin-2 after activation through engagement of the T cell receptor, in the presence of TPA. These results suggest that the PKC signaling pathway activated by TPA is defective in these cells. In an attempt to define and locate the defect present in the mutants, we have analysed the biochemical properties of PKC, the cellular receptor of TPA. The increase in kinase activity and the translocation of the enzyme to the plasma membrane after stimulation by TPA appeared to be normal in the mutants. We hypothesize that a metabolic step, critical for the completion of T cell activation, distinct from protein kinase C, is impaired in the mutant cells.

The BMI1 polycomb protein represses cyclin G2-induced autophagy to support proliferation in chronic myeloid leukemia cells.

The BMI1 polycomb protein regulates self-renewal, proliferation and survival of cancer-initiating cells essentially through epigenetic repression of the CDKN2A tumor suppressor locus. We demonstrate here for the first time that BMI1 also prevents autophagy in chronic myeloid leukemia (CML) cell lines, to support their proliferation and clonogenic activity. Using chromatin immunoprecipitation, we identified CCNG2/cyclin G2 (CCNG2) as a direct BMI1 target. BMI1 downregulation in CD34+ CML cells by PTC-209 pharmacological treatment or shBMI1 transduction triggered CCNG2 expression and decreased clonogenic activity. Also, ectopic expression of CCNG2 in CD34+ CML cells strongly decreased their clonogenicity. CCNG2 was shown to act by disrupting the phosphatase 2A complex, which activates a PKCζ-AMPK-JNK-ERK pathway that engages autophagy. We observed that BMI1 and CCNG2 levels evolved inversely during the progression of CML towards an acute deadly phase, and therefore hypothesized that BMI1 could support acute transformation of CML through the silencing of a CCNG2-mediated tumor-suppressive autophagy response.

L-type amino-acid transporter 1 (LAT1): a therapeutic target supporting growth and survival of T-cell lymphoblastic lymphoma/T-cell acute lymphoblastic leukemia.

The altered metabolism of cancer cells is a treasure trove to discover new antitumoral strategies. The gene (SLC7A5) encoding system L amino-acid transporter 1 (LAT1) is overexpressed in murine lymphoma cells generated via T-cell deletion of the pten tumor suppressor, and also in human T-cell acute lymphoblastic leukemia (T-ALL)/lymphoma (T-LL) cells. We show here that a potent and LAT1 selective inhibitor (JPH203) decreased leukemic cell viability and proliferation, and induced transient autophagy followed by apoptosis. JPH203 could also alter the in vivo growth of luciferase-expressing-tPTEN-/- cells xenografted into nude mice. In contrast, JPH203 was nontoxic to normal murine thymocytes and human peripheral blood lymphocytes. JPH203 interfered with constitutive activation of mTORC1 and Akt, decreased expression of c-myc and triggered an unfolded protein response mediated by the C/EBP homologous protein (CHOP) transcription factor associated with cell death. A JPH203-resistant tPTEN-/-clone appeared CHOP induction deficient. We also demonstrate that targeting LAT1 may be an efficient broad spectrum adjuvant approach to treat deadly T-cell malignancies as the molecule synergized with rapamycin, dexamethasone, doxorubicin, velcade and l-asparaginase to alter leukemic cell viability.

The protein tyrosine kinase p56(lck) regulates the serine-base exchange activity in Jurkat T cells.

Different classes of protein kinase inhibitors for protein kinase C, cAMP-dependent protein kinase or protein tyrosine kinases have been studied for their effect on phospholipid metabolism. The results show that among the compounds studied, only 4'-aminohydroxyflavone (AHF), previously described as a specific inhibitor of the protein tyrosine kinase p56(lck), markedly increased phosphatidylserine synthesis in Jurkat T cells. The biosyntheses of phosphatidylcholine and phosphatidylethanolamine were not affected. Also, the synthesis of phospholipids from tritium-labeled fatty acid as precursor was left unchanged by the p56(lck) inhibitor. The decreased phosphatidylserine synthesis induced when triggering the CD3-TCR complex was impaired by AHF, suggesting that p56(lck) could be implicated in the regulation of the serine-base exchange enzyme system. Direct evidence for the participation of p56(lck) in the regulation of the serine-base exchange enzyme system was obtained by using p56(lck)-deficient Jurkat cells (J.CaM 1.6) in which the basal base exchange activity was markedly increased and on the other hand AHF had no effect. In addition, transfection of J.Cam 1.6 cells with p56(lck)-cDNA allowed recovery of the AHF activity.

Stimulation of the T-cell antigen receptor-CD3 complex signaling pathway by the tyrosine phosphatase inhibitor pervanadate is mediated by inhibition of CD45: evidence for two interconnected Lck/Fyn- or zap-70-dependent signaling pathways.

The tyrosine phosphatase specific inhibitor pervanadate is a potent activator of T lymphocytes through induction of tyrosine phosphorylation and downstream events of the activation cascade. Using CD45- or CD3-negative variants of the Jurkat leukemic T-cell line we show that the different biochemical events induced by pervanadate appeared to be dependent on the presence at the cell surface of either CD45 or CD3. CD45-dependent events such as tyrosine phosphorylation of Shc, activation of nuclear factor-kappa B (NF-kappa B), activator protein-1 (AP-1), transcription factors, and stimulation of interleukin-2 (IL-2) promoter and of CD69 and CD25 surface expression paralleled activation of the tyrosine kinases lck and fyn. By contrast, stimulation of calcium influx, a CD3-dependent event, paralleled zap-70 activation. The data demonstrate that the T-cell antigen receptor-CD3 (TcR-CD3) complex is functionally linked to two different protein tyrosine kinase (PTK) modules with separate specific functions and that CD45 may be an important regulator of this coupling.

CD3-stimulated Jurkat T cells mediate IL-1 beta production in monocytic THP-1 cells. Role of LFA-1 molecule and participation of CD69 T cell antigen.

In this study we investigated the T cell signals required for monocyte activation. We used an in vitro co-culture system involving two human cell lines: Jurkat T cells and THP-1 monocytes. Monocyte activation was monitored by measuring IL-1 beta production, whereas IL-2 secretion reflected Jurkat activation. We showed that CD-3 -stimulated Jurkat cells delivered an IL-1-inductive signal to THP-1 cells through a cellular contact which was independent of THP-1 Fc receptors cross-linking. Stimulation of IL-1 beta production did not appear to require lymphokine secretion by T cell since a lymphokine defective mutant of Jurkat cell was able to deliver the stimulatory signal. The LFA-1 molecule was clearly shown to participate in the cooperation process, but its role was likely to be restricted to mediating initial adhesive interaction rather than to transducing the IL-1 -inductive signal. Interestingly, the co-culture stimulated by (Fab')2 fragments of CD3 mAb displayed an enhanced IL-1 beta production without any increase of IL-2 secretion. This result indicated that Jurkat cells could stimulate THP-1 cells even when they were only partially activated. The kinetics and conditions of IL-1 beta production called our attention to the early T cell activation antigen CD69. We then showed that CD69 mAb interfered with transmission of the IL-1 inductive signal (40-50% inhibition of IL-1 production). Our results are suggestive of a new role for CD69 molecule intervening in the T lymphocyte-dependent monocyte activation process.

[Biomodulation of transcriptional factor NF-kappa B by ionizing radiation]. / Biomodulation du facteur de transcription NF-kappaB par les radiations ionisantes.

NF-kappaB (Nuclear Factor-kappaB) was described for the first time in 1986 as a nuclear protein binding to the kappa immunoglobulin-light chain enhancer. Since then, NF-kappaB has emerged as an ubiquitous factor involved in the regulation of numerous important processes as diverse as immune and inflammatory responses, apoptosis and cell proliferation. These last two properties explain the implication of NF-kappaB in the tumorigenic process as well as the promise of a targeted therapeutic intervention. This review focuses on the current knowledge on NF-kappaB regulation and discusses the therapeutic potential of targeting NF-kappaB in cancer in particular during radiotherapy.

Pharmacological targeting of NF-kappaB potentiates the effect of the topoisomerase inhibitor CPT-11 on colon cancer cells.

NF-kappaB interferes with the effect of most anti-cancer drugs through induction of anti-apoptotic genes. Targeting NF-kappaB is therefore expected to potentiate conventional treatments in adjuvant strategies. Here we used a pharmacological inhibitor of the IKK2 kinase (AS602868) to block NF-kappaB activation. In human colon cancer cells, inhibition of NF-kappaB using 10 microM AS602868 induced a 30-50% growth inhibitory effect and strongly enhanced the action of SN-38, the topoisomerase I inhibitor and CPT-11 active metabolite. AS602868 also potentiated the cytotoxic effect of two other antineoplasic drugs: 5-fluorouracil and etoposide. In xenografts experiments, inhibition of NF-kappaB potentiated the antitumoural effect of CPT-11 in a dose-dependent manner. Eighty-five and 75% decreases in tumour size were observed when mice were treated with, respectively, 20 or 5 mg kg(-1) AS602868 associated with 30 mg kg(-1) CPT-11 compared to 47% with CPT-11 alone. Ex vivo tumour analyses as well as in vitro studies showed that AS602868 impaired CPT-11-induced NF-kappaB activation, and enhanced tumour cell cycle arrest and apoptosis. AS602868 also enhanced the apoptotic potential of TNFalpha on HT-29 cells. This study is the first demonstration that a pharmacological inhibitor of the IKK2 kinase can potentiate the therapeutic efficiency of antineoplasic drugs on solid tumours.

Phosphorylation of class I histocompatibility antigens in human B lymphocytes. Regulation by phorbol esters and insulin.

Phosphorylation of membrane proteins is one of the earliest steps in cell activation induced by growth-promoting agents. Since MHC (major histocompatibility complex) class I molecules are known to contain phosphorylation sites in their C-terminal intracellular domain, we have studied the regulation of HLA (human leucocyte antigen) phosphorylation in intact cells by two mitogens, namely TPA (12-O-tetradecanoylphorbol 13-acetate), a phorbol ester, and insulin, which are thought to exert their mitogenic effects through the stimulation of different protein kinases (protein kinase C and a tyrosine kinase respectively). Human B lymphoblastoid cells (526 cell line) were pulsed with [32P]Pi to label the intracellular ATP pool. Cells were then stimulated for 10 min with TPA, insulin, cyclic AMP or EGF (epidermal growth factor). The reaction was stopped by cell lysis in the presence of kinase and phosphatase inhibitors, and class I HLA antigens were immunoprecipitated with monoclonal antibodies. Analysis of labelled proteins by gel electrophoresis and autoradiography revealed that TPA increased the phosphorylation of the 45 kDa class I heavy chain by 5-7-fold, and insulin increased it by 2-3-fold. Cyclic AMP and EGF had no stimulatory effect. Analysis of immunoprecipitated HLA molecules by two-dimensional gel electrophoresis showed that TPA and insulin stimulated the incorporation of 32P into different 45 kDa molecular species, suggesting that different sites were phosphorylated by two agents. Moreover, incubation of purified class I MHC antigens with partially purified insulin-receptor tyrosine kinase and [gamma-32P]ATP revealed that class I antigens could also be phosphorylated in vitro by this tyrosine kinase. Altogether, these results therefore confirm that insulin receptors and HLA class I molecules are not only structurally [Fehlmann, Peyron, Samson, Van Obberghen, Brandenburg & Brossette (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8634-8637] but also functionally associated in the membranes of intact cells.

T cell activation: distinct pathways involve phosphorylation of different cellular proteins.

The murine T cell clone D10.G4.1 can be induced to proliferate by monoclonal antibodies (mAb) to the T cell receptor (TcR) or to Thy-1 molecules. When cells were stimulated by anti-TcR mAb, a group of 4 proteins (19-25 kDa) was specifically phosphorylated. This effect was completely mimicked by the Ca2+ ionophore A23187, whereas only two of these proteins (19 kDa and 25 kDa) were phosphorylated after cell exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. By contrast, anti-Thy-1 mAb had no effect on the phosphorylation of these proteins, but induced specifically the phosphorylation of a protein of 32 kDa. These results therefore demonstrate that distinct activating pathways in T cells involve the phosphorylation of different proteins, suggesting that the stimulation of protein kinases in T lymphocytes is an early event in cell activation.