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Prepublished online as a Blood First Edition Paper on October 31, 2002; DOI 10.1182/blood-2002-02-0456.
IMMUNOBIOLOGY
From the Laboratoire de Signalisation
Immunoparasitaire, Département d'Immunologie, Institut Pasteur,
Paris, France; Institute of Molecular Genetics, Prague,
Czech Republic; Institut National de la Santé et de la Recherche
Médicale (INSERM) U396, Immunogénetique Humaine, Institut
des Cordeliers, Paris, France; and Institute of Veterinary
Virology Faculty of Veterinary Medicine, Bern, Switzerland.
Infection of bovine T cells and B cells with the intracellular
protozoan parasite Theileria parva induces a transformed
phenotype with characteristics comparable to leukemic cells. The
transformed phenotype reverts on drug-induced parasite death, and the
cured lymphocytes acquire a resting phenotype and eventually die by apoptosis if not further stimulated. Here, we show that both lymphocyte proliferation and activation of the transcription factor AP-1 are
mediated by Src-family protein tyrosine kinases (PTKs) in a
parasite-dependent fashion. Src-family PTKs are known to be present in
glycolipid-enriched microdomains (GEMs), also called lipid rafts, and
to be negatively regulated by PTK Csk complexed to
tyrosine-phosphorylated transmembrane adapter protein PAG
(phosphoprotein associated with GEMs) also called Cbp (Csk-binding
protein). We, therefore, purified GEMs from proliferating infected B
cells and from growth-arrested cells that had been drug-cured of
parasites. Proliferation arrest led to a striking increase of PAG/Cbp
expression; correspondingly, the amount of Csk associated with
PAG/Cbp in GEMs increased markedly, whereas PTK Hck accumulation
in GEM fractions did not alter on growth arrest. We propose that
Theileria-induced lymphocyte proliferation and permanent
activation of Hck stems from down-regulation of PAG/Cbp and the
concomitant constitutive loss of the negative regulator Csk from the
GEMs of transformed B cells.
(Blood. 2003;101:1874-1881) Infection of bovine T cells and B cells with the
intracellular protozoan parasite Theileria parva induces a
transformed phenotype with characteristics comparable to tumor cells.
Hallmarks of transformation are the capacity of infected cells to
proliferate in vitro in the absence of exogenous growth factors or
antigenic stimulation.1 Moreover,
Theileria-infected leukocytes behave as invasive tumors in
both scid2 and in athymic, irradiated
mice,3,4 and they can form colonies in soft agar.
Parasite-dependent regulation of host cell signal transduction pathways
is thought to be critical in establishing the transformed phenotype of
infected leukocytes.5,6 Consistently, host cell c-Jun
N-terminal kinase (JNK), but not Erk or p38 mitogen-activated
protein (MAP) kinases, is activated constitutively in infected
leukocytes.7,8 JNK activation results in permanent
up-regulation of Jun and Fos family proteins and induction of the
transcription factors AP-18 and ATF-2.9
Importantly, Theileria-induced transformation can be
reverted by treatment with the theilericidal drug
BW720c,10 and the leukocytes then acquire a resting
phenotype, or die of apoptosis, as reviewed in Dobbelaere and
Heussler.11 Reversion of the transformed state is an
unusual feature for tumor cells, as cellular transformation is usually
associated with genomic alterations (rearrangements/mutations) or
stable insertion of foreign DNA.12 Because the
"transforming" stimulus can be eliminated by drug treatment,
lymphocytes infected with T parva offer a rare opportunity
to compare proliferative and antiapoptotic signaling pathways in
transformed cells with those of isogenic resting cells.
Src family members are a group of 9 nonreceptor tyrosine kinases that
share a high degree of structural homology. Their molecules are
organized into 3 major domains, which from the N- to the C-terminus, are a Src homology domain 3 (SH3) domain, a SH2 domain, and the tyrosine kinase (SH1) domain.13 Src family kinases are
widely expressed, but with cell type-specific expression patterns that differ between individual members. Src kinases are activated by a large
variety of receptors such as immunoreceptors (T-cell receptor [TCR],
B-cell receptor [BCR], Fc receptor [FcR]),14 growth
factor receptors,15 cytokine receptors,16
G-protein-linked receptors,17 or
integrins.18 Their function is to convey extracellular
signals through membrane-proximal compartments to cellular effector
pathways; for review, see Brown and Cooper.19
Src family kinases are modified by myristoylation and palmitoylation at
their N-termini; these fatty acid residues direct the enzymes to
membrane compartments20 and particularly to
Glycolipid Enriched Microdomains
(GEMs), also called membrane rafts.21 Recruitment to
nonlipidic subcellular locations, such as the cytoskeleton, requires
SH3 or SH2 domain-dependent interactions, which are thus important in
bringing the kinase into proximity with specific substrates, but also
in regulating their state of activation.22,23 Src family
kinases are negatively regulated by induction of a closed, inactive
conformation.13 Inhibitory phosphorylation of the
C-terminal tyrosine24 by the C-terminal Src kinase
Csk25-27 promotes binding of the SH2 domain and because of
steric hindrance inactivates the kinase as reviewed in Shalloway and
Taylor.28 Intramolecular interactions between the SH3
domain and the stretch of amino acids that link SH2 and catalytic
domains further reinforces the closed conformation.13
Activation is a stepwise process initiated by C-terminal
dephosphorylation,29,30 or SH3 domain displacement,17,22 resulting in an open conformation.
Opening of the catalytic cleft results in autophosphorylation of the
tyrosine in the activation loop within the kinase domain and full
activation of the kinase. In resting cells, or under conditions in
which activity is not required, Src kinases are maintained in an
inactive state through C-terminal phosphorylation by cytoplasmic
tyrosine kinase Csk.27,31 Csk is recruited to membrane and
activated by binding to the transmembrane adaptor protein called
PAG/Cbp phosphoprotine-associated with glycosphingolipid-enriched
microdomains/Csk-binding protein).32-34
In lymphocytes, activation of Src family kinases is a prerequisite to
activate a plethora of signaling pathways downstream of immune
receptors, cytokine receptors, and adhesion molecules, reviewed in
Thomas and Brugge.35 Their role in
Theileria-induced lymphocyte transformation, however, is
only poorly understood. Decreased electrophoretic mobility of Fyn and
Lck isolated from T parva-transformed T cells has been
suggested to reflect increased catalytic activity of these
kinases.5 Furthermore, parasite-dependent regulation of
Lyn, Fyn, and Lck has been observed in T parva-infected T-cell lines, with Fyn as the predominant Src kinase activity detected.36 Permanent activation of lymphocytes by
Theileria parasites induces production and secretion of
cytokines,37-39 and cell-cell contact is required for
infected cell growth,40 raising the possibility that
surface receptor-activated Src kinases are involved in this process.
We have demonstrated that proliferation of
Theileria-transformed lymphocytes is
phosphatidylinositiol-3-kinase (PI3-K) dependent.39 As Src
kinases can regulate both proliferation41-43 and cellular
transformation through activation of PI3-K,44 we decided
to examine the role of Src kinases in the proliferation of
Theileria-transformed B cells. We show that the
parasite-dependent uncontrolled proliferation of infected B cells is
associated with constitutive activation of the protein tyrosine kinase
(PTK) Hck apparently caused by exclusion of its negative regulator,
Csk, from Hck-positive GEMs.
Cells, cell culture
Preparation of resting B cells
Antibodies Antibodies (used in Western blotting usually at 1:1000 dilution) were the following: Hck, rabbit polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA, Sc-72) or mouse monoclonal (Transduction Laboratories); phosphotyrosine, mouse monoclonal 4G10 (Transduction Laboratories) and mouse monoclonal P-TYR-01 (the Prague laboratory); PAG/Cbp, rabbit polyclonal to human PAG (produced in the Prague laboratory); Csk, rabbit polyclonal (Santa Cruz Biotechnology). Mouse mAb PAG/Cbp-C6 (IgG2b) was prepared in the Prague laboratory; it is directed against the C-terminal peptide of human PAG/Cbp and cross-reacts with bovine PAG/Cbp. The hybridoma line secreting mAb CC21 recognizing the bovine CD21 molecule was purchased from the American Type Culture Collection (ATCC, Manassas, VA), and mAb was produced in the Bern lab.Proliferation assays Src kinase inhibitor PP2 (Calbiochem) was resuspended in dimethyl sulfoxide (DMSO) and used at different concentrations as indicated in figure legends. For DNA synthesis measurements, cells were washed in PBS and resuspended at 5 × 104 cells/mL in culture medium containing PP2 at different concentrations or solvent alone. [3H]-thymidine (1 µCi [0.037 MBq]) was added per sample for 17 hours, and incorporated radioactivity was quantified by -counting. When indicated, PMA (100 ng/mL) was added
with solvent or together with PP2.
Transient transfection experiments, luciferase and -galactosidase-expression vector (5 µg) for standardization of transfection efficiency was included in each sample. Luciferase assays
were performed following the manufacturer's (Promega) instructions. Transfection efficiency was standardized on the basis of
-galactosidase activity in each individual transfection: 10 µL
total cell lysate was mixed with 100 µL H2O, 700 µL
Z-buffer (100 mM Na-phosphate pH 7.5, 100 mM KCl, 1mM
MgSO4, 50 mM -mercaptoethanol), and 200 µL ONPG (4 mg/mL o-nitrophenyl -D-galactopyranoside in
100 mM Na-phosphate buffer, pH 7.5) and incubated for 1 to several
hours at 37°C. Reactions were stopped by the addition of 500 µL 1 M Na2CO3 and quantified by spectrophotometry at
420 nm. Results shown in figures are means of at least 3 independent experiments.
Detergent lysates and membrane preparation, kinase assays, SDS-PAGE, and Western blotting Crude plasma membranes were prepared by using the method based on gentle cell disruption in hypotonic buffer as described in detail elsewhere.48,49 For kinase assays, 2 × 107 PBS-washed TpM409 or cured cells or 1.5 × 108 Bcs or stimulated Bcs were lysed in ice-cold laurylmaltoside lysis buffer (150 mM NaCl, 50 mM Tris (tris(hydroxymethyl)aminomethane) [pH 7.5]) 1% laurylmaltoside [dodecylmaltoside; Calbiochem], 20 µg/mL leupeptin, 20 µg/mL aprotinin, 1 mM pefabloc, 1 mM Na-orthovanadate, 25 mM NaF) for 30 minutes on ice. Lysates were cleared by centrifugation and precleared with Protein A-Sepharose for 30 minutes at 4°C, and Hck was immunoprecipitated with 1.5 µg antibody per sample (Santa Cruz Biotechnology, SC-72) for 2 hours under rotation at 4°C. Immune complexes were fixed on Protein A-Sepharose for 30 minutes at 4°C and then washed as follows: 3 times in lysis buffer, once in ice-cold double-distilled H2O, and once in kinase buffer (20 mM Tris [pH 7.4]), 10 mM MgCl2). PP2 was added during the last 2 washing steps and during kinase reaction at 10 µM final concentration as indicated. Kinase assays were performed in the presence of 5 µM cold adenosine triphosphate (ATP), 10 µCi (0.37 MBq) [32P] -ATP, and 1.5 µg/sample
acid-activated enolase at 30°C for 15 minutes. Reactions were stopped
by adding 5× SDS-loading buffer. Proteins were fractionated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 8%
gels and electrophoretically transferred onto nitrocellulose membranes
(Schleicher & Schuell, Dassel, Germany), which allows
quantification of kinase activity and protein detection by Western
blotting on the same support. Incorporation of [32P]
-ATP was detected by phosphor imaging and analyzed and quantified by
using Image-quant software or exposure to X-ray films (Amersham). Equal
loading was confirmed by Western blotting using Hck-specific antibodies, and tyrosine phosphorylation status was determined with
phosphotyrosine-specific antibodies (for antibodies, see "Antibodies"). For Western blotting, the blots were
incubated with antibodies diluted in 5% nonfat milk (1% bovine serum
albumin [BSA]) when antiphosphotyrosine antibodies were used) in PBS, 0.1% Tween-20. Proteins were detected by horseradish
peroxidase-conjugated secondary antibodies and chemiluminescence-based
visualization (Super Signal detection kit, Interchim).
Quantitative immunoisolation of PAG/Cbp from plasma membranes Plasma membranes obtained from 3 × 107 cells were solubilized in 400 µL ice-cold laurylmaltoside (LM) lysis buffer (100 mM NaCl, 20 mM Tris buffer pH 8.2, 50 mM NaF, 10 mM Na-pyrophosphate, 10 mM EDTA [ethylenediaminetetraacetic acid], 1 mM Na-orthovanadate, 1 mM phenylmethyl sulfonyl fluoride [PMSF], 1% laurylmaltoside [dodecylmaltoside; Calbiochem]). The supernatant obtained after 3 minutes of centrifugation at 14 000 rpm was passed through 100-µL minicolumns of PAG-C6 mAb covalently immobilized (at 2 mg/mL gel) on CNBr-Sepharose (Pharmacia), the flow-through fractions were collected, the columns were washed with 1 mL lysis buffer, and the bound material was released by 2 column volumes of SDS sample buffer. The original cell lysates, flow-through, and SDS-eluted fractions were analyzed by SDS-PAGE and Western blotting.Ultracentrifugation in sucrose gradients (preparation of GEMs) TpM409 or cured cells (2 × 107) were collected and washed once in PBS. A volume of 500 µL MBS-Triton X-100 (Tx-100) (25 mM morpholinoethanesulfonic acid [MES], 150 NaCl [pH6.5], 0.5% Tx-100, 1 mM PMSF, 4 µg/µL PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin A, 1 mM orthovanadate, 10 mM NaF,) was added to cell pellet, and cells were lysed for 30 minutes on ice. An equal volume of 85% sucrose solution in metabisulfite (MBS) was gently mixed with the cellular lysate to generate a 42.5% solution (1 mL) at the bottom of an SW55ti ultracentrifugation tube. The lysate/sucrose mixture was subsequently overlayed on ice with 35% (2 mL) and 5% (1 mL) sucrose-MBS containing protease and phosphatase inhibitors described for MBS-T. Ultracentrifugation was performed in a SW55 Ti rotor at 47 000 rpm (200 000g) for 6 hours at 4°C. Samples (300 µL) were taken from the top to the bottom and fractionated by SDS-PAGE. The visible band at the 5% to 35% sucrose interface containing the GEMs corresponded to fraction 4.
Src kinase activity is essential for proliferation of T parva-transformed B cells To test the contribution of Src family kinases to the permanent proliferation of T parva-transformed B cells, we incubated the infected B cells with increasing concentrations of the specific inhibitor PP2 and measured DNA synthesis after 24, 48, and 72 hours of treatment. PP2 significantly reduces DNA synthesis in a dose-dependent manner with an IC50 (concentration that inhibits 50%) of approximately 10 µM (Figure 1A). Phorbol-ester (PMA) stimulation rescues the PP2-induced growth arrest (Figure 1B), albeit not to levels observed with PMA alone, which suggests that Src kinases do not stimulate proliferation through a PKC-dependent mechanism. Consistently, the PKC inhibitor bis-indolylmalemide (BIM) does not induce proliferation arrest of T parva-transformed B cells (data not shown) or T cells.7 We also investigated whether growth inhibition was due to induction of programmed cell death and found that PP2 treatment for 12 or 24 hours did not significantly increase annexin-V binding (data not shown). Thus, Src activity contributes to proliferation of Theileria-transformed lymphocytes, and, similar to PI3-K,39 the kinase inhibition does not result in apoptosis.
Hck is constitutively active in Theileria-transformed B cells The Src family kinase Hck is specifically expressed in cells of hemopoietic origin (reviewed in Thomas and Brugge35), and Western blot analysis revealed particularly high expression of Hck in T parva-transformed B lymphocytes (data not shown). We, thus, examined whether Hck might be responsible for proliferation of the infected B cells. To this end, we immunoprecipitated Hck from total cell extracts of TpM409 and determined Hck kinase activity in an in vitro kinase assay. Hck displayed significant kinase activity as estimated by autophosphorylation and by its capacity to phosphorylate enolase (Figure 2A). In parallel, we performed in vitro kinase assays in the presence of the Src kinase inhibitor PP2 to verify specificity of the kinase reaction (middle panel) and to determine tyrosine phosphorylation state of endogenous Hck prior to autophosphorylation (bottom panel). As expected, PP2 treatment completely blocked Hck autophosphorylation and phosphorylation of exogenous substrate enolase; tyrosine phosphorylation of endogenous Hck revealed by the PP2 treatment was relatively low. Another Src-family kinase expressed abundantly in the B cells, Lyn, was inactive under the conditions of the in vitro kinase assay and its basal level of tyrosine phosphorylation was high (M.B., unpublished data, 1999). Transformation of B cells, constitutive activation of signaling pathways, and permanent proliferation of the infected cells is dependent on the presence of live T parva in the host cell cytoplasm. To investigate whether Hck activity is increased by the intracellular parasite, we performed kinase assays with Hck immunoprecipitated from 2 × 107 transformed (TpM) or cured B cells (Figure 2B) and from 1.5 × 108 resting B cells purified from bovine blood (Figure 2C). Elimination of T parva clearly results in a decrease in Hck kinase activity, which is comparable to the inhibition of DNA synthesis in the presence of Src kinase inhibitor PP2 (Figure 1B), rescued by phorbol ester stimulation (Figure 2B). Compared with TpM, a much lower level of Hck kinase activity was detectable in resting B cells, and, as with cured cells, Hck activity is enhanced by PMA stimulation (Figure 2C). These results suggest that, although active in purified resting B cells, Hck activity is significantly higher in T parva-transformed B cells or B cells stimulated with PMA and ionomycin.
Hck kinase activity is required for constitutive activation of the transcription factor AP-1 The transcription factor AP-1 is constitutively induced in T parva-transformed B cells,8 and its transcriptional activity is regulated by PI3-K.39 As Src family kinases can contribute to AP-1 activation,43,50 we asked whether Hck is involved in activation of AP-1 in Theileria-transformed lymphocytes. To address this point, an AP-1-driven luciferase reporter construct was cotransfected into the TpM409 cells together with either kinase-inactive (267M/499F) or constitutively active (499F) Hck expression vectors (Figure 3). Transient expression of the kinase-dead Hck267M/499F reduced AP-1-driven luciferase activity by more than 50%, whereas its constitutively active counterpart 499F induced AP-1 activation significantly (Figure 3). These results imply that Hck contributes to AP-1 activation in Theileria-transformed lymphocytes.
Coordinated action of Hck and PI3-K is required for full AP-1 activation We have previously shown that permanent activation of AP-1 in Theileria-transformed B cells is under the control of PI3-K, as it can be blocked by overexpression of kinase inactive PI-3K39 and depends exclusively on the JNK kinase pathway.8 As Src family kinases can activate PI3-K directly,51 we examined whether Hck activates AP-1 in PI3-K-dependent manner. Transient expression of Hck499F in the TpM409 cells along with dominant-negative PI3-K ( p110) suppressed
Hck-induced AP-1 activity (Figure 3). This finding is consistent with
PI3-K being directly downstream of Hck in a pathway leading to AP-1
induction. However, parallel expression of a constitutively active form
of PI3-K (p110) did not overcome inhibition of AP-1 transcriptional
activity caused by Hck267M/499F (Figure 3). This result indicates that
the Hck- and PI3-K-mediated signals rather converge on an upstream
activator of the JNK pathway and that both kinases are at least
partially independently important in the induction of AP-1
transcriptional activity in the B cells transformed by T
parva. This hypothesis is compatible with the notion that
activation of PI3-K alone is not sufficient to induce AP-1 activation
in T cells.52
Parasite-dependent exclusion of Csk from Hck-positive GEMs Hck activation, proliferation, and AP-1 activation of Theileria-infected lymphocytes are parasite dependent and as both appear to be mediated by Hck, we asked by what mechanism the parasite could regulate Hck activity. An obvious possibility was regulation via the PAG/Cbp-Csk complex32,33 present, together with Hck, in GEMs. GEMs purified from Theileria-infected B cells and from B cells that had been drug-cured of parasites were analyzed by Western blotting. As demonstrated in Figure 4, Hck (and particularly the p56 isoform) was similarly concentrated in the GEM fractions prepared from both infected and BW720c drug-cured B lymphocytes, suggesting that parasite-mediated regulation of Hck does not involve its recruitment into an active signaling complex in the GEMs (Figure 4Ai). We confirmed the selectivity of the sucrose gradient purification by probing the GEM fraction with an antibody directed against the plasma membrane marker transferrin receptor (TfR), which is only detectable in the Tx-100 soluble fractions of TpM409 and cured cells but not in their GEM fractions (Figure 4B). Importantly, drug-induced parasite death leads to recruitment of Csk to the GEMs (Figure 4Aiii), to increased tyrosine-phosphorylation of Hck (Figure 4Aiv), and to marked increase of a tyrosine-phosphorylated protein of 80 kDa (Figure 4Aiv). This phosphoprotein was unambiguously identified as PAG/Cbp (Figure 4Aii,5A). Interestingly, the total amount of PAG/Cbp in the plasma membrane, rather than just the level of its tyrosine-phosphorylation, markedly increased as a result of the drug treatment; the amount of Csk associated with PAG/Cbp also increased correspondingly (Figure 5B). The effect of cellular activation on PAG/Cbp abundance and Csk recruitment can be mimicked by stimulating cured B cells with PMA/ionomycin, resulting in decreased PAG/Cbp expression and dissociation of Csk (Figure 5B). As expected and analogous to cellular activation by the parasite and decreased PAG/Cbp expression, stimulation of resting B cells with PMA/ionomycin results in PAG/Cbp downregulation (Figure 5C) and Hck activation (Figure 2C).
Reversible Csk recruitment to PAG/Cbp regulates AP-1 activation As Hck contributes to parasite-induced AP-1 activation (Figure 3) and most likely plays a key role in Src kinase-controlled proliferation of infected cells (Figure 1), we hypothesized that it is Csk exclusion from GEMs positive for Hck in transformed or PMA/ionomycin-stimulated cells that increases Hck activity (Figure 2). A comparison of Csk association with PAG/Cbp revealed comparable levels in cured and resting B cells, further arguing for a negative regulatory role of Csk in Hck activation (Figure 6A). We next established functional significance of Csk in negatively regulating transcriptional activation of AP-1 by assaying AP-1-driven luciferase activity following transfection of both wild-type and dominant-negative forms of Csk (Figure 6B). Csk expression clearly leads to a significant (53%) inhibition of the AP-1 activation. As both Hck and PI3-K are involved in signaling to AP-1, we also monitored the distribution of the p85 regulatory subunit and found that it remained largely unchanged in drug-cured lymphocytes (Figure 4Av). This finding is consistent with our previous observation that parasite death leads to a decrease in PI3-K activity, without any reduction in the amount of p85 associated with the plasma membrane.39
T parva orchestrates specific alterations in upstream signaling modules that lead to permanent activation of the transcription factor AP-1 in infected B cells,6,8,39 and we previously found that proliferation of these cells depends on parasite-induced PI3-K activity.39 In addition to directly demonstrating regulation of AP-1 by constitutively activated Hck in T parva-infected cells, the data presented herein reveal that Src family kinase activation is required for proliferation of these cells as well. Constitutive activation of Hck is parasite dependent, and elimination of the parasite leads to a drop in Hck activity to a level comparable to resting B cells (Figure 2). This minimal Hck kinase activity in both cured and resting cells is closely linked to the level of PAG/Cbp expression and phosphorylation and to the association of Csk with PAG/Cbp (Figure 5). Analogous to transient exclusion of Csk from GEMs in activated T cells,32,53,54 Csk associates with tyrosine-phosphorylated PAG/Cbp and accumulates in GEMs only in the absence of the transforming parasite, whereas Hck targeting in these particular membrane compartments appears to be independent of the activation status of the cell (Figures 4-5). Elimination of T parva, which results in growth arrest and decreased AP-1 activity in B cells,39 leads to increased expression of properly phosphorylated PAG/Cbp mediating clear redistribution of Csk into GEMs (Figures 4-5). Hck activation and PAG/Cbp expression can be mimicked by PMA/ionomycin stimulation of resting B cells, suggesting that PAG/Cbp is a critical mediator of B cell activation (Figure 5). Moreover, these data argue that in Theileria-transformed B cells constitutive Hck activation is due to low expression of PAG/Cbp, causing relative exclusion of Csk from GEMs leading further to decreased phosphorylation of negative regulatory C-terminal tyrosine of Hck and thus increased activity of this PTK. Increased phosphorylation of negative regulatory C-terminal tyrosine of Hck by Csk recruited to GEMs is likely to cause inhibition of Hck, which may be crucial in reversion of the transformed phenotype on drug-mediated parasite death. This notion is further underscored by the observation that in infected cells interruption of the spatial separation of Hck and its regulator Csk, by Csk overexpression, results in decreased AP-1 activation. We thus propose that in T parva-transformed B cells, Hck is constitutively and functionally active predominately in GEMs and most probably as a result of permanent exclusion of Csk from this membrane compartment. It cannot be ruled out, however, that additional regulatory mechanisms contribute to the final outcome of Hck kinase activity in infected cells. The relatively low level of endogenous Hck tyrosine phosphorylation (Figure 2) suggests that, in addition to Csk, a phosphotyrosyl-specific phosphatase is permanently involved in Hck regulation. A fraction of PI3-K, which is required in synergy with Hck to induce full activation of AP-1 (Figure 3), colocalizes with Hck in GEMs (Figure 4). Concomitant accumulation of activated Hck and PI3-K in GEMs may thus result in permanent activation of AP-1 described herein and in previous studies.8,39 Because activation of PI3-K and Src family kinases and induction of AP-1 appears to be a common feature of Theileria-infected leukocytes,8,9,11,36,39 it is tempting to speculate that regulation of these kinases in GEMs is a key mechanism of host cell transformation induced by Theileria parasites. Importantly, activities of both these kinases may be jointly regulated by means of the PAG/Cbp-Csk system, as PI3-K is activated by Src PTKs.51 Thus, a crucial step in Theileria-mediated lymphocyte transformation may be down-modulation of PAG/Cbp expression, which results in relaxation of Csk-mediated Src family PTK inhibition and subsequently also Src-family PTK (Hck in B cells)-mediated PI3-K activation. Activated Hck and PI3-K then apparently jointly substantially contribute to the transformed phenotype of the infected cells. Another potential contribution to the transformed phenotype related to modified PAG/Cbp expression could be mediated by the recently described interaction of the C-terminus of PAG/Cbp with a cytoskeleton linker EBP50.54,55 Taken together, our data exemplify that the same molecular mechanisms regulating the fate of B lymphocytes in health may become a target of pathologic alterations caused by an intracellular parasite, and they illustrate how B-lymphocyte activation may be subjected to fine-tuned regulation that is not a direct result of extracellular stimuli. Future work will be directed toward understanding how Theileria parasites regulate expression of PAG/Cbp, as one can speculate that down-modulation of PAG/Cbp expression (or altered phosphorylation) might be involved in other types of B-lymphocyte transformation.
The authors are grateful to Glen Scholz for kindly providing Hck expression vectors and helpful advice. We thank Marie-Françoise Moreau and Brigitte Blumen for technical support and Marie Chaussepied and Alphonse Garcia for stimulating discussions.
Submitted February 12, 2002; accepted September 17, 2002.
Prepublished online as Blood First Edition Paper, October 31, 2002; DOI 10.1182/ blood-2002-02-0456.
Supported by fellowships of the Swiss National Science Foundation and the Roche Research Foundation (M.B.) and by the Center of Molecular and Cellular Immunology LN00A026 (Ministry of Education of the Czech Republic; V.H.) and The Wellcome Trust (J1116W24Z; V.H.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Gordon Langsley, Laboratoire de Signalisation Immunoparasitaire, Departement d'Immunologie, Institut Pasteur, 25-28 rue du Dr Roux, 75724 Paris Cedex 15, France; e-mail: langsley{at}pasteur.fr.
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Abstract
Introduction
5 M 2-mercaptoethanol, and 100 IU/mL gentamycin
(Sigma Chemical). B cells (Bcs) were isolated from PBMCs after staining
with monoclonal antibody (mAb) to CD21. Thereafter, cells were
incubated with antimouse immunoglobulin G1 (IgG1) superparamagnetic
particles (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany), and
labeled cells were isolated using MiniMacs columns (Miltenyi Biotech) following the manufacturer's instructions. The purity of the cells was
evaluated by flow cytometry and shown to be more than 98%. Cell
viability, assessed by exclusion of trypan blue, was more than 95%.
Sorted Bcs were either left untreated or stimulated in medium
supplemented with 50 ng/mL phorbol 12 myristate 13-acetate (PMA) and 1 µg/mL ionomycin for 4 hours, for the last 4 hours of the
24-hour incubation period, or for the last 24 hours of the 48-hour
incubation period. After the times indicated, cells were pelleted
(6000g, 2 minutes, 4°C), and washed twice with ice-cold phosphate-buffered saline (PBS; 6000g, 2 minutes, 4°C).
Thereafter, the supernatant was removed, and the pellets were stored at
80°C until analysis.
