The SH3 Domain of Lck Modulates T-Cell Receptor-Dependent Activation of Extracellular Signal-Regulated Kinase through Activation of Raf-1

Cell cultures and transfection and establishment of stable cell lines.The human Jurkat T leukemia cell line (clone E6-1) and the Lck-deficient Jurkat mutant J.CaM1.6 were obtained from the American Type Culture Collection (ATCC). All cells were maintained at logarithmic growth at 37°C and 5% CO₂ in RPMI 1640 medium supplemented with 7.5% fetal bovine serum, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, and 50 U/ml penicillin G and 50 μg/ml streptomycin. For transient transfections, DNA was introduced into cells by electroporation using a Cell-Porator (Life Technologies). J.CaM1.6 cells at a density of 4 × 10⁵ to 6 × 10⁵ cells/ml were harvested and resuspended in RPMI 1640 medium supplemented with only 7.5% fetal bovine serum to a final density of 1 × 10⁷ cells/500 μl. For each transfection, 5 μg of Lck-enhanced green fluorescent protein (EGFP) DNA (WTLck or W97ALck) or 15 μg of myc-Raf-1 DNA was preincubated with 500 μl cell suspension at room temperature for 15 min. Electroporation was then performed at a setting of 800 μF, 250 V. After electroporation, the cells were kept at room temperature for 15 min and transferred into complete medium. Cells were harvested 24 to 36 h after transfection and prepared for further analysis. Jurkat cells were transfected with 20 μg of EGFP-N-Ras. J.CaM1.6 cell lines stably expressing either WTLck or the W97ALck mutant (14) were generated by cotransfecting cells with a pCAGGS plasmid containing either WTLck or W97ALck (15 μg DNA) and the pBabe-puro plasmid (5 μg DNA), which confers resistance to the antibiotic puromycin. Twenty-four hours following transfection, transfected cells were selected for growth in media containing 0.25 μg/ml puromycin for 14 days. Selected cells were screened for Lck expression by anti-Lck immunoblotting and cloned by limiting dilution. Cloned cell lines expressing either WTLck or W97ALck at similar levels were identified and used in the study. Equivalent TCR expression levels in all cell lines were verified by fluorescence-activated cell sorter (FACS) analysis (Purdue Cancer Center Analytical Cytology Facility). Experiments were conducted primarily using W97ALck clone D3.A2 and verified using W97ALck clone G2.B5 and W97ALck clone H6.B3.

Plasmids.Murine Lck cDNA was obtained from Andrey Shaw (Washington University) and subcloned into the pCAGGS vector (39) by use of EcoRI sites, resulting in the expression plasmid pCAGGS-Lck. The generation of pCDNA3-LckGFP constructs was described previously (14). The human Raf-1 cDNA sequence was subcloned from the construct p627 (ATCC 41050) into pCS2+MT, resulting in a plasmid (pCS2+MT-myc-Raf-1) encoding five copies of the myc tag followed by the human Raf-1 cDNA sequence. The myc-Raf-1 sequence was subcloned into pCAGGS by use of EcoRI sites, resulting in the expression plasmid pCAGGS-myc-Raf-1. The YY340/341FFRaf-1 mutant was generated using a Transformer site-directed mutagenesis kit (Clontech). All plasmids were verified by DNA sequencing. The pGEX-RBD vector, encoding the glutathione S-transferase (GST)-RBD (Ras-binding domain of Raf-1) fusion protein, was obtained from Elizabeth Taparowsky (Purdue University); the yellow fluorescent protein (YFP)-GalT and the EGFP-N-Ras vectors were obtained from Mark Philips (New York University School of Medicine); the pGEX-MEKKD vector, encoding the kinase-deficient GST-MEK fusion protein, was obtained from Curtis Ashendel (Purdue University); and the pGEX-CD3-ζ vector, encoding the GST-CD3-ζ fusion protein, was obtained from Janet Smith (GlaxoSmithKline).

Antibodies and reagents.Mouse anti-CD3-ε antibody (UCHT1, NA/LE; BD Pharmingen) was used to ligate the TCR and to determine the TCR expression level by FACS analysis. The goat anti-mouse immunoglobulin G (IgG)-fluorescein isothiocyanate used in FACS analysis was purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-Lck antibody (2102; Santa Cruz Biotechnology) was used for anti-Lck immunoblotting. The rabbit polyclonal anti-Lck antiserum used in anti-Lck immunoprecipitations was described previously (28, 35). The anti-phospho-ZAP-70 (Tyr319) rabbit polyclonal antibody, anti-phospho-MEK1/2 (Ser217/221) rabbit polyclonal antibody, and anti-phospho-ERK1/2 (Thr202/Tyr204) rabbit polyclonal antibody were purchased from Cell Signaling Technology. The anti-phospho-Raf-1 (Ser338) mouse monoclonal antibody, anti-ZAP-70 rabbit polyclonal antibody, and mixed anti-PLC-γ1 mouse monoclonal antibodies were from Upstate Biotechnology. The anti-Raf-1 mouse monoclonal antibody was from BD Transduction Laboratories. The anti-phospho-PLC-γ1 (Tyr783) rabbit polyclonal antibody was from Biosource. The mouse anti-CD3-ζ antibody (6B10.2), anti-Raf-1 rabbit polyclonal antibody (C-12), anti-c-myc tag mouse monoclonal antibody (9E10), anti-MEK1 rabbit polyclonal antibody (12-B), anti-ERK1 rabbit polyclonal antibody (691), and anti-N-Ras mouse monoclonal antibody (sc-31) were purchased from Santa Cruz Biotechnology. The anti-pan-Ras mouse monoclonal antibody (Ab-3) was from Oncogene Research Products. The antiphosphotyrosine mouse monoclonal antibody (4G10) was purchased from Upstate Biotechnology. The antigiantin antibody was from Covance Research Products, Inc. Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated goat anti-mouse antibodies were purchased from Molecular Probes. Rhodamine-conjugated goat anti-rabbit IgG was purchased from Jackson ImmunoResearch. The Src family protein tyrosine kinase inhibitor PP2 was purchased from Calbiochem.

Cell stimulation.Cells in logarithmic growth were harvested and resuspended at 1.0 × 10⁷ cells/ml in serum-free RPMI medium and placed on ice. Anti-CD3-ε antibody was added at the indicated concentrations. Cells were incubated with the antibody on ice for 15 min, followed by a 3-min incubation at 37°C. Stimulated cells were returned to ice for 3 min prior to centrifugation at 180 × g for 10 min at 4°C. Pelleted cells were resuspended in 1% NP-40 lysis buffer (1% Nonidet P-40, 25 mM HEPES, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin and leupeptin, pH 7.4) and incubated on ice for 15 min. Lysates were cleared by centrifugation at 18,000 × g for 10 min at 4°C. Cell lysates were either boiled with equal volumes of sodium dodecyl sulfate (SDS) sample buffer (0.25 g/ml sucrose, 0.025 g/ml SDS, 25 mM Tris, 2.5 mM EDTA, 0.025 mg/ml pyronin Y, 5% 2-mercaptoethanol, pH 8.0) and resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or left for further analysis.

Immunoblotting and immunoprecipitation.Protein samples were resolved by SDS-PAGE on 8% gels (or as otherwise specified) and transferred to polyvinylidene difluoride membranes (Immun-Blot PVDF; Bio-Rad). Membranes were blocked with 5% goat serum and incubated with primary antibody at room temperature for 1 to 2 h (or at 4°C overnight for phosphospecific antibodies), followed by incubation with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG. Membranes were developed using enhanced chemiluminescence reagents (ECL; Amersham Pharmacia Biotech) and autoradiography film. Quantitative immunoblotting was performed using a ChemiImager system (Alpha Innotech) or, where specified, using alkaline phosphatase-conjugated goat anti-mouse IgG+IgM (Amersham Pharmacia Biotech) and enhanced chemifluorescence (ECF; Amersham Pharmacia Biotech) and analyzed using a Molecular Dynamics Storm 860 imaging system.

For immunoprecipitations, cell lysates (in 1% NP-40 lysis buffer) were incubated with antibody and protein A-conjugated Sepharose 4B beads at 4°C for 2 h. After incubation, the beads were washed four times with prechilled 1% NP-40 lysis buffer and either boiled with SDS sample buffer and resolved by SDS-PAGE or left for further analysis.

In vitro Lck and Raf-1 kinase assays.Anti-Lck immunoprecipitates (polyclonal anti-Lck antibody [28, 35]) prepared from 1 × 10⁷ cells were washed four times with prechilled 1% NP-40 lysis buffer and once with prekinase buffer (25 mM HEPES, 1 mM sodium vanadate, 20 μg/ml aprotinin and leupeptin, pH 7.4) and incubated with 5 μg GST-CD3-ζ fusion protein in 50 μl kinase assay buffer (25 mM HEPES, 1 mM sodium vanadate, 20 μg/ml aprotinin and leupeptin, 10 mM MnCl₂, 5 μM ATP, 5 mM p-nitrophenyl phosphate, 20 μCi [γ-³²P]ATP/reaction, pH 7.4) at 30°C for the indicated times. The reactions were stopped by the addition of equal volumes of SDS sample buffer. The samples were boiled and resolved by SDS-PAGE, transferred to PVDF membranes, and quantified by using a Molecular Dynamics Storm 860 imaging system.

For Raf-1 kinase assays, exogenously expressed myc-Raf-1 was precipitated using anti-myc antibody (3 × 10⁶ transfected cells/sample) and endogenous Raf-1 was precipitated using anti-Raf-1 rabbit polyclonal antibody (1 × 10⁷ cells/sample). The Raf-1 immunoprecipitates were washed four times with prechilled 1% NP-40 lysis buffer and once with the prekinase buffer and incubated with 5 μg kinase-deficient GST-MEK fusion protein in 50 μl kinase assay buffer at 30°C for 20 min (myc-Raf-1) or 10 min (endogenous Raf-1), and the reactions were stopped by the addition of equal volumes of SDS sample buffer. Raf-1 kinase activity was analyzed following the same procedure as that for the Lck kinase activity.

ERK phosphatase assay.ERK phosphatase activity was determined as previously described, with minor modifications (19). Whole-cell lysates (80 μg/sample) prepared in 1% NP-40 lysis buffer (without phosphatase inhibitors, containing 10 μM MEK inhibitor U0126) were diluted 1:4 in phosphatase assay buffer (10 mM MgCl₂, 10 mM HEPES, pH 7.4, and 10 μM MEK inhibitor U0126). Recombinant phosphorylated GST-ERK2 (Upstate Biotechnology) was added at 30 ng/sample and incubated for various times at room temperature. Samples were resolved by SDS-PAGE and transferred to PVDF membranes. The remaining amount of phosphorylated GST-ERK2 was determined by immunoblotting using antibodies against phospho-ERK1/2. Endogenous ERK2 was used as a protein loading control. The exposed film was developed using ECLplus and quantified using a Typhoon 9400 variable-mode imager and ImageQuant software (GE Healthcare Life Sciences). The data were analyzed using Microsoft Excel software and are expressed as the decrease in ERK phosphorylation as a function of time.

Ras activity assays.Cells (2.5 × 10⁶) were lysed in 500 μl Mg²⁺-containing lysis buffer (1% Nonidet P-40, 25 mM HEPES, 150 mM NaCl, 0.25% sodium deoxycholate, 10% glycerol, 10 mM MgCl₂, 1 mM EDTA, 1 mM sodium vanadate, 10 mM NaF, 20 μg/ml aprotinin and leupeptin, pH 7.4) on ice. The lysates were cleared by centrifugation at 18,000 × g for 10 min at 4°C. The supernatants were mixed with GST-RBD fusion protein (10 μg/sample) bound to glutathione-Sepharose 4B beads for 30 min at 4°C. The beads were collected and washed with prechilled Mg²⁺-containing lysis buffer three times. The samples were boiled with SDS sample buffer, resolved by SDS-PAGE on 12% gels, and transferred to PVDF membranes. Membranes were blotted with anti-pan-Ras antibody and quantified.

Fluorescence microscopy.J.CaM1.6 cells were transiently transfected with either WTLck-EGFP or W97ALck-EGFP. The data in Fig. 8B were visualized by fluorescence microscopy as previously described (23). All other image collections and analyses were performed in the Purdue University Cytometry Laboratory (see below). Jurkat cells were transiently transfected with YFP-GalT as described above. For all immunofluorescence studies, cells were washed with phosphate-buffered saline and overlaid onto poly-l-lysine-coated coverslips (100 μg/ml). Cells were fixed with 3.7% formaldehyde solution for 10 min. For indirect immunostaining, cells were incubated with blocking buffer (10% goat serum in phosphate-buffered saline), followed by incubation with primary antibody overnight at 4°C and then with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated goat anti-mouse IgG or rhodamine-conjugated goat anti-rabbit IgG. Images were collected on a Nikon e1000 fluorescence microscope, and confocal analysis was performed with a Bio-Rad 2100 MP multiphoton microscope in the Purdue University Cytometry Laboratories in the Bindley Biosciences Center. All images were captured using identical camera settings. Image processing was performed using Image Pro-Plus package 6.2 (Media Cybernetics, Silver Spring, MD). The statistical data analysis and plots were prepared using R, a public-domain computer language and programming environment for statistical computing (R Foundation for Statistical Computing, Vienna, Austria). Image analysis was performed by segmenting the rhodamine channel (giantin) and using it as a mask to extract the Golgi membrane regions in the GFP channel (LckGFP). The segmentation threshold was constant for all images and was determined experimentally. The outline of the cells was found by binary segmentation of green-channel images. The extracted binary masks were smoothed using morphological filters (opening and closing filter with a 5 by 5 octagonal kernel). The background of the green images was flattened and removed by fitting a two-dimensional polynomial before segmentation. To eliminate the contribution of autofluorescence, cells that contained less than 50 arbitrary units/pixel were excluded from the analysis. Mann-Whitney analysis was performed to determine the ratio of GFP fluorescence intensity associated with the Golgi body to the total fluorescence intensity of the cell.

Generation of cell lines stably expressing either WTLck or W97ALck.WTLck and a point mutant crippling the binding capability of the SH3 domain (W97ALck) were expressed in the Lck-deficient cell line J.CaM1.6, a derivative of the human leukemia T-cell line Jurkat (12, 34). Several stable cell lines expressing similar levels of WTLck and W97ALck were isolated (Fig. 1A) and analyzed for TCR expression by flow cytometry (Fig. 1B). The expression level of WTLck and W97ALck was approximately one-fifth of the level of Lck present in parental Jurkat cells (Fig. 1A). We found that the widely used anti-Lck mouse monoclonal antibody clone 3A5 (Santa Cruz Biotechnology, Inc.) poorly recognized W97ALck in immunoblotting protocols (data not shown). Therefore, a rabbit polyclonal antibody was used throughout this study. Cell lines expressing similar levels of WTLck or W97ALck and equivalent levels of TCR were chosen for further study.

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FIG. 1.

Stable cell lines expressing WTLck or W97ALck. (A) Cell lysates from Jurkat (5 × 10⁵ cells), J.CaM1.6 (2.5 × 10⁶ cells), and generated W97ALck (2.5 × 10⁶ cells) cell lines were assayed for Lck expression by immunoblot analysis using anti-Lck polyclonal antibodies. (B) TCR expression level was determined by staining cells with anti-CD3-ε mouse monoclonal antibody followed by goat anti-mouse IgG-fluorescein isothiocyanate secondary antibody. Stained cells were analyzed by flow cytometry. Control cells were stained with secondary antibody in the absence of the primary antibody.

TCR-induced ERK and MEK activation were reduced in W97ALck-expressing cells.In agreement with results originally reported by Denny et al. (8), we found that cells expressing W97ALck failed to support the activation of ERK following TCR ligation (Fig. 2A). Additionally, however, we found that this failure was critically dependent on the level of stimulation through the TCR. At high concentrations of anti-TCR stimulating antibody, the activation of ERK in W97ALck-expressing cells was similar to that seen in cells expressing WTLck. At lower concentrations of stimulating antibody, however, loss of binding though the SH3 domain of Lck had a profound effect on ERK activation. W97ALck cells, however, showed normal activation following stimulation with phorbol myristate acetate (data not shown). As shown in Fig. 2A, a similar but less dramatic result was obtained when the activity of MEK was measured. These results were obtained with three independently derived W97ALck-expressing cell lines (Fig. 2B). One potential explanation for the decreased ERK MAP kinase activity in W97ALck-expressing cells was that these cells contained elevated dephosphorylating activity toward ERK MAP kinase. This does not seem to be the case, as, if anything, there appeared to be a slight decrease in phosphatase activity toward exogenous phospho-ERK MAP kinase in cell lysates from W97ALck-expressing cells compared with the activity in lysates from cells expressing WTLck (Fig. 2C).

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FIG. 2.

Decreased activation of ERK1/2 and MEK1/2 in cell lines expressing W97ALck. (A) WTLck (WT)- and W97ALckD3.A2 (97A)-expressing cells were incubated with the indicated concentrations of anti-CD3-ε antibody. Cell lysates were assayed for the presence of phosphorylated ERK1/2 by immunoblotting using anti-phospho-ERK1/2 antibody (top panel) and phosphorylated MEK1/2 by immunoblotting using anti-phospho-MEK1/2 antibody (third panel). The membranes were stripped and reprobed with anti-ERK1/2 antibody (second panel) and anti-MEK1/2 antibody (bottom panel) to confirm equal loading of samples. The activation of ERK1/2 and MEK1/2 was determined in separate experiments. The experiments were repeated three times with similar results. Pixel intensities were determined by NIH Image J software and were normalized to maximal activated ERK or MEK. (B) Three independently derived cell lines expressing W97ALck as well as cells expressing WTLck were incubated in the absence (−) or presence (+) of anti-CD3-ε antibody (1 μg/ml). The phosphorylation of ERK1/2 was determined as described above. The experiment was repeated three times with similar results. (C) WTLck- and W97ALck-expressing cells were stimulated with 1 μg/ml anti-CD3-ε antibody. Whole-cell lysates were assayed for phosphatase activity for the indicated times by use of phosphorylated GST-ERK2 as an exogenous substrate, followed by anti-phospho-ERK1/2 immunoblotting (bottom panel). Data were corrected for endogenous ERK2 levels (loading control, top panel) and are presented graphically. The data are the averages of three independent experiments. mAb, monoclonal antibody.

The Lck substrates CD3-ζ and ZAP-70 were constitutively tyrosine phosphorylated in cells expressing W97ALck.Another possible explanation for the failure of W97ALck to support the activation of ERK was that the kinase activity of W97ALck was impaired. We considered this unlikely since structural studies predict that disruption of the intramolecular binding of the SH3 domain would actually activate kinase activity. In fact, it had been reported that W97ALck did indeed exhibit increased catalytic activity (8). W97ALck immunoprecipitated from W97ALckD3.A2 cells showed a kinase activity similar to that of immunoprecipitated WTLck (Fig. 3A). Despite the similar in vitro kinase activities, cells expressing W97ALck contained constitutively elevated levels of tyrosine-phosphorylated Lck substrates CD3-ζ and ZAP-70 (Fig. 3B and C). The constitutively high level of ZAP-70 tyrosine phosphorylation was further increased following TCR engagement (Fig. 3C and D). Elevated levels of tyrosine-phosphorylated ZAP-70 were seen in three independently derived cell lines expressing W97ALck (Fig. 3D). The elevation in the constitutive tyrosine phosphorylation of CD3-ζ in W97ALck-expressing cells was abolished when the cells were incubated in the presence of the Src family kinase inhibitor PP2 (Fig. 3B). These results indicated that the failure of W97ALck to support ERK activation was not because the mutant failed to initiate signaling through the TCR. Rather, they revealed that in addition to activating signaling through the TCR, Lck was needed at a step distal to the phosphorylation and activation of ZAP-70.

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FIG. 3.

W97ALck displays increased catalytic activity in cells. (A) WTLck and W97ALck from unstimulated cells were immunoprecipitated and incubated with GST-CD3-ζ in the presence of [γ-³²P]ATP for the indicated times. The reaction mixtures were resolved by SDS-PAGE and transferred to a PVDF membrane, and phosphorylation was detected (left) and quantified (right) using a Storm 860 phosphorimager. Arbitrary units of activity are shown in the right panel. (B) Untransfected J.CaM1.6 cells or J.CaM1.6 cells expressing either WTLck or W97ALck were preincubated in the absence (−) or the presence (+) of the Src family kinase inhibitor PP2 (10 μM) at 37°C for 30 min. CD3-ζ was immunoprecipitated (I.P.) from cell lysates and resolved by SDS-PAGE. Following transfer to a PVDF membrane, the tyrosine phosphorylation of CD3-ζ was detected by immunoblotting (I.B.) using the antiphosphotyrosine antibody 4G10 (top). The membrane was stripped and reprobed with anti-CD3-ζ antibody to confirm equal levels of immunoprecipitation of CD3-ζ (bottom). (C) WTLck (WT)- and W97ALck (97A)-expressing cells were stimulated with the indicated concentrations of anti-CD3-ε antibody. The tyrosine phosphorylation of ZAP-70 in cell lysates was determined using anti-phospho-ZAP-70 antibody (top). The membrane was stripped and reprobed with anti-ZAP-70 antibody to confirm equal loading of samples (bottom). (D) Independently derived W97ALck-expressing cell lines and cells expressing WTLck were incubated in the absence (−) or presence (+) of anti-CD3-ε antibody (1 μg/ml), and the phosphorylation of ZAP-70 in cell lysates was determined by immunoblotting using anti-phospho-ZAP-70 antibody (top). The membrane was stripped and reprobed with anti-ZAP-70 antibody to confirm equal loading of samples (bottom). mAb, monoclonal antibody.

Tyrosine phosphorylation of PLC-γ1 was elevated in W97ALck-expressing cells.Following TCR engagement, activated ZAP-70 phosphorylates tyrosine residues on the membrane-bound adapter protein LAT, creating a binding site for the SH2 domain of PLC-γ1. Membrane-recruited PLC-γ1 is phosphorylated and activated by both ZAP-70 and Itk. To determine whether the lesion in the activation of the ERK pathway that was seen in cells expressing W97ALck was at the level of phosphorylation of PLC-γ1 on tyrosine, we immunoblotted lysates of activated cells by using antibody recognizing phospho-Y783, which is phosphorylated in active PLC-γ1 (18, 21). As can be seen in Fig. 4A, like CD3-ζ and ZAP-70, the tyrosine phosphorylation of PLC-γ1 was constitutively elevated in cells expressing W97ALck relative to the level in cells expressing WTLck. This level was further enhanced following TCR engagement with concentrations of anti-CD3-ε antibody that failed to trigger the activation of ERK in cells expressing W97ALck. These results indicated that the additional step in the ERK pathway where Lck is needed lay downstream of ZAP-70 and PLC-γ1.

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FIG. 4.

Tyrosine phosphorylation of PLC-γ1 and activation of Ras following TCR engagement are enhanced in W97ALck-expressing cells. (A) WTLck- and W97ALck-expressing cells were incubated in the absence (−) or presence (+) of anti-CD3-ε antibody (1 μg/ml). The phosphorylation of PLC-γ1 at Y783 was detected by immunoblotting cell lysates with anti-phospho-PLC-γ1 (Y783) antibody (top). The membrane was stripped and reprobed with anti-PLC-γ1 antibody to confirm equal loading of samples (bottom). (B) WTLck (WT)- and W97ALck (97A)-expressing cells were incubated in the presence of the indicated concentrations of anti-CD3-ε antibody. The GTP-bound active form of Ras was precipitated from cell lysates by use of a GST-Raf-1-RBD fusion protein bound to glutathione-linked Sepharose beads. Precipitated Ras was detected by immunoblotting using anti-pan-Ras antibodies (top). The membrane was stripped and reprobed using antibodies specific for N-Ras (middle). Cell lysates from the same samples were immunoblotted with anti-pan-Ras antibody (bottom). Ras activation was quantified by using a ChemiImager system (histogram). mAb, monoclonal antibody.

Ras activation following TCR ligation was normal in cells expressing W97ALck.In T lymphocytes, the major pathway for the activation of Ras is through RasGRP, a guanine nucleotide exchange factor that is recruited and activated by diacylglycerol, a product of PLC-γ1. To test the integrity of Ras activation following TCR ligation in W97ALck-expressing cells, we examined cell lysates for the presence of activated Ras by using RBD pull-down assays. As can be seen in Fig. 4B, at levels of anti-TCR stimulating antibody where ERK activation is not supported in W97ALck-expressing cells, levels of activated Ras were normal. When the immunoblot shown in the top panel of Fig. 4B was stripped and reprobed with antibodies to the N-Ras isoform of Ras, it was found that under the stimulation conditions used in this study N-Ras was activated and that the extent of activation in WTLck-expressing cells was similar to that in W97ALck-expressing cells (Fig. 4B, second panel). These results indicated that the second, distal site in the ERK pathway where Lck is needed was likely to be downstream of Ras.

Raf-1 activation was impaired in cells expressing W97ALck.Following TCR engagement, the activation of Ras results in the membrane recruitment and activation of Raf-1, which, in turn, phosphorylates and activates MEK1/2, the upstream activator of ERK1/2. To test whether the activation of Raf-1 was impaired in cells expressing W97ALck, a myc-Raf-1 fusion protein was transiently expressed in the WTLck- and W97ALck-expressing cell lines. Following stimulation through the TCR, myc-Raf-1 was immunoprecipitated and tested for kinase activity in an in vitro kinase assay using kinase-deficient GST-MEK as a substrate. As shown in Fig. 5A, the ability of W97ALck-expressing cells to activate myc-Raf-1 was significantly reduced relative to that of cells expressing WTLck. To verify that this result was not a function of Raf-1 overexpression, the activity of endogenous Raf-1 was measured in WTLck- and W97ALck-expressing cells and similar results were obtained (Fig. 5B). Since the activation of Ras was fully supported by W97ALck, it was likely that the recruitment of Raf-1 to the membrane occurred normally in W97ALck-expressing cells. This led to the hypothesis that the lesion in W97ALck-expressing cells resulted from an inability of membrane-bound Raf-1 to be fully activated.

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FIG. 5.

Lck regulates the activation of Raf-1 following TCR engagement through phosphorylation of Y340/Y341. (A) Myc-tagged Raf-1 was transiently expressed in WTLck- and W97ALck-expressing cells. Cells were stimulated with anti-CD3-ε antibody (+) (4 μg/ml) or left untreated (−). Myc-Raf-1 was immunoprecipitated from cell lysates by use of anti-myc antibody and incubated with a GST-tagged kinase-dead version of MEK [GST-MEK(KD)] and [γ-³²P]ATP. Samples were resolved by SDS-PAGE, transferred to PVDF membranes, and analyzed by autoradiography (left). The kinase activity of myc-Raf-1 from three independent experiments was determined and expressed relative to the activity of myc-Raf-1 in resting WTLck-expressing cells (right). (B) J.CaM1.6 cells expressing either WTLck or W97ALck were stimulated with anti-CD3-ε antibody (+) (1 μg/ml) or left untreated (−). Endogenous Raf-1 was immunoprecipitated using anti-Raf-1 antibody and assayed for kinase activity as described above. (C) Myc-tagged wild-type Raf-1 (WTRaf-1) or a myc-tagged YY340/341FF Raf-1 mutant (FFRaf-1) was transiently expressed in Jurkat cells. Cells were stimulated with anti-CD3-ε antibody (4 μg/ml) or left untreated. Myc-Raf-1 was assayed for kinase activity as described above. (D) Myc-tagged YY340/341FF Raf-1 (Myc-FFRaf-1) was transiently expressed in J.CaM1.6 cells expressing either WTLck or W97ALck. Cells were stimulated with anti-CD3-ε antibody (4 μg/ml) or left untreated. The YY340/341FF mutant Myc-Raf-1 was assayed for kinase activity as described above. mAb, monoclonal antibody.

W97ALck fails to support the phosphorylation of Raf-1.Although the mechanism of activation of Raf-1 is complex and involves several phosphorylation events and sites, mutational studies show that phosphorylation at tyrosine 340/341 and serine 338 is a prerequisite for full activation of Raf-1 in fibroblasts (11, 17, 25). Consistent with the current model of Raf-1 activation, a Raf-1 construct in which phenylalanines were substituted for tyrosines 340 and 341 (FFRaf-1) showed significantly reduced activity following TCR ligation compared to that of Raf-1 (Fig. 5C). However, the activation of FFRaf-1 was not completely abolished. We reasoned that if the lesion in the activation of the ERK pathway in W97ALck-expressing cells was due to a failure of Raf-1 phosphorylation at tyrosines 340 and 341, then the tyrosine 340/341-independent activation of FFRaf-1 should not be compromised in W97ALck-expressing cells. This was indeed the case, as demonstrated in Fig. 5D.

Prior phosphorylation of Y340/Y341 is necessary for activation-induced phosphorylation of S338 in Raf-1.Phosphorylation of S338 positively regulates the activation of Raf-1 (4, 9). Mutation of Raf-1 Y341 to phenylalanine blocks the phosphorylation of S338 when Raf-1 is activated by coexpression with oncogenic Ras and active Src but not when Raf-1 is activated by stimulation of the epidermal growth factor receptor (25). To test if the prior phosphorylation of Y340 and/or Y341 was required for the phosphorylation of S338 following TCR engagement, we transiently expressed wild-type Raf-1 or FFRaf-1 in the WTLck-expressing J.CaM1.6 cells. The cells were stimulated through the TCR and tested for Raf-1 S338 phosphorylation by use of an antibody specific to phospho-S338. As seen in Fig. 6A, the TCR-induced phosphorylation of S338 in FFRaf-1 relative to that in wild-type Raf-1 was severely impaired, suggesting that the TCR-induced phosphorylation of Raf-1 on S338 largely depends on the prior phosphorylation of Y340/Y341. If, as our data suggested, the Lck SH3 domain mutation impairs the phosphorylation of Y340/Y341, we reasoned that the phosphorylation of S338 would be reduced significantly in cells expressing W97ALck. To test this, we stimulated cells expressing either WTLck or W97ALck and measured the phosphorylation of S338 by immunoblotting. As suspected, the phosphorylation of S338 of Raf-1 in the W97ALck-expressing cells was defective compared to that in WTLck-expressing cells (Fig. 6B).

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FIG. 6.

Raf-1 Ser338 phosphorylation following TCR engagement is dependent on the prior phosphorylation of Tyr340/Tyr341. (A) Myc-tagged wild-type Raf-1 (WTRaf-1) or a myc-tagged YY340/341FF Raf-1 mutant (FFRaf-1) was transiently expressed in J.CaM1.6 cells expressing WTLck. Cells were stimulated with anti-CD3-ε antibody (+) (1 μg/ml) or left untreated (−). The phosphorylation of either WTRaf-1 or FFRaf-1 at Ser338 was detected by immunoblotting whole-cell lysates using anti-phospho-Raf-1 (Ser338) antibody. The membrane was stripped and reprobed with antibodies to Raf-1 to verify equal loading of samples. (B) Three independent clones of J.CaM1.6 cells expressing W97ALck as well as cells expressing WTLck were stimulated with anti-CD3-ε antibody (+) (1 μg/ml) or left untreated (−). The phosphorylation of endogenous Raf-1 at Ser338 was determined as described above. mAb, monoclonal antibody.

Trafficking of Lck through the Golgi apparatus.One of the major phenotypic differences between J.CaM1.6 cells expressing WTLck and those expressing W97ALck is that the SH3 domain mutant demonstrates an aberrant intracellular localization (14). As shown in Fig. 7A (top left), in addition to its presence at the plasma membrane, WTLck was found localized at an intracellular structure containing giantin, a marker for the cis-medial Golgi apparatus. In contrast, however, a significantly reduced amount of W97ALck appeared to colocalize with giantin (Fig. 7A, bottom left). Golgi apparatus-associated Lck was also seen in the parental Jurkat cells, where Lck was found to colocalize with both giantin and YFP-1,4-galactosyltransferase (GalT), a marker for the trans-Golgi apparatus (Fig. 7B). In addition, we analyzed 43 Jurkat cells and found that the mean value of fluorescence from Golgi apparatus-associated Lck as a proportion of total cellular Lck was 0.18 ± 0.02, indicating that approximately 18% of Lck is localized to the Golgi apparatus in Jurkat cells (data not shown). We next investigated whether the intracellular distribution of either WTLck or W97ALck changed following stimulation through the TCR and found that there was no significant redistribution of either WTLck or W97ALck following low-grade stimulation (Fig. 7A, left).

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FIG. 7.

W97ALck displays decreased localization to the Golgi apparatus. (A) J.CaM1.6 cells transiently transfected with the indicated Lck-GFP constructs were either unstimulated or stimulated with α-CD3 antibodies. Cells were fixed and stained with antibodies to giantin (rhodamine) and visualized by fluorescence microscopy. Fifty-nine W97ALckGFP- and 103 WTLckGFP-expressing cells were analyzed. The median total fluorescence intensity for WTLckGFP-expressing cells was (5.42 ± 1.87) × 10⁵ arbitrary units (AUs) and that for W97ALckGFP-expressing cells was (4.88 ± 2.51) × 10⁵ AUs, indicating that expression levels of the two constructs were similar. The distribution of the fluorescence intensity both for WTLckGFP-expressing cells and for W97ALckGFP-expressing cells was non-Gaussian and therefore the quantified data are represented by a Tukey box plot (right). Mann-Whitney analysis was used to generate the Tukey box plot (P < 0.05). The median ratio of fluorescence intensity (Golgi apparatus-associated Lck to total cellular Lck) for WTLck was 0.201 ± 0.026 and that for W97ALck was 0.142 ± 0.025. The bars in the Tukey box plot demarcate the 75th percentile and the 25th percentile, respectively. (B) (Top) Jurkat cells were fixed and costained using antibodies to Lck (Alexa Fluor 488, green) and giantin (cis-medial Golgi apparatus, rhodamine). (Bottom) Jurkat cells were transiently transfected with YFP-GalT (trans-Golgi apparatus marker). After 24 h, cells were fixed and stained for Lck (Alexa Fluor 594, red) and visualized by fluorescence confocal microscopy.

The demonstration that Lck colocalizes with markers for the Golgi apparatus coupled with the findings described by Perez de Castro et al. (29) predicted that a portion of intracellular Lck would colocalize with Golgi apparatus-associated N-Ras. In order to determine whether W97ALck localized less readily with Golgi apparatus-associated N-Ras, J.CaM1.6 cells expressing W97ALck-GFP or WTLck-GFP were immunostained for N-Ras. As can be seen in Fig. 8A, considerably less W97ALck than WTLck was observed to colocalize with N-Ras on the Golgi apparatus, despite equal levels of Golgi apparatus-associated N-Ras in WTLck- and W97ALck-expressing cells (Fig. 8B). This is consistent with the results shown in Fig. 7 demonstrating a reduced colocalization of W97ALck with giantin.

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FIG. 8.

Reduced colocalization of W97ALck with Golgi apparatus-associated N-Ras. (A) J.CaM1.6 cells were transiently transfected with the indicated GFP constructs, fixed and stained with antibodies to N-Ras, and visualized by fluorescence microscopy. The images are representative of a population of cells. (B) Fluorescence images of J.CaM1.6 cells stably expressing either WTLck or W97ALck were fixed and costained with antibodies to giantin (cis-medial Golgi apparatus marker) and N-Ras. The relative ratio of Golgi apparatus-localized N-Ras fluorescence to total cell fluorescence was calculated and graphed as the mean of Golgi apparatus-localized N-Ras (n = 18).