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The Journal of Immunology, 1999, 162: 7224-7232.
Copyright © 1999 by The American Association of Immunologists

Fyn Membrane Localization Is Necessary to Induce the Constitutive Tyrosine Phosphorylation of Sam68 in the Nucleus of T Lymphocytes1

Valérie Lang*, Monique Semichon{dagger}, Frédérique Michel{ddagger}, Cédric Brossard*, Hélène Gary-Gouy* and Georges Bismuth2,*

* Laboratoire d’Immunologie Cellulaire, Centre National de la Recherche Scientifique UMR 7627, Centre Hospitalier Pitié-Salpêtrière, CERVI, Paris, France; {dagger} Unité Claude Bernard C20, Département d’Hématologie, Centre Hospitalier Pitié-Salpêtrière, Centre d’Examen et de Recherche en Virologie et Immunologie, Paris, France; and {ddagger} Laboratoire d’Immunologie Moléculaire, Département d’Immunologie, Institut Pasteur, Paris, France


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


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sam68 is a ubiquitous protein, reported to be predominantly nuclear in fibroblasts or epithelial cells (1, 2, 3, 4). The molecule contains in its central part a K homology domain (KH),3 a particular conserved module that is a landmark of a family of proteins acting as a direct contact with RNA (5) and also necessary for the self-association of different members of this family (6). In vitro RNA-binding properties of Sam68 mediated by its KH domain have been reported (1, 7, 8), as well as the capacity of this domain to influence Sam68 localization inside the cell (8). Deletion of the KH domain also antagonizes cell cycle progression (9). The function of Sam68 is unknown, but all of these characteristics strongly suggest that this protein is involved in some aspects of RNA synthesis and/or transport essential for cell metabolism and cell division.

Analysis of the amino acid sequence of Sam68 reveals additional typical features of a signaling protein with numerous tyrosine in its carboxyl-terminal domain and at least five proline-rich regions (1). Initially described as a tyrosine-phosphorylated protein associated with Src both in normal and in Src-transformed fibroblasts blocked in mitosis (7, 10), numerous other partnerships with different Src homology (SH) 2 and SH3 domain-containing signaling molecules were documented afterward. They include phospholipase C{gamma}-1, Grb2, Grap (a Grb2-like protein), the p21ras GTPase-activating protein, the regulatory subunit of PI3-kinase, p47phox, Tec kinase family, SHP-1, Cbl, Jak3, and Nck (11, 12, 13, 14, 15, 16, 17, 18, 19, 20). The functional consequences of these interactions are still unclear, but an adaptor function for Sam68 downstream of Src kinase-associated receptors has been proposed. One can thus speculate about a function for Sam68 as a link between Src kinase-dependent pathways on the one hand and RNA metabolism on the other. The finding that Sam68 phosphorylation can affect its binding to RNA further supports this hypothesis (21).

Sam68 tyrosine phosphorylation is induced in T lymphocytes after stimulation of the Ag-specific receptor (TCR) (16, 20, 22). It was suggested that in activated T cells, tyrosine-phosphorylated Sam68 may also serve as an adaptor protein, linking the protein tyrosine kinase (PTK) of the Src family Fyn and Lck and the second class of T cell-specific PTK ZAP-70 to some of the downstream effectors listed above (11, 16). However, the phosphorylation status of Sam68 in T cells is presumably complex since a constitutive tyrosine phosphorylation of the protein has also been seen in different transformed T cell lines (16, 22, 23). Whether Sam68 phosphorylation is involved in the determination of the transformed phenotype is unknown, but interestingly, as has been evoked in one study, it seems to be able to affect the cell cycle in these T lymphocytes (16).

Taken together, these findings led us to consider the study of the mechanisms involved in this constitutive phosphorylation of Sam68 in T cells as important. Our recent report suggested a distinct relationship between the two Src kinases and Sam68 phosphorylation in Jurkat T cells (22). We demonstrate in this work that only overexpression of Fyn triggers the constitutive tyrosine phosphorylation of Sam68 in T cells in vivo. Investigating the subcellular distribution of tyrosine-phosphorylated Sam68 in these cells, we also show that phospho-Sam68 molecules are present in the nucleus. In parallel, we demonstrate that Fyn localization at the cell membrane is essential for the constitutive phosphorylation of Sam68 in this cell compartment and also that a Sam68 mutant unable to localize in the nucleus is still phosphorylated. Overall, our results suggest that the specific participation of Fyn in the constitutive tyrosine phosphorylation of Sam68 in T cells originally occurs in a cell membrane compartment and is necessary for phospho-Sam68 to be expressed in the nucleus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

The hybridoma T cell line, T8.1, has already been described (24). It was maintained in DMEM medium (Life Technologies, Cergy-Pontoise, France) supplemented with 10% FCS, antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin, 1 mg/ml geneticin), 400 nM methotrexate, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µM 2-ME. Tag Jurkat cells, a derivative of the human T cell leukemia Jurkat stably transfected with the SV40 large T Ag (25), were grown in RPMI 1640 medium (Flow Laboratories, Irvine, CA) supplemented with 10% FCS, antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin), 2 mM L-glutamine, and 1 mM sodium pyruvate.

Antibodies

The CD3{epsilon}-specific mAb UCHT1 was produced as ascitic fluid. The anti-phosphotyrosine mAb 4G10 and the polyclonal Ab specific for the N-terminal region of Lck were from Upstate Biotechnology (UBI, Lake Placid, NY). The rabbit polyclonal Ab specific for Sam68 used for immunoprecipitation experiments has already been described (22). A polyclonal Ab against the C terminus of the molecule, obtained from Santa Cruz Biotechnology (Santa Cruz, CA), was used for blotting experiments. The Fyn mAb used for Western blotting experiments was from Transduction Laboratories (Lexington, KY), and the Fyn polyclonal Ab used for immunofluorescence and immunoprecipitation experiments from UBI.

Plasmids and constructs

A full-length cDNA encoding wild-type murine Fyn in pBluescript SK+ vector was a kind gift from S. Richard (McGill University, Montreal, Canada) (11). The Fyn insert was isolated with EcoRI and XhoI, blunted with the klenow fragment of DNA polymerase I, and subcloned into the mammalian vector pSR{alpha}-puro (26), which conferred resistance to puromycine. A cDNA encoding dead kinase Fyn with a point mutation at the ATP binding site (K299 M) in pSR{alpha} was kindly given by Dr. N. Fusaki (Science University of Tokyo, Chiba, Japan) (27). A cDNA encoding wild-type mouse Lck, kindly provided by S. Fischer (INSERM U363, Institut Cochin de Génétique Moléculaire, Paris, France), was subcloned into the EcoRI site of pSR{alpha}-puro. The Fyn-{Delta}N-ter mutant (without the first six amino acids at the N terminus) was generated by PCR using the following oligonucleotides as primers, 5'-GGAATTCGATGAAGGATAAAGAAGCAGCGAA-3' and 5'-GGAATTCTCACAGGTTTTCACCGGGCTG-3', and wild-type Fyn as a template. The PCR product was digested with EcoRI and then inserted into pSR{alpha}-puro. A full-length cDNA encoding human Sam68 in pSV2 vector was a kind gift from D. F. Schweighoffer (Rhône-Poulenc Rorer, Vitry sur Seine, France). It was digested with KpnI and SacI, blunted with the klenow fragment of DNA polymerase I, and subcloned into the SmaI site of the pEGFP-C1 vector (Clontech Laboratories, Palo Alto, CA) containing the gene encoding the green fluorescent protein (GFP). Sam68-{Delta}NLS fused to EGFP was constructed by digesting the plasmid containing Sam68-EGFP with HindIII and cloning the resulting fragment in HindIII restriction site of pEGFP-C1. This removed the DNA sequence C terminal of the HindIII site in Sam68 containing the nuclear localization signal (NLS) (3).

Cell transfection

For stable transfections of T8.1 hybridoma cells, 10 x 106 cells were mixed with 30 µg of plasmid DNA in 0.5 ml of a buffer containing 120 mM KCl, 150 µM CaCl2, 10 mM K2HPO4/KH2PO4, 2 mM EGTA, 5 mM MgCl2, 1 mM ATP, 5 mM glutathione, and 25 mM HEPES, and electroporated at 250 V, 960 µF in a Gene Pulse cuvette (Bio-Rad, Ivry sur Seine, France). After 48 h in growth medium, cells were seeded at 104 cells/well in flat-bottom 96-well plates and placed under selection in the presence of 1 µg/ml puromycin (Sigma-Aldrich Chimie, St Quentin Fallavier, France). Puromycin-resistant clones were analyzed by SDS-PAGE for protein expression. Transient transfections of Jurkat cells were performed by electroporating (at 320 V, 960 µF) 10 x 106 cells with 20 µg of plasmid DNA. Transfected cells were cultured in 6 ml of growth medium for 48 h before use.

Whole cell lysate and subcellular fractionation

For whole cell lysate preparation, T cells (10 x 106) were washed once in 1 ml of RPMI medium and lysed at 4°C for 1 h in a lysis buffer (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA, 50 U/ml aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate, 50 mM sodium fluoride) containing 1% (v/v) Nonidet P-40 detergent. Nuclei and cellular debris were removed by centrifugation at 10,000 x g for 10 min, and the amount of proteins present in each postnuclear supernatant was determined. In some experiments, cells were stimulated before lysis. After a wash in RPMI medium, cells (10 x 106) were equilibrated for 15 min at 37°C and stimulated for 2 min at 37°C in the presence of the CD3{epsilon}-specific mAb UCHT1 (1/500 dilution of an ascitic fluid) in a final volume of 500 µl. Activation was stopped by brief centrifugation before lysis. For subcellular fractionation, cells were harvested by centrifugation and washed once with cold PBS. The pellet was resuspended in ice-cold hypotonic buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 50 U/ml aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate, 50 mM sodium fluoride), incubated on ice for 15 min, and homogenized with a Dounce homogenizer (15 strokes, pestle B). Nuclei were collected by centrifugation at 400 x g for 15 min at 4°C. The supernatant (cytoplasmic and particulate fractions) was centrifuged at 100,000 x g for 30 min at 4°C. The high speed supernatant was designed as cytoplasmic fraction. The pellet, designed as membrane-enriched fraction, was lysed at 4°C for 1 h in the Nonidet P-40-containing buffer. Pelleted nuclei were resuspended in a buffer containing 20 mM HEPES, pH 7.9, 25% glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 U/ml aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and nuclear protein extracted on ice for 30 min. Nuclear extract was obtained by centrifugation at 100,000 x g for 30 min at 4°C. All samples were diluted in Laemmli buffer (500 mM Tris-HCl, pH 6.8, 10% SDS, 10% glycerol, 5% 2-ME, 10% bromophenol blue) and boiled for 3 min.

Western blot analysis and immunoprecipitation

Proteins (60 µg) were loaded onto 10% SDS-polyacrylamide gels, and electrophoretically transferred for 75 min at 65 V to a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). Immunoblotting was then performed with the anti-phosphotyrosine mAb 4G10 (0.2 µg/ml), anti-Fyn mAb (1 µg/ml), anti-Sam68, or anti-Lck polyclonal Abs (1/800 dilution), followed by peroxidase-labeled goat anti-mouse or goat anti-rabbit antiserum (Bio-Rad). Reaction was revealed with an enhanced chemoluminescence system (ECL; Amersham, Paris, France), according to the supplier’s instructions. Scanning densitometry of the films was performed with the Bio-Rad’s densitometer GS-670, and results were analyzed with the Molecular Analyst/PC image analysis software (Bio-Rad).

For Sam68 immunoprecipitation, whole cell lysates from 10 x 106 cells or from 500 µg of nuclear extract were incubated for 2 h at 4°C with 30 µl of protein A-Sepharose beads (Sigma-Aldrich Chimie) previously incubated 2 h at 4°C with the Sam68 rabbit antiserum. After four washes in the lysis buffer, immune complexes were recovered by boiling for 3 min in Laemmli buffer and analyzed by Western blot.

In vitro kinase assay

Fyn or Lck was immunoprecipitated from 20 x 106 cells lysed in the Nonidet P-40 lysis buffer. After a preclearing on protein A-Sepharose, lysates were precipitated with the specific antisera and 30 µl of protein A-Sepharose. After four washes in the lysis buffer, beads were kept on ice until use. GST-Sam68 fusion protein, obtained as reported (22), was used as a substrate. It was purified by affinity chromatography on glutathione-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden). Fusion protein complexes were washed three times in a lysis buffer (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA) containing 1% (v/v) Nonidet P-40 detergent. Kinase immunoprecipitates and fusion protein complexes (1 µg of GST-Sam68) were then resuspended in a phosphorylation buffer containing 50 mM PIPES, pH 6.8, 10 mM MnCl2, 10 mM MgCl2, 50 µM sodium orthovanadate, 50 U/ml aprotinin, and 1 mM PMSF, mixed together, and briefly centrifuged. The reaction was started by the addition on the pellet of 30 µl of phosphorylation buffer supplemented with 10 µM ATP and 10 µCi [{gamma}-32P]ATP (4500 Ci/mmol; ICN Biomedicals France, Orsay, France). After 10 min at room temperature, the reaction was stopped by the addition of 6 µl of Laemmli sample buffer 6x and boiling. Proteins were separated by 10% SDS-PAGE, and the gel was dried and subjected to autoradiography on x-ray film.

Immunofluorescence

For Fyn immunofluorescence analysis, cells were allowed to adhere in RPMI 1640 without serum on slide coverslips for 1 h at 37°C. Cells were then fixed for 10 min at room temperature in 3% paraformaldehyde, followed by three washes in PBS. Slide coverslips were then incubated with 0.1% Triton X-100 in PBS for 10 min at room temperature to permeabilize the cells. After three washes in PBS, anti-Fyn (1/100 dilution) diluted in PBS-BSA (1 mg/ml) was added for a 1-h incubation period at room temperature, followed by an FITC-conjugated F(ab')2 fragment donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. After extensive washing in PBS, the slide coverslips were then mounted onto glass slide using a poly(vinyl alcohol) solution (Mowiol, Sigma-Aldrich Chimie). Fluorescence microscopy was performed with a Leitz DM IRB inverted microscope (Leica, Wetzlar, Germany) equipped with fluorescein filters using a x100 oil objective. Fluorescence images were collected with a cooled CDD camera (Sensys 400; Photometrics, Tucson, AZ) and the Image Pro software (Media Cybernetics, Silver Spring, MD). For image processing and presentation, digital images (12 bits scale) were deconvolved using the Slidebook software (Intelligent Imaging Innovations, Denver, CO). Digital files were printed directly. Fluorescence of cells transfected with EGFP constructs was directly analyzed with the inverted microscope on viable cells seeded in their culture medium on a glass coverslip mounted on 30-mm petri dishes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The constitutive phosphorylation of Sam68 in T cells directly correlates with intracellular Fyn levels

A constitutive tyrosine phosphorylation of Sam68 has already been observed in different established T cell lines (16, 22, 23). T cell Src kinases are believed to play a key role in this phenomenon, especially Fyn, as suggested by the observation that Sam68 is highly phosphorylated in a human T cell leukemia virus type 1-transformed T cell overexpressing Fyn (16). To directly investigate this issue, we transfected wild-type Fyn in the murine hybridoma T cell line T8.1 and established stable transfectants overexpressing the PTK in which we analyzed the constitutive phosphorylation of Sam68. A series of clones with different levels of Fyn was obtained. Shown in Fig. 1GoA are the blots with three clones, Fyn-WT11, Fyn-WT3, and Fyn-WT7, in which we analyzed Fyn and Sam68 expression as well as the phosphorylation of Sam68 after immunoprecipitating the molecule. The constitutive phosphorylation of Sam68 was low in T8.1 cells, but a marked tyrosine phosphorylation of the molecule was observed in the cells overexpressing Fyn. Sam68 levels, measured in whole cell lysates and in Sam68 immunoprecipitates, showed no difference between the three clones (see Fig. 1GoA). By plotting the values obtained by scanning densitometry for the Fyn protein and phospho-Sam68, we could see a close relationship between the fold increase of the two parameters in the different clones (see Fig. 1GoB). Taken together, these findings demonstrate a direct relationship between Fyn expression and the constitutive tyrosine phosphorylation of Sam68 in T lymphocytes.



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  		FIGURE 1. The constitutive phosphorylation of Sam68 in T cells correlates with Fyn level. Different clones were obtained by stably transfecting T8.1 hybridoma cells with murine wild-type Fyn (Fyn-WT11, Fyn-WT3, Fyn-WT7). A, After lysis in Nonidet P-40 lysis buffer, solubilized proteins (60 µg) were resolved by SDS-PAGE on a 10% polyacrylamide gel and subjected to Western blot analysis with anti-Fyn and anti-Sam68 Abs (two upper panels). In the two lower panels, Sam68 was immunoprecipitated from the same lysates with a specific polyclonal antiserum. After blotting, the membrane was probed with anti-phosphotyrosine mAb 4G10 or anti-Sam68 Abs. B, Fyn protein and Sam68 tyrosine phosphorylation levels were measured by scanning densitometry from the blots shown in A and the values plotted in parallel.

 
Lck cannot promote the constitutive phosphorylation of Sam68 in vivo

A role for Lck, the second Src kinase present in T cells, in mediating the constitutive tyrosine phosphorylation of Sam68 is more elusive since Sam68 phosphorylation is apparently maintained in Jurkat T cells without Lck (22). As for Fyn, we investigated whether phospho-Sam68 levels were induced by overexpressing Lck in the T8.1 cell model. Stable transfectants expressing different levels of Lck were established. Shown in Fig. 2Go are the results obtained with five different clones, which we compared with clone Fyn-WT7, used in this study as a positive control. After measuring the levels of Lck and Sam68 by Western blot analysis of whole cellular lysates (Fig. 2GoA), the constitutive phosphorylation of Sam68 was analyzed by blotting the lysates with the anti-phosphotyrosine mAb 4G10 (Fig. 2GoB). The highly phosphorylated band (arrow) corresponding to Sam68 was evident in Fyn-WT7 lysates. It was clearly missing in the clones transfected with Lck, especially in Lck-G3 and Lck-H8, two clones exhibiting a clear phosphoprotein increase after Lck overexpression. This finding was confirmed by blotting Sam68 immunoprecipitates with the anti-phosphotyrosine mAb (Fig. 2GoA).



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  		FIGURE 2. In vivo phosphorylation of Sam68 is not induced in cells overexpressing Lck. Different clones were obtained by stably transfecting T8.1 hybridoma cells with murine wild-type Lck (Lck-A1, Lck-E9, Lck-F5, Lck-G3, Lck-H8). A, After lysis in Nonidet P-40 lysis buffer, solubilized proteins (60 µg) were resolved by SDS-PAGE on a 10% polyacrylamide gel subjected to Western blot analysis with anti-Lck or anti-Sam68 Abs (two upper panels). The clone Fyn-WT7, overexpressing wild-type Fyn, was used in parallel. In the two lower panels, Sam68 was immunoprecipitated from the same lysates with a specific polyclonal antiserum. After blotting, the membrane was probed with anti-phosphotyrosine mAb 4G10 or anti-Sam68 Abs. B, Whole cell lysates were also subjected to Western blot analysis with anti-phosphotyrosine mAb 4G10. Sam68 position is shown by an arrow.

 
We also investigated this issue by evaluating phospho-Sam68 levels in immunoprecipitates from Nonidet P-40 lysates of Jurkat cells transfected 48 h with cDNAs encoding Fyn or Lck. A sharp increase in phospho-Sam68 level was only observed in cells overexpressing Fyn, as compared with the slight labeling of the molecule obtained when Lck was transfected (Fig. 3GoA). Several phosphoproteins were coimmunoprecipitated with Sam68 only in the Fyn-transfected cells. As shown in Fig. 3GoB, the whole pattern of tyrosine-phosphorylated proteins was strongly induced by the two PTKs. The expression of Fyn and Lck in the transfected cells is also shown. Control experiments were also performed with a dead kinase mutant form of Fyn. As shown in Fig. 3GoC, only a slight increase was observed with the dead kinase, suggesting that wild-type Fyn directly phosphorylates Sam68 in the transfected cells. By blotting cellular lysates with anti-Fyn Abs, we checked that the two forms of Fyn were overexpressed similarly (data not shown). We conclude that, contrary to its Fyn counterpart, overexpression of Lck does not promote the constitutive phosphorylation of Sam68 in vivo.



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  		FIGURE 3. Sam68 is highly tyrosine phosphorylated only in Jurkat cells overexpressing Fyn. Jurkat T cells (10 x 106) were transiently transfected with 20 µg of the pSR{alpha}-puro empty vector or containing the cDNA of Lck wild type or Fyn wild type. After a 48-h culture, cells were lysed in Nonidet P-40 lysis buffer. A, Sam68 was immunoprecipitated from the different cell lysates with a specific polyclonal antiserum, and immune complexes were resolved by SDS-PAGE on a 10% polyacrylamide gel. After blotting, the membrane was probed with the anti-phosphotyrosine mAb 4G10 or anti-Sam68 Abs. B, A total of 60 µg of the lysates was also subjected to Western blot analysis with either 4G10, anti-Fyn, or anti-Lck Abs. C, Jurkat cells were transfected as above with the empty vector or vectors encoding Lck wild-type, Fyn wild-type, or a Fyn dead kinase mutant. Sam68 immunoprecipitates were then analyzed as in A.

 
Both Fyn and Lck phosphorylate Sam68 in vitro

We also performed in vitro kinase assays with Fyn and Lck immunoprecipitates. The purpose of this experiment was to control the kinase activities in the clones overexpressing the two Src PTKs and also to evaluate their capacity to phosphorylate Sam68 in vitro by using as a substrate Sam68, itself produced as a GST fusion protein. Fig. 4GoA shows an autoradiography of the gel obtained after immunoprecipitating Fyn and Lck from Fyn-WT7 and Lck-G3, the two clones expressing the highest level of Fyn and Lck, respectively, and untransfected T8.1 cells. We observed a phosphorylation of the recombinant protein only when Fyn or Lck was immunoprecipitated from the clones overexpressing the corresponding PTKs. A marked autophosphorylation of the PTKs was also noticeable in these samples. The 66-kDa 32P-labeled protein strongly phosphorylated in Fyn immunoprecipitates from clone Fyn-WT7 most likely corresponds to endogeneous Sam68. A Coomassie blue staining of the same gel in Fig. 4GoB shows that the same amount of GST-Sam68 fusion protein was loaded in the different lanes. This finding demonstrates that both Fyn and Lck are able to phosphorylate Sam68 in kinase assays in sharp contrast to the in vivo results.



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  		FIGURE 4. In vitro phosphorylation of Sam68 by Fyn and Lck in cells overexpressing the src kinases. A, Lysates of T8.1, Fyn-WT7, or Lck-G3 cells (20 x 106 cells) were subjected to Fyn or Lck immunoprecipitation with specific Abs. Immune complexes were incubated with 1 µg of GST-Sam68 recombinant protein (used as an exogenous substrate) immobilized on glutathione Sepharose beads. They were then subjected to in vitro kinase assay using [{gamma}-32P]ATP. Autoradiography of a 10% SDS-polyacrylamide is shown. B, Coomassie blue staining of the gel shown in A.

 
Fyn membrane localization is required for the constitutive phosphorylation of Sam68

Sam68 has a NLS (3), and it has been shown to accumulate in the nucleus of different cell types (1, 2, 3, 4, 17). Furthermore, the N-terminal region of Fyn contains residues that are necessary for Fyn myristylation (Gly-2), and also palmitylation (Cys-3 and Cys-6) of the molecule, both required to target the protein to membranes (28, 29, 30, 31, 32). It was therefore interesting to investigate in which cell compartment phosphorylated Sam68 was expressed or accumulated in T cells overexpressing Fyn, and whether Fyn membrane localization was essential for this process. To investigate these issues, we constructed a Fyn mutant without the first six amino acids. This mutant (Fyn-{Delta}N-ter) was stably transfected in T8.1 cells, and two clones (Fyn-{Delta}N-ter A6 and Fyn-{Delta}N-ter A12) overexpressing the molecule (see Fig. 5GoB, middle panel) were studied. We first analyzed Fyn localization by immunofluorescence. As shown in Fig. 5GoA, Fyn exhibited a main localization at the cell membrane and in the adjacent cortical region in clone Fyn-WT7 surexpressing wild-type Fyn. Similar results were obtained with the other clones overexpressing Fyn (data not shown). A punctate staining was also detected intracellularly in many cells in a region most probably corresponding to the microtubule organization center. Note that the Fyn labeling was barely detectable in untransfected T8.1 cells (not shown). A diffuse cytoplasmic labeling was obtained with the two clones overexpressing Fyn-{Delta}N-ter surrounding the nucleus and replacing the usual membrane staining. We found no significant increase in Sam68 phosphorylation in the whole cell lysates of cells overexpressing Fyn-{Delta}N-ter (Fig. 5GoB, upper panel) or after immunoprecipitating the molecule (Fig. 5GoC). We checked in parallel experiments that the Fyn-{Delta}N-ter mutant was fully active in in vitro kinase assays (not shown).



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  		FIGURE 5. Membrane targeting of Fyn is required for constitutive tyrosine phosphorylation of Sam68 in the nucleus. Two clones were obtained from T8.1 hybridoma cells stably transfected with a Fyn mutant without the first six amino acids at the N terminus (Fyn-{Delta}N-ter A6 and Fyn-{Delta}N-terA 12). A, Fyn localization was analyzed in clones Fyn-WT7, Fyn-{Delta}N-terA6, and Fyn-{Delta}N-ter A12 by indirect immunofluorescence of fixed permeabilized cells. B, Nonidet P-40 whole cell lysates (60 µg) from clones Fyn-{Delta}N-ter A6 and Fyn-{Delta}N-ter A12 were subjected to Western blot analysis with anti-phosphotyrosine (upper panel), anti-Fyn (middle panel), or anti-Sam68 (lower panel) Abs. Untransfected T8.1 cells and clone Fyn-WT7 overexpressing wild-type Fyn were used in parallel (C). In a separate experiment, Sam68 was immunoprecipitated from whole cell lysates of untransfected T8.1 cells, Fyn-WT7, or Fyn-{Delta}N-ter A12 cells with a specific polyclonal antiserum. After blotting, the membrane was probed with the anti-phosphotyrosine mAb 4G10. D, Cytosol (C), membrane-enriched (M), and nuclear (N) fractions were prepared from T8.1 cells, Fyn-WT7, and Fyn-{Delta}N-ter A12 cells, as described under Materials and Methods. Subcellular fractions (60 µg) were resolved by SDS-PAGE on a 10% polyacrylamide gel. After blotting, the membrane was probed with anti-Sam68 Abs. E, Nuclear fractions were prepared from untransfected T8.1 cells and Fyn-WT7 or Fyn-{Delta}N-ter A12 clones, as described under Materials and Methods. Sam68 was immunoprecipitated from the nuclear extracts and subjected to Western blot analysis with anti-phosphotyrosine mAb 4G10.

 
Sam68 blots of cellular fractions from T8.1, Fyn-WT7, and Fyn-{Delta}N-ter A12 were also performed to compare the cellular distribution of the molecule in the different clones (Fig. 5GoD). Not surprisingly, we found that Sam68 was strongly expressed into the nucleus. As reported in fibroblasts, a significant amount of the molecule was also present in a particulate fraction enriched in cell membranes. However, the protein was barely detectable in the cytosol. Similar results were obtained with Jurkat T cells (not shown). No clear alteration in the distribution of Sam68 between the different cell types was found, indicating that the predominant expression of Sam68 in the nucleus was insensitive to Fyn overexpression. Essentially, large amounts of phosphorylated Sam68 molecules were only present in the nuclear extract (Fig. 5GoE) and the membrane-enriched fraction (not shown) obtained from clone Fyn-WT7.

These data demonstrate that Fyn membrane targeting is required to trigger the constitutive phosphorylation of Sam68 in T cells, including in the nucleus. They also show that the phosphorylation of Sam68 in this cell compartment, where it is predominantly expressed, is dependent of Fyn cellular levels.

The delocalization of Sam68 from the nucleus does not impair Sam68 phosphorylation

Truncated Sam68 molecules fused to GFP have been used successfully to demonstrate the role of the NLS of Sam68 in the predominant localization of the protein into the nucleus (3). Since our previous data strongly suggested that Sam68 phosphorylation was a process occurring outside the nucleus, studies were performed to analyze the tyrosine phosphorylation of a mutated form of Sam68 without the NLS (Sam68-{Delta}NLS). The mutated protein was fused to GFP to follow first its localization in vivo. Constructs encoding wild-type Sam68 fused to GFP and GFP alone were used as controls.

Shown in Fig. 6Go are images obtained by analyzing the fluorescence of living Jurkat T cells 48 h after electroporation with the different constructs. As expected, Sam68-GFP gave a strong staining inside the nucleus (overlapping images were obtained by parallel staining with a cell-permeant bisbenzimide DNA stain, not shown). Note the punctate nuclear staining observed in some cells (less than 5%). Sam68-{Delta}NLS-GFP was clearly expressed outside the nucleus and frequently in vesicular and randomly distributed structures. A diffuse staining in the whole cell was obtained when using the control GFP construct.



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  		FIGURE 6. Fluorescence analysis of Jurkat cells transfected with Sam68-GFP constructs. Jurkat T cells (10 x 106) were transiently transfected with 20 µg of the empty pEGFP-C1 vector (coding for GFP alone) or pEGFP-C1 containing, fused to the GFP gene, the cDNA coding for Sam68 (Sam68-GFP) or for a mutated form of Sam68 without the NLS (Sam68{Delta}NLS-GFP). Fluorescence of transfected cells was directly analyzed after a 48-h culture with the inverted microscope on viable cells. Transmitted light images of the corresponding fluorescent cells are also shown.

 
We next analyzed the tyrosine phosphorylation of the Sam68-{Delta}NLS-GFP protein after immunoprecipitating the molecule from Jurkat cells stimulated or not with a CD3{epsilon}-specific mAb (Fig. 7Go). The constitutive as well as the CD3-induced phosphorylation of the molecule were obvious in the transfected cells and clearly increased as compared with the control. This increase was correlated with the level of the protein in the detergent-soluble fraction, as shown after probing the same immunoprecipitates with Sam68-specific Ab. The Sam68-blotting Ab used throughout this work recognized the deleted part of the molecule. The weak labeling of Sam68 in this experiment was thus explained by the use, as a blotting reagent, of the second polyclonal Ab against Sam68, which poorly recognized the molecule in Western blot. In conclusion, our results demonstrate that the CD3-induced as well as the constitutive phosphorylation of Sam68 are triggered outside the nucleus.



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  		FIGURE 7. The delocalization of Sam68 from the nucleus does not impair Sam68 phosphorylation. Forty-eight-hour transfected cells with GFP or Sam68{Delta}NLS-GFP were stimulated or not with mAb UCHT1 (CD3{epsilon} specific) for 2 min and lysed in Nonidet P-40 lysis buffer. Sam68 was immunoprecipitated from the different cell lysates, and immune complexes were resolved by SDS-PAGE on a 10% polyacrylamide gel. After blotting, membranes were probed with the anti-phosphotyrosine mAb 4G10 (upper panel) or with the anti-Sam68 antiserum (lower panel).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sam68 is spontaneously tyrosine phosphorylated in murine fibroblasts overexpressing Src. A functional relationship has been suggested between this phenomenon and the regulation of cell metabolism by the PTK, especially during mitosis (7, 10, 33, 34). In T cells also, Sam68 can be found constitutively tyrosine phosphorylated (16, 22, 23), and it was suggested that the constitutive phosphorylation of Sam68 may also affect the T cell cycle, especially in human T cell leukemia virus type 1-transformed cells (16). However, this constitutive phosphorylation of Sam68 is blurred in T cells by the presence of two distinct kinases of the Src family, Lck and Fyn, that may have some overlapping functions since both are presumed to interact with Sam68. The present study was therefore undertaken initially to better understand the respective contribution of the two Src PTKs expressed in T cells in this constitutive tyrosine phosphorylation of Sam68.

PTKs from the Src family can physically interact with Sam68 (7, 10, 11, 16, 23, 35). This has been documented clearly by in vitro experiments performed with GST-fusion proteins, and showing that the SH3 and the SH2 domains of Src PTKs, including Fyn and Lck, precipitate Sam68. Different studies have also shown a direct relationship between Src PTK expression in a given cell system and Sam68 phosphorylation. Different examples can be cited as NIH3T3 cells overexpressing Src (7, 10), Hela cells cotransfected with constructs encoding Fyn and Sam68 (11), or Hayai T cells expressing high levels of Fyn (16). On this basis, Sam68 is actually considered as a privileged substrate of Src kinases. Accordingly, we show in the present study that it is a very good substrate for Fyn and Lck in vitro. Nevertheless, it is clear from our data that Fyn, but not Lck, is implicated in the constitutive tyrosine phosphorylation of Sam68 in T cells in vivo, further supporting our previous observations in JCaM1 cells, a Jurkat cell mutant without Lck (22). How can we explain this difference?

Transfected Lck could be negatively regulated in vivo. Indeed, some T8.1 cell clones overexpressing the PTK displayed no obvious change in their whole tyrosine phosphorylation pattern. However, numerous proteins were phosphorylated in others with no parallel induction of Sam68 phosphorylation (see Fig. 2GoB). This hypothesis was further excluded by the experiments performed with Jurkat cells, in which both kinases induced intense phosphorylations, while only Fyn triggered a strong phosphorylation of Sam68 (see Fig. 3Go, A and B). We found interestingly a slight increase of phospho-Sam68 levels in Jurkat cells transfected with a Fyn dead kinase mutant. Although very low as compared with the phosphorylation induced by the wild type, it may reveal some kinase-independent function of Fyn in regulating the phosphorylation status of Sam68 in T cells.

In contrast to Lck, instead of being concentrated at the cell membrane of T lymphocytes, Fyn was reported to be expressed also inside the cell, in the centromeric region (36). We suspected initially that this particular localization might account for the specific role played by Fyn in vivo. We found, however, that the PTK was mainly localized at the cell membrane of the transfected cells. It can be further speculated that Fyn and Lck are differently distributed in the cell membrane, but no evidence has yet been provided confirming this assumption. On the contrary, it was reported recently that both are present in membrane detergent-soluble and insoluble fractions of T lymphocytes (37), especially in the so-called glycolipid-enriched microdomains enriched in Src family kinases (38, 39). It is thus conceivable that some physical constraints at the molecular level would favor an interaction of Sam68 with Fyn. Accordingly, Lck was not consistently present in Sam68 immunoprecipitated from cells overexpressing the PTK (not shown). It is worth noting that the SH3 domains of Fyn and Lck have not exactly the same basic fold (40). Thus, they may differ in their ability to bind proline-rich-containing proteins such as Sam68 in in vivo situations. Moreover, Fyn and Lck may be not obligatory adaptable to different substrates, as clearly demonstrated recently in the case of Pyk2, a member of the focal adhesion kinase family PTK, which is a specific substrate of Fyn (41).

Sam68 is very rapidly tyrosine phosphorylated after CD3 stimulation. By using Lck-negative cells, we previously reported that Lck plays a role in this tyrosine phosphorylation of Sam68 after CD3 triggering, probably activating ZAP-70 to phosphorylate the molecule (22). This suggests that two distinct pathways are involved in the constitutive and the CD3-induced phosphorylation of Sam68 in T cells, and also possibly that Fyn does not participate in Sam68 phosphorylation in activated T cells. But we do not know if Fyn is really dispensable in such circumstances. We cannot exclude for instance that a role for Fyn may be to facilitate the recruitment of Sam68 in the activated receptors in the close proximity of activated ZAP-70 molecules, since both Fyn and ZAP-70 have been shown to bind TCR activation motifs (42, 43) and since phosphorylated ZAP-70 can also bind Fyn directly (44). Whatever the case may be, as discussed above, one should admit that in this situation Sam68 is presumably not phosphorylated by Lck. This remains to be demonstrated. An adaptor function for Sam68, after TCR activation, in early signaling pathways downstream of nonreceptor PTK like Fyn or Lck in T cells has been put forward (11, 16, 20). This is based on our knowledge that Sam68 in its tyrosine-phosphorylated form has the capacity to bind several essential SH2-containing signaling molecules in vitro, but also in vivo, as recently shown for phospholipase C{gamma}-1 and p21ras GTPase-activating protein (19, 20). An influence of Sam68 on the corresponding metabolic pathways has not been firmly proven yet and we did not find any clear effect of Sam68 expression on CD3/TCR-induced calcium response in preliminary experiments (V. L., unpublished results); it is nevertheless tempting to imagine a distinct participation of constitutively phosphorylated Sam68 molecules in these pathways compared with the phosphorylated form that appears after CD3/TCR stimulation.

Sam68 is a member of the GRP33-Sam68-GLD-1 (GSG) family of protein (also known as STAR, for signal transduction and activator of RNA, or SGQ, for Sam68, GLD-1, and Qk1 family) characterized by a larged conserved domain (the so-called GSG domain) comprising a unique KH domain (8, 45). The GSG domain is necessary to mediate RNA binding (1, 7, 8, 21, 46, 47, 48) and oligomerization of the molecule (6, 49). Its exact function is unknown, but is probably essential since genetic alterations of different members of the GSG family in this domain, affecting RNA binding and/or dimerization, are accompanied by an altered phenotype (49, 50). No such alterations have been reported for Sam68, but studies have demonstrated that both RNA binding and multimerization of the molecule are inhibited after phosphorylation (6, 21), showing that it is probably crucial in regulating Sam68 biological function(s). Since Sam68 has a dual localization in a cell membrane compartment and mainly in the nucleus, our knowledge of where the molecule is phosphorylated is therefore essential. By performing immunoblotting experiments on fractionated T cells, we observed that Sam68 was expressed both in the highly enriched cell membrane fraction, where Fyn is present, and in the nucleus. From a quantitative point of view, we estimated that a majority of Sam68 was in the nuclear fraction, a conclusion that was further supported by fluorescence analysis showing a very bright staining of the nucleus with the Sam68-GFP construct (see Fig. 6Go) or by using Sam68-specific Abs (data not shown). One should note that, using fluorescence, Sam68 was difficult to detect outside the nucleus, especially at the plasma membrane. Moreover, the Sam68{Delta}NLS-GFP molecule used in this work was mainly expressed in vesicular structures, suggesting that Sam68 is not specifically targeted to the plasma membrane when its NLS is missing. Whatever it may be, we show that the nuclear fraction of Sam68 was highly tyrosine phosphorylated in cells overexpressing Fyn. We also demonstrate that Fyn membrane localization is required. Posttranslational addition of myristate or palmitate is known to target Src PTKs to membranes (51). Consequently, our results using a truncated form of the molecule lacking the first N-terminal residues necessary for these modifications also suggest that the constitutive phosphorylation of Sam68 initially occurs in this cell compartment and not into the nucleus. Our observation that a Sam68 mutant unable to localize in the nucleus is still phosphorylated also strongly supports this assumption. It should be noted that Fyn overexpression does not modify the level of Sam68 in the different cellular fractions (see Fig. 5GoD). This may indicate that the level of Sam68 inside and outside the nucleus is tightly regulated by a mechanism that does not involve Fyn itself. Interestingly, it was reported recently that the localization of Sam68 in the nucleus of fibroblasts from Src knockout mice was unchanged (4), suggesting that the localization of the molecule is independent of Src expression. However, more careful analysis would be necessary to formally conclude on this point and also to determine whether Sam68 phosphorylation at or near the cell membrane may influence its relocalization in the nucleus. When considering this problem, it is interesting to note that the NLS in Sam68 lies near the C terminus in the tyrosine-rich region of the molecule. Like other NLS, whose function is regulated by protein-protein interaction (52), the function of the NLS in Sam68 may be thus directly regulated by the state of phosphorylation of the molecule and its subsequent interaction with SH2-signaling proteins. Using our T cell models, experiments are now under progress to study these aspects of Sam68 metabolism. We believe that such approach would help to understand the relationship between the proposed adaptor function of Sam68 and its potential role in RNA metabolism.


   Acknowledgments
 
We thank Dr. J. Delon and Dr. P. Hubert for their critical reading of the manuscript, and also N. Delon for her comments on the text.


   Footnotes
 
1 This work was supported by grants from the Association de la Recherche sur le Cancer, the Ligue Nationale contre le Cancer, and the Fédération Nationale des Groupements des Entreprises Françaises et Monégasques dans la Lutte contre le Cancer. Back

2 Address correspondence and reprint requests to Dr. Georges Bismuth, Centre National de la Recherche Scientifique UMR 7627, Centre Hospitalier Pitié-Salpétrière/Centre d’Examen et de Recherche en Virologie et Immunologie, 83 Blvd de l’Hopital, 75013, Paris, France. E-mail address: Back

3 Abbreviations used in this paper: KH, K homology domain; GFP, green fluorescent protein; GSG, GRP33-Sam68-GLD-1; NLS, nuclear localization signal; PTK, protein tyrosine kinase; SH, Src homology. Back

Received for publication December 21, 1998. Accepted for publication April 5, 1999.


   References
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 Discussion
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