This information is current as
of June 18, 2017.
Fyn Membrane Localization Is Necessary to
Induce the Constitutive Tyrosine
Phosphorylation of Sam68 in the Nucleus of T
Lymphocytes
Valérie Lang, Monique Semichon, Frédérique Michel,
Cédric Brossard, Hélène Gary-Gouy and Georges Bismuth
J Immunol 1999; 162:7224-7232; ;
http://www.jimmunol.org/content/162/12/7224
Subscription
Permissions
Email Alerts
This article cites 51 articles, 33 of which you can access for free at:
http://www.jimmunol.org/content/162/12/7224.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
References
Fyn Membrane Localization Is Necessary to Induce the
Constitutive Tyrosine Phosphorylation of Sam68 in the Nucleus
of T Lymphocytes1
Valérie Lang,* Monique Semichon,† Frédérique Michel,‡ Cédric Brossard,*
Hélène Gary-Gouy,* and Georges Bismuth2*
S
am68 is a ubiquitous protein, reported to be predominantly
nuclear in fibroblasts or epithelial cells (1– 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 ty-
*Laboratoire d’Immunologie Cellulaire, Centre National de la Recherche Scientifique
UMR 7627, Centre Hospitalier Pitié-Salpêtrière, CERVI, Paris, France; †Unité
Claude Bernard C20, Département d’Hématologie, Centre Hospitalier Pitié-Salpêtrière, Centre d’Examen et de Recherche en Virologie et Immunologie, Paris, France;
and ‡Laboratoire d’Immunologie Moléculaire, Département d’Immunologie, Institut
Pasteur, Paris, France
Received for publication December 21, 1998. Accepted for publication April 5, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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.
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: gbismuth@ ccr.jussieu.fr
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.
Copyright © 1999 by The American Association of Immunologists
rosine 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 Cg-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 (11–20).
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
0022-1767/99/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
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 phosphoSam68 molecules can thereafter accumulate into the nucleus. The Journal of Immunology, 1999, 162: 7224 –7232.
The Journal of Immunology
7225
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
Cells
Antibodies
The CD3e-specific mAb UCHT1 was produced as ascitic fluid. The antiphosphotyrosine mAb 4G10 and the polyclonal Ab specific for the Nterminal 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
1
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 pSRa-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 pSRa 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 pSRa-puro. The Fyn-DN-ter mutant (without the first six amino
acids at the N terminus) was generated by PCR using the following oligonucleotides as primers, 59-GGAATTCGATGAAGGATAAAGAAG
CAGCGAA-39 and 59-GGAATTCTCACAGGTTTTCACCGGGCTG-39,
and wild-type Fyn as a template. The PCR product was digested with
EcoRI and then inserted into pSRa-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-DNLS 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 3 106 cells were
mixed with 30 mg of plasmid DNA in 0.5 ml of a buffer containing 120
mM KCl, 150 mM 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 mF in a Gene Pulse cuvette (Bio-Rad, Ivry sur Seine,
France). After 48 h in growth medium, cells were seeded at 104 cells/well
Whole cell lysate and subcellular fractionation
For whole cell lysate preparation, T cells (10 3 106) 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 3 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 3 106) were equilibrated for 15 min at 37°C and stimulated for
2 min at 37°C in the presence of the CD3e-specific mAb UCHT1 (1/500
dilution of an ascitic fluid) in a final volume of 500 ml. 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 3 g for 15 min at 4°C. The supernatant (cytoplasmic and particulate fractions) was centrifuged at
100,000 3 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 3 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 mg) 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 mg/ml), antiFyn mAb (1 mg/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 3 106 cells
or from 500 mg of nuclear extract were incubated for 2 h at 4°C with 30 ml
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 3 106 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 ml 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 glutathioneSepharose 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 mg of
GST-Sam68) were then resuspended in a phosphorylation buffer containing
50 mM PIPES, pH 6.8, 10 mM MnCl2, 10 mM MgCl2, 50 mM 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 ml of phosphorylation buffer supplemented with 10 mM ATP and 10
mCi [g-32P]ATP (4500 Ci/mmol; ICN Biomedicals France, Orsay, France).
After 10 min at room temperature, the reaction was stopped by the addition
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
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
mg/ml streptomycin, 1 mg/ml geneticin), 400 nM methotrexate, 2 mM
L-glutamine, 1 mM sodium pyruvate, and 50 mM 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 mg/ml streptomycin), 2 mM L-glutamine, and 1 mM sodium
pyruvate.
in flat-bottom 96-well plates and placed under selection in the presence of
1 mg/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 mF) 10 3 106 cells with 20 mg of plasmid
DNA. Transfected cells were cultured in 6 ml of growth medium for 48 h
before use.
7226
Fyn MEMBRANE LOCALIZATION AND Sam68 PHOSPHORYLATION IN T CELLS
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 (FynWT11, Fyn-WT3, Fyn-WT7). A, After lysis in Nonidet P-40 lysis buffer, solubilized proteins (60 mg)
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.
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(ab9)2 fragment donkey antirabbit 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 3100 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
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. 1A are the blots with three clones, FynWT11, 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 phos-
phorylation 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. 1A). 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. 1B). Taken together,
these findings demonstrate a direct relationship between Fyn expression and the constitutive tyrosine phosphorylation of Sam68 in
T lymphocytes.
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. 2 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. 2A), the constitutive phosphorylation of
Sam68 was analyzed by blotting the lysates with the antiphosphotyrosine mAb 4G10 (Fig. 2B). 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. 2A).
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. 3A). Several phosphoproteins were coimmunoprecipitated with Sam68 only in the
Fyn-transfected cells. As shown in Fig. 3B, 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
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
of 6 ml of Laemmli sample buffer 63 and boiling. Proteins were separated
by 10% SDS-PAGE, and the gel was dried and subjected to autoradiography on x-ray film.
The Journal of Immunology
7227
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 mg) 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 antiphosphotyrosine mAb 4G10. Sam68 position is
shown by an arrow.
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. 4A 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. 4B 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.
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– 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 –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-DN-ter) was stably transfected in T8.1
cells, and two clones (Fyn-DN-ter A6 and Fyn-DN-ter A12) overexpressing the molecule (see Fig. 5B, middle panel) were studied.
We first analyzed Fyn localization by immunofluorescence. As
shown in Fig. 5A, 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-DN-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-DN-ter (Fig. 5B,
upper panel) or after immunoprecipitating the molecule (Fig. 5C).
We checked in parallel experiments that the Fyn-DN-ter mutant
was fully active in in vitro kinase assays (not shown).
Sam68 blots of cellular fractions from T8.1, Fyn-WT7, and FynDN-ter A12 were also performed to compare the cellular distribution of the molecule in the different clones (Fig. 5D). 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. 5E) 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
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
also shown. Control experiments were also performed with a dead
kinase mutant form of Fyn. As shown in Fig. 3C, only a slight
increase was observed with the dead kinase, suggesting that wildtype 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.
7228
Fyn MEMBRANE LOCALIZATION AND Sam68 PHOSPHORYLATION IN T CELLS
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-DNLS). 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. 6 are images obtained by analyzing the fluorescence of living Jurkat T cells 48 h after electroporation with the
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 3. Sam68 is highly tyrosine phosphorylated only in Jurkat cells overexpressing Fyn. Jurkat
T cells (10 3 106) were transiently transfected with
20 mg of the pSRa-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 antiphosphotyrosine mAb 4G10 or anti-Sam68 Abs. B,
A total of 60 mg 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.
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-DNLS-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.
We next analyzed the tyrosine phosphorylation of the Sam68DNLS-GFP protein after immunoprecipitating the molecule from
The Journal of Immunology
Jurkat cells stimulated or not with a CD3e-specific mAb (Fig. 7).
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 Sam68specific 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.
Discussion
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 con-
tribution 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. 2B). 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. 3, 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 socalled 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
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
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 3 106 cells) were subjected to Fyn or Lck immunoprecipitation
with specific Abs. Immune complexes were incubated with 1 mg of GSTSam68 recombinant protein (used as an exogenous substrate) immobilized
on glutathione Sepharose beads. They were then subjected to in vitro kinase assay using [g-32P]ATP. Autoradiography of a 10% SDS-polyacrylamide is shown. B, Coomassie blue staining of the gel shown in A.
7229
7230
Fyn MEMBRANE LOCALIZATION AND Sam68 PHOSPHORYLATION IN T CELLS
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
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-DN-ter A6 and Fyn-DN-terA
12). A, Fyn localization was analyzed in clones Fyn-WT7, Fyn-DN-terA6,
and Fyn-DN-ter A12 by indirect immunofluorescence of fixed permeabilized cells. B, Nonidet P-40 whole cell lysates (60 mg) from clones FynDN-ter A6 and Fyn-DN-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-DN-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-DN-ter A12 cells, as described under Materials and Methods. Subcellular fractions (60 mg) 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 FynWT7 or Fyn-DN-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.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Fluorescence analysis of Jurkat cells transfected with
Sam68-GFP constructs. Jurkat T cells (10 3 106) were transiently transfected with 20 mg 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
(Sam68DNLS-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.
The Journal of Immunology
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 tyrosinephosphorylated form has the capacity to bind several essential
SH2-containing signaling molecules in vitro, but also in vivo, as
recently shown for phospholipase Cg-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 – 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. 6) 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
Sam68DNLS-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. 5D). 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.
References
1. Wong, G., O. Müller, R. Clark, L. Conroy, M. F. Moran, P. Polakis, and
F. McCormick. 1992. Molecular cloning and nucleic acid binding properties of
the GAP-associated tyrosine phosphoprotein p62. Cell 69:551.
2. McBride, A. E., A. Schlegel, and K. Kirkegaard. 1996. Human protein Sam68
relocalization and interaction with poliovirus RNA polymerase in infected cells.
Proc. Natl. Acad. Sci. USA 93:2296.
3. Ishidate, T., S. Yoshihara, Y. Kawasaki, B. C. Roy, K. Toyoshima, and
T. Akiyama. 1997. Identification of a novel nuclear localization signal in Sam68.
FEBS Lett. 409:237.
4. McBride, A. E., S. J. Taylor, D. Shalloway, and K. Kirkegaard. 1998. KH domain
integrity is required for wild-type localization of Sam68. Exp. Cell Res. 241:84.
5. Siomi, H., M. J. Matunis, W. M. Michael, and G. Dreyfuss. 1993. The pre-mRNA
binding K protein contains a novel evolutionarily conserved motif. Nucleic Acids
Res. 21:1193.
6. Chen, T., B. B. Damaj, C. Herrera, P. Lasko, and S. Richard. 1997. Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and
Qk1: role of the KH domain. Mol. Cell. Biol. 17:5707.
7. Taylor, S. J., and D. Shalloway. 1994. An RNA-binding protein associated with
Src through its SH2 and SH3 domains in mitosis. Nature 368:867.
8. Lin, Q., S. J. Taylor, and D. Shalloway. 1997. Specificity and determinants of
sam68 RNA binding: implications for the biological function of k homology
domains. J. Biol. Chem. 272:27274.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 7. The delocalization of Sam68 from the nucleus does not impair Sam68 phosphorylation. Forty-eight-hour transfected cells with GFP
or Sam68DNLS-GFP were stimulated or not with mAb UCHT1 (CD3e
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).
7231
7232
Fyn MEMBRANE LOCALIZATION AND Sam68 PHOSPHORYLATION IN T CELLS
30. Shenoy-Scaria, A. M., D. J. Dietzen, J. Kwong, D. C. Link, and D. M. Lublin.
1994. Cysteine 3 of Src family protein tyrosine kinase determines palmitoylation
and localization in caveolae. J. Cell Biol. 126:353.
31. Wolven, A., H. Okamura, Y. Rossenblatt, and M. D. Resh. 1997. Palmitoylation
of p59fyn is reversible and sufficient for plasma membrane association. Mol. Cell.
Biol. 8:1159.
32. Gauen, L. K. T., M. E. Linder, and A. S. Shaw. 1996. Multiple features of the
p59fyn src homology 4 domain define a motif for immune-receptor tyrosine-based
activation motif (ITAM) binding and for plasma membrane localization. J. Cell
Biol. 133:1007.
33. Pillay, I., H. Nakano, and S. V. Sharma. 1996. Radicicol inhibits tyrosine phosphorylation of the mitotic Src substrate Sam68 and retards subsequent exit from
mitosis of Src-transformed cells. Cell Growth Differ. 7:1487.
34. Laird, A. D., and D. Shalloway. 1997. Oncoprotein signalling and mitosis. Cell.
Signal. 9:249.
35. Weng, Z., S. M. Thomas, R. J. Rickles, J. A. Taylor, A. W. Brauer,
C. Seidel-Dugan, W. M. Michael, G. Dreyfuss, and J. S. Brugge. 1994. Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of
SH3 domains. Mol. Cell. Biol. 14:4509.
36. Ley, S. C., M. Marsh, C. R. Bebbington, K. Proudfoot, and P. Jordan. 1994.
Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes. J. Cell Biol. 125:639.
37. Xavier, R., T. Brennan, Q. Li, C. McCormack, and B. Seed. 1998. Membrane
compartmentation is required for efficient T cell activation. Immunity 8:723.
38. Stefanova, I., V. Horejsi, I. J. Ansotegui, W. Knapp, and H. Stockinger. 1991.
GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science 254:1016.
39. Thomas, P. M., and L. E. Samelson. 1992. The glycophosphatidylinositol-anchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells.
J. Biol. Chem. 267:12317.
40. Hiroaki, H., W. Klaus, and H. Senn. 1996. Determination of the solution structure
of the SH3 domain of human p56 Lck tyrosine kinase. J. Biomol. NMR 8:105.
41. Qian, D., S. Lev, N. S. van Oers, I. Dikic, J. Schlessinger, and A. Weiss. 1997.
Tyrosine phosphorylation of Pyk2 is selectively regulated by Fyn during TCR
signaling. J. Exp. Med. 185:1253.
42. Gauen, L. K. T., Y. Zhu, F. Letourneur, Q. Hu, J. B. Bolen, L. A. Matis,
R. D. Klausner, and A. S. Shaw. 1994. Interactions of p59fyn and ZAP-70 with
T-cell receptor activation motifs: defining the nature of a signalling motif. Mol.
Cell. Biol. 14:3729.
43. Timson Gauen, L. K., A. N. Kong, L. E. Samelson, and A. S. Shaw. 1992. p59fyn
tyrosine kinase associates with multiple T-cell receptor subunits through its
unique amino-terminal domain. Mol. Cell. Biol. 12:5438.
44. Fusaki, N., S. Matsuda, H. Nishizumi, H. Umemori, and T. Yamamoto. 1996.
Physical and functional interactions of protein tyrosine kinases, p59fyn and ZAP70, in T cell signaling. J. Immunol. 156:1369.
45. Vernet, C., and K. Artzt. 1997. STAR, a gene family involved in signal transduction and activation of RNA. Trends Genet. 13:479.
46. Arning, S., P. Gruter, G. Bilbe, and A. Kramer. 1996. Mammalian splicing factor
SF1 is encoded by variant cDNAs and binds to RNA. RNA 2:794.
47. Ebersole, T. A., Q. Chen, M. J. Justice, and K. Artzt. 1996. The quaking gene
product necessary in embryogenesis and myelination combines features of RNA
binding and signal transduction proteins. Nat. Genet. 12:260.
48. Jones, A. R., and T. Schedl. 1995. Mutations in gld-1, a female germ cell-specific
tumor suppressor gene in Caenorhabditis elegans, affect a conserved domain also
found in Src-associated protein Sam68. Genes Dev. 9:1491.
49. Zorn, A. M., and P. A. Krieg. 1997. The KH domain protein encoded by quaking
functions as a dimer and is essential for notochord development in Xenopus
embryos. Genes Dev. 11:2176.
50. Sidman, R. L., M. M. Dickie, and S. H. Appel. 1964. Mutant mice (quaking and
jimpy) with deficient myelination in the central nervous system. Science 144:309.
51. Resh, M. D. 1994. Myristylation and palmitylation of Src family members: the
fats of the matter. Cell 76:411.
52. Jans, D. A., and S. Hubner. 1996. Regulation of protein transport to the nucleus:
central role of phosphorylation. Physiol. Rev. 76:651.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
9. Barlat, I., F. Maurier, M. Duchesne, E. Guitard, B. Tocque, and F. Schweighoffer.
1997. A role for Sam68 in cell cycle progression antagonized by a spliced variant
within the KH domain. J. Biol. Chem. 272:3129.
10. Fumagalli, S., N. F. Totty, J. J. Hsuan, and S. A. Courtneidge. 1994. A target for
Src in mitosis. Nature 368:871.
11. Richard, S., D. Yu, K. J. Blumer, D. Hausladen, M. W. Olszowy, P. A. Connelly,
and A. S. Shaw. 1995. Association of p62, a multifunctional SH2- and SH3domain binding protein, with src family tyrosine kinases, Grb2, and phospholipase Cg-1. Mol. Cell. Biol. 15:186.
12. Finan, P. M., A. Hall, and S. Kellie. 1996. Sam68 from an immortalized B-cell
line associates with a subset of SH3 domains. FEBS Lett. 389:141.
13. Bunnell, S. C., P. A. Henry, R. Kolluri, T. Kirchhausen, R. J. Rickles, and
L. J. Berg. 1996. Identification of Itk/Tsk Src homology 3 domain ligands. J. Biol.
Chem. 271:25646.
14. Trüb, T., J. D. Frantz, M. Miyazaki, H. Band, and S. E. Shoelson. 1997. The role
of a lymphoid-restricted, Grb2-like SH3-SH2-SH3 protein in T cell receptor signaling. J. Biol. Chem. 272:894.
15. Jabado, N., A. Pallier, F. Le Deist, F. Bernard, A. Fischer, and C. Hivroz. 1997.
CD4 ligands inhibit the formation of multifunctional transduction complexes involved in T cell activation. J. Immunol. 158:94.
16. Fusaki, N., A. Iwamatsu, M. Iwashima, and J. Fujisawa. 1997. Interaction between Sam68 and Src family tyrosine kinases, Fyn and Lck, in T cell receptor
signaling. J. Biol. Chem. 272:6214.
17. Lawe, D. C., C. Hahn, and A. J. Wong. 1997. The Nck SH2/SH3 adaptor protein
is present in the nucleus and associates with the nuclear protein SAM68. Oncogene 14:223.
18. Guinamard, R., M. Fougereau, and P. Seckinger. 1997. The SH3 domain of
Bruton’s tyrosine kinase interacts with Vav, Sam68 and EWS. Scand. J. Immunol. 45:587.
19. Guitard, E., I. Barlat, F. Maurier, F. Schweighoffer, and B. Tocque. 1998. Sam68
is a Ras-GAP-associated protein in mitosis. Biochem. Biophys. Res. Commun.
245:562.
20. Jabado, N., S. Jauliac, A. Pallier, F. Bernard, A. Fischer, and C. Hivroz. 1998.
Sam68 association with p120GAP in CD41 T cells is dependent on CD4 molecule expression. J. Immunol. 161:2798.
21. Wang, L. L., S. Richard, and A. S. Shaw. 1995. p62 association with RNA is
regulated by tyrosine phosphorylation. J. Biol. Chem. 270:2010.
22. Lang, V., D. Mege, M. Semichon, H. Gary-Gouy, and G. Bismuth. 1997. A dual
participation of ZAP-70 and scr protein tyrosine kinases is required for TCRinduced tyrosine phosphorylation of Sam68 in Jurkat T cells. Eur. J. Immunol.
27:3360.
23. Vogel, L. B., and D. J. Fujita. 1995. p70 phosphorylation and binding to p56lck
is an early event in interleukin-2-induced onset of cell cycle progression in Tlymphocytes. J. Biol. Chem. 270:2506.
24. Blank, U., B. Boitel, D. Mege, M. Ermonval, and O. Acuto. 1993. Analysis of
tetanus toxin peptide/DR recognition by human T cell receptors reconstituted into
a murine T cell hybridoma. Eur. J. Immunol. 23:3057.
25. Clipstone, N. A., and G. R. Crabtree. 1992. Identification of calcineurin as a key
signalling enzyme in T-lymphocyte activation. Nature 357:695.
26. Takebe, Y., M. Seiki, J. Fujisawa, P. Hoy, K. Yokota, K. Arai, M. Yoshida, and
N. Arai. 1988. SR a promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5
segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell.
Biol. 8:466.
27. Fusaki, N., K. Semba, T. Katagiri, G. Suzuki, S. Matsuda, and T. Yamamoto.
1994. Characterization of p59fyn-mediated signal transduction on T cell activation. Int. Immunol. 6:1245.
28. Shenoy-Scaria, A. M., L. K. Timson Gauen, J. Kwong, A. S. Shaw, and
D. M. Lublin. 1993. Palmitylation of an amino-terminal cysteine motif of protein
tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins. Mol. Cell. Biol. 13:6385.
29. Alland, L., S. M. Peseckis, R. E. Atherton, L. Berthiaume, and M. D. Resh. 1994.
Dual myristylation and palmitylation of Src family member p59fyn affects subcellular localization. J. Biol. Chem. 269:16701.