From the Imperial Cancer Research Fund, P. O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX, United Kingdom and the Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095
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ABSTRACT |
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Treatment of quiescent Swiss 3T3 cells with bombesin rapidly increased focal adhesion kinase (FAK)-associated tyrosine kinase activity in immune complexes. The effect was rapid (maximum at 2.5 min) and dose dependent (half-maximum response at 0.05 nM). Addition of vasopressin, lysophosphatidic acid, and sphingosylphosphorylcholine also elicited a rapid increase in FAK-associated tyrosine kinase activity. Addition of the selective Src inhibitor pyrazolopyrimidine directly to the in vitro kinase assay potently inhibited Src kinase activity induced by bombesin but did not affect the kinase activity of FAK measured by autophosphorylation or by synthetic substrate phosphorylation in paralell assays. In addition, Src activity was not detected in FAK immunoprecipitates using an optimal Src peptide substrate. Thus, agonist-induced tyrosine kinase activity measured in FAK immunoprecipitates is mediated by FAK activation rather than by co-immunoprecipitating Src. Bombesin-induced FAK activation is not dependent either on protein kinase C or Ca2+ mobilization but was completely blocked by treatment with cytochalasin D or by placing the cells in suspension. These findings indicate that FAK activation requires an intact actin cytoskeleton. Our results demonstrate that agonists that act via 7-transmembrane domain receptors stimulate FAK kinase activation.
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INTRODUCTION |
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Neuropeptides stimulate DNA synthesis and cell proliferation in
cultured cells and are implicated as growth factors in a variety of
fundamental processes including development, inflammation, tissue
regeneration, and tumorigenesis (1, 2). In particular, bombesin and its
mammalian counterpart gastrin-releasing peptide bind to a
G-protein-coupled receptor (3, 4) that promotes Gq-mediated activation of
isoforms of phospholipase
C (5, 6) to produce two second messengers: inositol
1,4,5-trisphosphate, that mobilizes Ca2+ from internal
stores and diacylglycerol that activates protein kinase C
(PKC)1 (7-9). Subsequently,
bombesin induces activation of phosphorylation cascades including
p42MAPK/P44MAPK and p70S6K (10-13)
leading to increased expression of immediate early response genes,
stimulation of cell cycle events, and subsequent cell proliferation (9,
14-17).
The binding of bombesin to its receptor also induces rapid tyrosine phosphorylation of multiple substrates in Swiss 3T3 cells (9, 18, 19). Focal adhesion kinase (FAK) (20, 21) has been identified as a prominent tyrosine-phosphorylated protein in cells stimulated with bombesin (22, 23) and other neuropeptides (22, 24, 25). Tyrosine phosphorylation of FAK is also increased by diverse signaling molecules that mediate cell growth and differentiation, including bioactive lipids such as lysophosphatidic acid (LPA) and sphingosylphosphorylcholine (SPC) (26-28), polypeptide growth factors (29, 30), bacterial toxins (31, 32), activated variants of pp60src (33, 34), and extracellular matrix proteins (21, 35-37). These results indicate that FAK is a point of convergence in a variety of signal transduction pathways (38, 39). The importance of FAK-mediated signal transduction is underscored by recent experiments showing that this tyrosine kinase is implicated in embryonic development (40) and in the control of cell migration (41-43), proliferation (42, 44), and apoptosis (45-47).
While an increase in the tyrosine phosphorylation of FAK is recognized as an early event in the action of multiple agents, much less is known about the regulation of the tyrosine kinase activity of FAK in response to extracellular stimuli. Integrin engagement with fibronectin and v-Src transformation have been shown to increase FAK-associated tyrosine kinase activity in immunoprecipitates (33, 36). Subsequent studies, however, showed that these conditions also promote the formation of a complex between autophosphorylated FAK at Tyr-397 and the SH2 domain of Src (48-53). Consequently, it is not clear whether the increase in the kinase activity of FAK immune complexes is mediated by FAK or by co-immunoprecipitating Src. Furthermore, the effect of neuropeptide agonists or bioactive lipids on FAK-associated tyrosine kinase activity has not been examined.
In the present study we demonstrate that stimulation of Swiss 3T3 cells with bombesin, vasopressin, LPA, and SPC induces a rapid increase in FAK-associated tyrosine kinase activity. Our results show that this increase in FAK activity can be dissociated from the presence of Src kinase members in the FAK immunoprecipitates. Bombesin induces FAK activation through a PKC- and Ca2+-independent pathway that requires the integrity of the actin filament network.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- Stock cultures of Swiss 3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum, in a humidified atmosphere containing 10% CO2 and 90% air at 37 °C. For experimental purposes, Swiss 3T3 cells were plated in 35-mm Nunc Petri dishes at 105 cells/dish in DMEM containing 10% fetal bovine serum and used after 6-8 days when the cells were confluent and quiescent (14).
Immunoprecipitation-- Quiescent cultures of Swiss 3T3 cells (1-2 × 106 cells) were washed twice with DMEM, equilibrated in the same medium at 37 °C for at least 15 min, and then treated with peptide factors in 1 ml of DMEM for the times indicated. The stimulation was terminated by aspirating the medium and solubilizing the cells in 1 ml of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.35, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 2 mM sodium orthovanadate, 1% Triton X-100, 50 µg/ml aprotinin, 50 µg/ml leupeptin, 5 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride). Lysates were clarified by centrifugation at 14,000 rpm for 10 min and the pellets were discarded. After centrifugation, supernatants were transferred to fresh tubes and proteins were immunoprecipitated at 4 °C for 3 h with either protein A-agarose linked polyclonal anti-FAK (C-20) or polyclonal anti-Src family (SRC-2) antibodies or with protein G-agarose linked mAb directed against FAK (mAb 2A7) as described previously (18, 22, 54, 55). Immunoprecipitates were washed three times with lysis buffer, extracted in 2 × SDS-PAGE sample buffer (200 mM Tris-HCl, pH 6.8, 0.1 mM sodium orthovanadate, 1 mM EDTA, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol), by boiling 5 min, fractionated by one-dimensional SDS-PAGE, and further analyzed as described under "Results" and in the figure legends. Immunoprecipitates obtained with C-20, 2A7, or SRC-2 were also used for in vitro kinase reactions.
In Vitro Kinase Reactions--
FAK immunoprecipitates were
washed and pelleted (2,500 rpm for 10 min in the cold) 3 times in lysis
buffer. When C-20 polyclonal antibody was used, immunoprecipitates were
washed twice with FAK kinase buffer A (20 mM Hepes, pH
7.35, 3 mM MnCl2). Pellets were dissolved in 40 µl of kinase buffer and reactions were started by adding 10 µCi of
[-32P]ATP. The reactions were carried out at 30 °C
for 15 min, and stopped on ice by adding 10 mM EDTA. In
some experiments poly(Glu-Tyr) (4:1) (40 µg) was added to the C-20
immunocomplex. The incorporation of 32P label into
poly(Glu-Tyr) (4:1) was stopped by removing the supernatant from the
agarose beads and adding 2 × SDS-PAGE sample buffer. Samples were
then analyzed by SDS-PAGE and autoradiography.
Phosphorylation of Src Peptide and Raytide
Substrates--
Phosphorylation of the highly specific Src peptide,
p-Ala-Glu-Glu-Glu-Ile-Tyr-Gly-Glu-Phe-Glu-Ala-Lys-Lys-Lys-Lys-NH2
(56), and RaytideTM substrate were performed as follows.
Immunoprecipitates were washed three times with lysis buffer as above
and twice with kinase assay buffer (50 mM Hepes, pH 7.5, 0.1 mM EDTA, 0.01% Brij) and resuspended in 20 µl of
this buffer. Kinase reactions were initiated by the addition of 100 µM ATP, 10 mM MgCl2, and 2 µCi
of [-32P]ATP, in the presence of 100 µM
Src peptide or RaytideTM substrate, in a total volume of 30 µl. After incubation at 30 °C for 5 min (Src peptide) or 30 min
(RaytideTM substrate), peptide phosphorylation was stopped
by the addition of 120 µl of 10% phosphoric acid, and the reaction
mixture was then applied onto P-81 ion exchange chromatography paper.
Papers were washed five times in 0.5% phosphoric acid, once with
acetone, dried, and counted in a scintillation counter.
Western Blotting-- Treatment of quiescent cultures of cells with factors, cell lysis, and immunoprecipitations were performed as described above. After SDS-PAGE, proteins were transferred to Immobilon membranes for 2 h at 400 mAmp in transfer buffer (192 mM glycine, 25 mM Tris base, 0.075% SDS, 0.6 mM sodium orthovanadate, and 20% methanol). After transfer, membranes were blocked using 5% nonfat dried milk in phosphate-buffered saline, pH 7.2, and incubated for 2 h at 22 °C either with the anti-FAK mAb from Transduction Laboratories, or with the polyclonal anti-FAK C-20 antibody, both diluted 1:500 in phosphate-buffered saline containing 5% nonfat dried milk. After incubating membranes with secondary antibodies (horseradish peroxidase-conjugated goat antibodies to rabbit or mouse immunoglobulin), immunoreactive bands were visualized using ECL reagents.
Phosphoamino Acid Analysis-- This analysis was carried according to Ref. 57.
Materials--
Bombesin, vasopressin, SPC, phorbol
12,13-dibutyrate (PDBu), cytochalasin D, poly(Glu-Tyr) (4:1),
o-phospho-DL-threonine, o-phospho-DL-tyrosine,
o-phospho-DL-serine, agarose-linked anti-mouse IgG were obtained from Sigma and [-32P]ATP (5000 Ci/mmol) and ECL reagents were from Amersham (Buckinghamshire, UK).
Protein A-agarose and protein G-agarose were from Boehringer Mannheim.
The highly specific Src peptide substrate was synthesized at the
Imperial Cancer Research Fund. RaytideTM was from Oncogene
Science, Inc. The PKC inhibitor GF109203X, the Src inhibitor PP-1, and
thapsigargin were obtained from Calbiochem-Novabiochem Ltd.,
Nottingham, United Kingdom. The 2A7 mAb anti-FAK was from Upstate
Biotechnology Inc., Lake Placid, NY. FAK anti-peptide polyclonal
antibody C-20 and Src-family affinity-purified anti-peptide polyclonal
antibody (SRC-2) were from Santa Cruz Biotechnology Inc. Anti-FAK mAb
used in Western blotting was from Transduction Laboratories, Lexington,
KY. All other reagents used were of the purest grade available.
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RESULTS |
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Bombesin Stimulation of FAK-associated Tyrosine Kinase Activity in
Swiss 3T3 Cells--
To examine the effect of bombesin on
FAK-associated tyrosine kinase activity, quiescent cultures of Swiss
3T3 cells were treated with or without 10 nM bombesin for
2.5 min and lysed. The lysates were incubated with the 2A7 mAb, which
recognizes the C-terminal sequence of FAK (58). The resulting immune
complexes were incubated with [-32P]ATP and analyzed
by SDS-PAGE and autoradiography. As shown in Fig.
1A (2A7, upper)
bombesin induced an increase in the phosphorylation of a 125-kDa band
that exactly co-migrated with immunoreactive FAK. In order to confirm
that the radiolabeled 125-kDa band was FAK, the phosphorylated band was
eluted by denaturation and incubated with a different anti-FAK mAb that
recognizes an epitope in the kinase domain of FAK (corresponding to
amino acids 354-533 of the chicken protein). The
re-immunoprecipitation of the 125-kDa radiolabeled band (Fig. 1A,
a-FAK) confirmed that it is phosphorylated FAK.
Immunoblotting with anti-FAK mAb of anti-FAK immunoprecipitates
prepared in parallel with those used for the assays of kinase activity
verified that similar amounts of FAK were recovered after bombesin
treatment (Fig. 1A, ip:2A7; wb:a-FAK).
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Bombesin, Vasopressin, LPA, and SPC Rapidly Increase FAK-associated Tyrosine Kinase Activity-- An increase in FAK-associated tyrosine kinase in anti-FAK immunoprecipitates was a rapid consequence of the addition of bombesin to intact Swiss 3T3 cells, reaching a maximum between 1 and 2.5 min of incubation (Fig. 2A). Immunoblotting with anti-FAK mAb of anti-FAK immunoprecipitates prepared in parallel with those used for the assays of kinase activity verified that similar amounts of FAK were recovered after different times of bombesin treatment (Fig. 2A). Bombesin induced FAK phosphorylation in a concentration-dependent manner reaching half-maximum and maximum effects at 0.05 and 1 nM, respectively (Fig. 2B).
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Dissociation of FAK Kinase Activity from Src Kinases in Swiss Cells-- Autophosphorylation of FAK at Tyr-397 either in v-Src transformed cells or in cells replated on fibronectin, creates a binding site for the SH2 domain of members of the Src family that leads to the formation of a signaling complex in which Src kinases are active (48-53). Consequently, the increase in tyrosine kinase activity measured in FAK immunocomplexes under these conditions could be due to co-immunoprecipitating Src rather than to FAK. Similarly, the increase in FAK-associated tyrosine kinase activity induced by bombesin and other agonists shown in the present study could be mediated by co-precipitating Src kinases rather than to FAK activation and autophosphorylation. Here, we provide several lines of evidence indicating that bombesin and other agonists induce FAK activation in Swiss 3T3 cells.
We found that lysis of Swiss 3T3 cells in a buffer solution containing 1% Triton (the composition of the lysis buffer is given under "Experimental Procedures") rather than in modified RIPA buffer greatly diminished complex formation between FAK and Src family members, as judged by Western blot analysis with anti-FAK C-20 antibody of Src immunoprecipitates (results not shown). To examine further whether Src family members were present in FAK immunocomplexes under our experimental conditions, Triton lysates from control and bombesin-stimulated Swiss 3T3 cells were immunoprecipitated with the 2A7 mAb and the immunocomplexes were incubated with [
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Role of PKC and Ca2+ on Stimulation of FAK Activity-- Next, we examined the signaling pathways leading to FAK activation in response to bombesin in Swiss 3T3 cells. As a rapid activation of PKC is a prominent early event elicited by bombesin in these cells, we examined the role of PKC in bombesin-stimulated FAK activation. Direct stimulation of PKC with PDBu for 2.5 min increased FAK autophosphorylation (Fig. 6A). To examine whether PKC was required for stimulation of FAK activity in response to bombesin, quiescent Swiss 3T3 cells were incubated with or without 3.5 µM bisindolylmaleimide I (also known as GF 109203X), an inhibitor of PKC (60), prior to stimulation with either PDBu or bombesin. Treatment with GF 109203X abrogated PDBu-induced stimulation of immunoprecipitable FAK kinase activity but did not prevent bombesin stimulation of FAK autophosphorylation (Fig. 6). Similarly, pretreatment with GF 109203X did not affect LPA stimulation of FAK autophosphorylation (not shown). These results indicated that activation of PKC could not account for the stimulation of FAK activity by bombesin or LPA.
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Treatment with Cytochalasin D and Suspension of Cells Inhibit Bombesin-stimulated FAK Activity-- Given the localization of FAK to focal adhesions which form at the end of actin stress fibers (20, 21), we examined whether the stimulation of FAK activity induced by bombesin depends on the integrity of the actin cytoskeleton. Quiescent cultures of Swiss 3T3 cells were treated for 2 h with cytochalasin D at 2.5 µM, conditions known to depolymerize the actin cytoskeleton in these cells (23). The cultures, treated with or without cytochalasin D, were subsequently stimulated with bombesin. As illustrated by Fig. 7 (upper panel), treatment with cytochalasin D completely blocked the increase in FAK activity in response to bombesin.
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DISCUSSION |
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An increase in the tyrosine phosphorylation of the non-receptor tyrosine kinase FAK has been extensively documented as an early event in the action of multiple agonists that modulate cell growth, differentiation, and motility in a variety of cell types. In contrast, little is known about the regulation of FAK activity in response to these agents. The results presented here demonstrate that bombesin rapidly increases FAK-associated tyrosine kinase activity in Swiss 3T3 cells. Other agonists that act via 7-transmembrane domain receptors including vasopressin, LPA, and SPC also elicited a rapid increase in FAK-associated kinase activity in these cells.
Previous studies demonstrated that integrin engagement or v-Src transformation not only induces FAK tyrosine phosphorylation but also enhances kinase activity of FAK immunoprecipitates (33, 36). However, subsequent studies from several laboratories demonstrated that the major site of FAK autophosphorylation (i.e. Tyr-397) is a high affinity binding site for the SH2 domain of members of the Src kinase family. Association of Src to phosphorylated FAK has been demonstrated in v-Src transformed cells and in cells replated on fibronectin (48, 50, 51, 53). Consequently, the increase in kinase activity measured in FAK immunoprecipitates in previous studies could be due to the presence of co-precipitating Src rather than to FAK activation. The agonist-mediated increases in FAK-associated kinase demonstrated in the present study could also reflect the presence of Src in the immunoprecipitates rather than an increase in the activity of FAK. Assays of FAK phosphorylation, as performed here do not exclude this possibility because active Src has been shown to phosphorylate FAK at multiple sites in vitro (62, 63). Consequently, it was important to elucidate whether increases in the tyrosine kinase activity detected in FAK immunoprecipitates were due to FAK activation or reflected co-precipitating Src.
To determine the contribution of Src kinases to the activity of FAK immunoprecipitates, we used PP-1, a novel inhibitor of Src kinase family members, in the in vitro phosphorylation assay. This compound inhibits Src kinases at nanomolar concentrations but does not affect other tyrosine kinases such as ZAP70 or JAK-1 (59). Our results demonstrate that PP-1 does not inhibit FAK-associated tyrosine kinase activity but, at similar concentrations, virtually abolishes bombesin-stimulated Src kinase activity measured in parallel Src immunoprecipitates. Furthermore, under the conditions of cell lysis used here (i.e. Triton only buffer) we could not detect Src kinase activity in FAK immunoprecipitates from bombesin-stimulated Swiss 3T3 cells. Our results dissociate the increase in FAK-associated tyrosine kinase induced by bombesin from the presence of Src kinases in the immunoprecipitates. We conclude that neuropeptides and bioactive lipids not only elevate FAK tyrosine phosphorylation but also stimulate FAK kinase activation.
Bombesin is known to stimulate the rapid hydrolysis of inositol phospholipids to generate the second messengers diacylglycerol and inositol 1,4,5-trisphosphate which activate PKC and mobilize Ca2+, respectively. The results presented here demonstrate that direct activation of PKC by treatment of intact cells with biologically active phorbol esters increases FAK tyrosine kinase activity and imply that PKC stimulation is a potential signaling pathway leading to FAK kinase activation. However, our results also show that bombesin rapidly stimulates FAK activation through a signal transduction pathway that is independent of PKC.
In view of the rapid kinetics of neuropeptide-stimulated Ca2+ mobilization and FAK kinase activation, we also tested whether Ca2+ could be responsible for the effect of bombesin on FAK activation. We found that neither Ca2+-ionophore nor thapsigargin caused a Ca2+-dependent increase in FAK kinase activity. Furthermore, depletion of the intracellular Ca2+ pool by treating cells with thapsigargin did not prevent bombesin-induced FAK stimulation. We conclude that increases in the cytoplasmic Ca2+ concentration do not mediate bombesin-induced FAK kinase activation. Thus, neither of the two major signals generated by activation of phospholipase C is responsible for neuropeptide stimulation of FAK kinase activation. These findings distinguish the regulation of FAK activity by G protein-coupled receptor agonists from that of the FAK homologue Pyk2/CAKb/FAFTK/CaDTK, which is believed to be a Ca2+-regulated tyrosine kinase (64, 65).
Agonist-mediated increase in FAK tyrosine phosphorylation is accompanied by profound alterations in the organization of the actin cytoskeleton and in the assembly of focal adhesions (26, 27, 29, 66-68), the distinct areas of the plasma membrane where FAK is localized (20, 21). Treatment of the cells with cytochalasin D, which disrupts actin filaments, prevents the increase in FAK tyrosine phosphorylation in response to multiple agents, suggesting a mechanism involving the actin cytoskeleton and the focal adhesion plaques (23, 27-29, 31, 32, 55). The small G protein p21rho, a member of the Ras superfamily of small GTP-binding proteins (68), has been implicated in mitogen-stimulated formation of focal adhesions and actin stress fibers as well as in the tyrosine phosphorylation of FAK (28, 66, 69, 70). It is thought that translocation of FAK into nascent focal adhesions would induce its activation and autophosphorylation, as a result of clustering and/or conformational change (39, 71).
Here we show that cytochalasin D, which disrupts the organization of the actin cytoskeleton, profoundly inhibited FAK activation induced by bombesin and other agonists. Previous studies demonstrated that treatment with cytochalasin D does not inhibit production of inositol phosphates, Ca2+ mobilization, and stimulation of PKC, Src, and p42MAPK/p44MAPK activation (23, 27, 44, 54, 55), indicating that disruption of the actin cytoskeleton prevents FAK activation in a selective manner. Furthermore, bombesin also failed to induce FAK activation in Swiss 3T3 cells placed in suspension, a condition that also causes disruption of the actin cytoskeleton and disassembly of focal adhesion plaques (55) but does not interfere with bombesin receptor signaling, as judged by assays of Ca2+ mobilization (9, 23, 72), p42MAPK activation (44), and Src kinase activation (55). These findings are consistent with a model in which FAK activation, like tyrosine phosphorylation, requires the recruitment of FAK into intact focal adhesion plaques.
In conclusion, our results demonstrate that stimulation of intact cells with bombesin, vasopressin, LPA, or SPC induces a rapid increase in the tyrosine kinase activity of FAK. Our studies demonstrate that the increase in FAK activity can be dissociated from the presence of Src kinase family members in the immunoprecipitates by the use of PP-1, a compound that potently inhibited Src but not FAK. Bombesin induces FAK activation, like tyrosine phosphorylation, through a PKC- and Ca2+-independent pathway that is critically dependent on the integrity of the focal adhesions and the actin cytoskeleton.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom all correspondence should be addressed: 900 Veteran Ave.,
Warren Hall, Rm. 11-124, Dept. of Medicine, UCLA School of Medicine,
Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399.
1 The abbreviations used are: PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; FAK, focal adhesion kinase; LPA, lysophosphatidic acid; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PDBu, phorbol 12,13-dibutyrate; PP-1, pyrazolopyrimidine; SPC, sphingosylphosphorylcholine; SH2, Src homology domain 2.
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REFERENCES |
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