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CSK Controls Retinoic Acid Receptor (RAR) Signaling: a RAR-c-SRC Signaling Axis Is Required for Neuritogenic Differentiation

Section of Pediatric Hematology/Oncology, Department of Pediatrics, Aflac Cancer Center and Blood Disorders Services, Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia 30022,¹ Department of Pediatrics, Wells Center for Pediatrics Research, Riley Hospital for Children, Indiana University Medical Center, Indianapolis, Indiana 46202,³ Department of Biochemistry and Molecular Biology, School of Medicine, Indiana University, Indianapolis, Indiana 46202²

Received 24 July 2006/ Returned for modification 12 September 2006/ Accepted 14 February 2007

	ABSTRACT

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Herein, we report the first evidence that c-SRC is required for retinoic acid (RA) receptor (RAR) signaling, an observation that suggests a new paradigm for this family of nuclear hormone receptors. We observed that CSK negatively regulates RAR functions required for neuritogenic differentiation. CSK overexpression inhibited RA-mediated neurite outgrowth, a result which correlated with the inhibition of the SFK c-SRC. Consistent with an extranuclear effect of CSK on RAR signaling and neurite outgrowth, CSK overexpression blocked the downstream activation of RAC1. The conversion of GDP-RAC1 to GTP-RAC1 parallels the activation of c-SRC as early as 15 min following all-trans-retinoic acid treatment in LA-N-5 cells. The cytoplasmic colocalization of c-SRC and RAR was confirmed by immunofluorescence staining and confocal microscopy. A direct and ligand-dependent binding of RAR with SRC was observed by surface plasmon resonance, and coimmunoprecipitation studies confirmed the in vivo binding of RAR to c-SRC. Deletion of a proline-rich domain within RAR abrogated this interaction in vivo. CSK blocked the RAR-RA-dependent activation of SRC and neurite outgrowth in LA-N-5 cells. The results suggest that transcriptional signaling events mediated by RA-RAR are necessary but not sufficient to mediate complex differentiation in neuronal cells. We have elucidated a nongenomic extranuclear signal mediated by the RAR-SRC interaction that is negatively regulated by CSK and is required for RA-induced neuronal differentiation.

	INTRODUCTION

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Retinoic acid (RA) is an active form of vitamin A. As a morphogen, it induces cellular differentiation in various cell types. RA or its derivatives have been shown to cause profound morphological differentiation in embryonic stem cells, embryonal carcinoma (EC) cell lines, and neuroblastoma (NB) cell lines (3, 6, 65). RA action is initiated through its binding to two members of the class II family of nuclear hormone receptors, RA receptors (RARs) (, ß, and ) and retinoid X receptors (, ß, and ) (2, 43, 44). RA associates with nuclear RARs to form a heterodimeric complex that binds to the RA-responsive element (RARE) in the promoter regions of the target genes (43). Characteristic effects of RA in tumor cells (growth inhibition and differentiation) are known to be mediated through the transcriptional activation of its signature genes (classical genomic effect) (34, 56). Other modes of RA action in malignant cells include the inhibition of the AP-1 protein (18, 33), the inhibition of c-Jun NH₂-terminal kinase (38), the regulation of histone acetylation (53), the expression of transforming growth factor 2 (TGF-2) and insulin-like growth factor binding protein 3 IGFBP-3 (27), and the upregulation of the PEPCK gene (39). Although much work has been done on the effects of RA on gene expression, little is known concerning the potential for nongenomic extranuclear cellular signaling downstream of RAR engagement.

NB is the most common malignant extracranial solid tumor diagnosed in children and is responsible for 15% of pediatric cancer deaths (39, 45, 63, 70). As an embryonal tumor (12) of neural crest origin, the NB tumor consists of typically undifferentiated neuroectodermal cells (63) that have essentially lost their differentiation cues. NB cell lines continue to serve as a useful model for neuritogenic differentiation. Several studies have reported an improved prognosis for this disease following treatment of high-risk NB patients with retinoids (14). Moreover, RA is clinically one of the most effective inducers of differentiation in NB, and retinoids are routinely used in the treatment of high-risk NB (2, 67). Profound neuritogenesis observed in NB cell lines following RA administration is due to the activation of endogenous differentiation signaling and indicates the relevance of RA in this process. We and others reported an in vitro induction of postmitotic phenotypes in human NB cells lines following all-trans-RA (ATRA) administration (28-30, 59, 66). It is generally held that RARs function as DNA binding transcription factors to induce NB differentiation. This aspect of RAR function has been the major focus of studies devoted to the study of RA-RAR function in neuronal cells. The possibility that other functions may exist for the RAR protein, separate from the DNA binding domain, has not been explored to date. Herein, we have explored evidence that another domain within RAR may functionally interact with tyrosine kinases and play a critical role in neuronal differentiation.

RA-RAR signaling involves both the induction of genes required for neuronal differentiation and dramatic changes in cell shape and function normally ascribed to the posttranslational modification of proteins, e.g., phosphorylation, tubulin acetylation, actin polymerization, etc. Therefore, we hypothesized that there might be a more direct mechanism by which the RARs may regulate and/or coordinate nuclear and cytoplasmic events. One such modification would be the direct interaction with extranuclear protein kinase activities. This led to our investigation of the role for the src family of kinases (SFKs) and CSK in RAR signaling. The SFKs are one of the oldest, largest (comprised of 11 members in humans, of which c-SRC, FYN, and YES are ubiquitously expressed), and most studied family of nonreceptor protein tyrosine kinases (8, 47, 68, 73). The activation of SFKs is known to occur by the "domain displacement" mechanism involving their SH2 and SH3 domains (9, 16, 46, 61). The catalytic activity of SFKs is tightly regulated by the state of phosphorylation of their conserved C-terminal regulatory tyrosine (Y⁵²⁷ in c-SRC) residue (20, 57, 60) by a family of nonreceptor protein tyrosine kinases comprised of C-terminal SRC kinase, CSK, and CSK-homologous kinase (19, 22, 41, 60, 72, 75). Phosphorylation by CSK leads to the conformational changes in the SFK molecule through intramolecular contacts involving the SH2 domain (72). Although enzymatic regulation of SFKs by CSK is well documented (41), the role of CSK in neuronal differentiation and/or nuclear hormone receptor signaling has not been studied.

Our data provide evidence for a direct and functionally relevant connection between RAR, c-SRC, and CSK. Our results suggest a mechanism by which RARs might coordinate their interaction with cytoplasmic and membrane extranuclear events linked to the process of neuronal differentiation by directly binding to and activating protein tyrosine kinases en route to the nucleus, where they mediate transcription. Our results demonstrate that (i) the inhibition of SFKs by CSK or PP1 inhibits RA-induced neurite outgrowth in NB cell lines, (ii) RAR binds to and catalytically activates c-SRC in an RA-dependent manner, (iii) CSK overexpression in LA-N-5 cells blocks the activation of c-SRC and RA-induced activation of the small GTPase RAC1, and (iv) a search of the RAR amino acid sequence identified a highly conserved proline-rich region in the N terminus as being a potential binding site for the SFK-SH3 domain. Taken together, these data suggest that ligand-dependent signaling of the RAR involves c-SRC and that SRC kinase is necessary for RA-induced neuritogenesis of NB cells. The results suggest a paradigm by which nuclear hormone receptors integrate membrane/cytoplasmic events in concert with nuclear transcriptional effects to orchestrate the complex differentiation program required for neuritogenesis.

	MATERIALS AND METHODS

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Antibodies and reagents. ATRA, 9-cis-RA, 13-cis-RA, and monoclonal antibody against human ß-actin were obtained from Sigma-Aldrich (St. Louis, MO). A pan-SFK kinase inhibitor, PP1, was purchased from Biomole (Plymouth Meeting, PA). Rabbit polyclonal antibodies against SRC (SC-19), FYN (SC-16), and YES (SC-14) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and used for immunoprecipitation (IP), kinase assays, and Western blot analyses. Horseradish peroxidase-tagged anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG were obtained from Amersham Biosciences (Buckinghamshire, England). Goat anti-mouse and anti-rabbit IgG (heavy plus light chains)-AP (human adsorbed) were obtained from Southern Biotechnology, Inc. (Birmingham, AL). Recombinant c-SRC and RAR proteins were procured from Upstate Biotechnology (Lake Placid, NY) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. This full-length recombinant RAR of human origin (amino acids 1 to 454) is expressed in Escherichia coli as a 75-kDa tagged fusion protein. The product RAR (corresponding to amino acids 1 to 454) was purified from bacterial lysates by glutathione agarose affinity chromatography (as specified by Santa Cruz Biotechnology, Inc.). Commercially obtained recombinant SRC (p60^c-src) is an approximately 60-kDa protein that is expressed in Sf9 insect cells by recombinant baculovirus containing the human c-src gene. The protein is purified by sequential chromatography on hydroxyapatite (HA) and affinity columns (as specified by Upstate Biotechnology). PAK-1 PBD (RAC1 assay reagent, agarose for the pull-down of the activated RAC1) and monoclonal RAC1 antibody were obtained from Upstate Biotechnology (Lake Placid, NY). Rabbit antiserum (4301.3) raised against a CSK peptide fragment (31) was used for immunoblotting (1:1,000). Pansorbin was purchased from Calbiochem (La Jolla, CA). Nitroblue tetrazolium, 5-bromo-4-chloro-3-indolylphosphate (BCIP) p-toluidine salt, aprotinin, and bovine serum albumin (BSA) were obtained from Sigma (St Louis, MO). Geneticin (G418 sulfate) was procured from the Invitrogen Corporation (Carlsbad, CA). A protein assay kit was obtained from Bio-Rad (Hercules, CA). A chemiluminescence kit was obtained from Amersham Biosciences (Buckinghamshire, England). For immunofluorescence studies, rabbit polyclonal anti-RAR (C-19) and mouse monoclonal anti-c-SRC (H-12) antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), were used. For fluorescence visualization, rhodamine-conjugated goat anti-rabbit and fluorescein isothiocyanate-conjugated goat anti-mouse IgGs from Molecular Probes, Inc. (Eugene, OR), were used as secondary antibodies against RAR and c-SRC primary antibodies, respectively. DAPI (4',6'-diamidino-2-phenylindole) for counterstaining was also purchased from Molecular Probes, Inc. (Eugene, OR). SRC, FYN, and YES immunokinase assays were done using an SRC assay kit (catalog no. 17-131) from Upstate Biotechnology (Lake Placid, NY). Radioactive [-³²P]ATP (specific activity of 3,000 Ci/mmol) was purchased from Perkin-Elmer Life and Analytical Sciences (Boston, MA). Affi-Gel 15 (activated affinity medium) was bought from Bio-Rad Laboratories (Hercules, CA). Lipofectamine 2000 reagent was procured from Invitrogen Corporation (Life Technologies, Carlsbad, CA). Protein G-agarose Fast Flow beads were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-HA monoclonal antibody (HA.11, clone 16B12) and anti-HA polyclonal antibody (Y-11) were obtained from Covance, the Development Service Company (Berkeley, CA), and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. Anti-FLAG antibody (anti-FLAG-M2 monoclonal antibody peroxidase conjugate) and anti-FLAG polyclonal antibody were obtained from Sigma-Aldrich (St. Louis, MO). Protease inhibitor cocktail tablets were procured from Roche Diagnostics (Mannheim, Germany). Mutagenesis was carried out using a Stratagene (La Jolla, CA) QuikChange II XL site-directed mutagenesis kit.

Construction of plasmids. All plasmid constructions were prepared according to standard procedures. The sequences and orientations of inserted DNA fragments in plasmid constructs were verified by restriction enzyme analysis and automated standard DNA sequencing. The wild-type human RAR1 (kindly provided by Ron Evans) gene was amplified by PCR using two primers, 5'-GATCGCGGATCCATGTACCCATACGATGTTCCAGATTACGCTCTTGCGGCCACCAATAAGGAGCGACTC-3' and 5'-CTAGCGGAATCTCAGGCTGGGGACTTCAGGC-3' (with a 2x FLAG tag at the N terminus), and then subcloned into plasmid pcDNA3.1 between BamHI and EcoRI sites. Deletion of a proline-rich domain (from positions P75 to R85) in the N-terminal A/B domain of wild-type human RAR was done by site-directed mutagenesis (QuikChange II XL site-directed mutagenesis kit; Stratagene) using primers 5'-CAGCTCAGAGGAGATGGTGGTCTACAAGCCATGCTTCGTG-3'and 5'-CACAAGCATGGCTTGTAGACCACCATCTCCTCTGAGCTG-3' and confirmed by DNA sequencing (Davis Sequencing, Davis, CA). Chicken SRC (c-SRC) (from H. Fu) was in plasmid pCSA with an HA tag at the N terminus of the protein. A QuikChange II XL site-directed mutagenesis kit (Stratagene) was used to delete the proline-rich domain of RAR (11 amino acids from positions 75 to 85). Oligonucleotides used for deletion were 5'-CAGCTCAGAGGAGATGGTGGTCTACAAGCCATGCTTCGTG-3' and 5'-CACGAAGCATGGCTTGTAGACCACCATCTCCTCTGAGCTG-3'. The wild-type FLAG-RAR gene in the pcDNA3.1 vector was used as the template. DNA sequencing ensured the successful deletion of the domain at positions 75 to 85 (75-85) of RAR. A point mutation of SRC (Y527F) was made by using a QuikChange II XL site-directed mutagenesis kit according to the manufacturer's conditions. Oligonucleotides containing the mutation were designed according to the manufacturer's instructions, and they were 5'-GACAGAGCCCCAGTTCCAGCCTG GAGAGAACC-3' and 5'-GGTTCTCTCCAGGCTGGAACTGGGGCTCTGTC-3'. Wild-type HA-SRC in vector pCSA was used as the template. The mutation was confirmed by DNA sequencing (Davis Sequencing). The c-src gene was amplified by PCR using primers 5'-CATCGCGGATCCACTAGTAACGGCCGCCAG-3' with a BamHI site and 5'-GTCATGCCATGGCGAGGTTCTCTCCAGGCTG-3' with an NcoI site and subcloned into plasmid pcDNA3-EGFP (from our laboratory) between BamHI and NcoI sites with enhanced green fluorescent protein (EGFP) at the C-terminal end of c-SRC. For hRAR, PCR was carried out using primers 5'-GATCCGGAATTCCATGGCCACCAATAAGGAGCG-3', containing an EcoRI site, and 5'-CGGGATCCCCCGGGGAAATAAGTTAGCACAATCAT-3', containing a BamHI site. The amplified gene was then inserted into vector pDsRed2-C1 (Invitrogen) between EcoRI and BamHI sites with DsRed protein at the N terminus of hRAR. The integrity of all the constructs was confirmed by DNA sequencing (Davis Sequencing).

Cell culture and treatments. The human NB cell lines LA-N-5, LA-N-6, and SK-N-BE(2) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) (HyClone, UT) with 100 units/ml penicillin and streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Cells were treated with 10^–5 to 10^–6 M ATRA in 100% ethanol or dimethyl sulfoxide (DMSO) (vehicles for ATRA) under amber-light conditions (29, 30). Cells were pretreated with pan-SRC inhibitor PP1 at a final concentration of 4 µM for 1 h. Culture medium (containing PP1 and ATRA) was changed every 48 h. Similar culture conditions and treatment regimens were maintained for morphological studies and kinase assays.

Retroviral vectors and stable overexpression of wild-type CSK in LA-N-5, LA-N-6, and SK-N-BE(2) cells. Stable clones of NB cells overexpressing wild-type CSK were produced using a retroviral construct (pLXSN). Cells were plated in log phase at a density of 5 x 10⁶ cells per 10-cm dish. The fresh supernatants from stable virus producer PG13 cells were filtered (0.22 µm) and were used for infection. The infected cells were selected in G418 (1 mg/ml), and the expression levels of CSK in different clones derived from the bulk population were evaluated by Western blot analysis. The clones (5P, 10P, 15P, 8, 10, 1, and 2) were maintained in 300 to 500 µg/ml of G418 in cultures, and expression was confirmed by Western blot analysis before every experiment (data not shown).

Neurite outgrowth assay. For the neurite outgrowth assay, cells were seeded (3 x 10⁶ cells) in the above-described growth medium in a 10-cm-diameter tissue culture treated dish and allowed to attach for 24 h. The cells were then either treated with ATRA (10^–5 M) under amber-light conditions or left untreated in control medium (25 µl of 100% ethanol in 10 ml) as described elsewhere previously (30). The medium was changed every 48 h. Neurite-bearing cells were semiquantified morphologically after 7 to 9 days of culture after staining them with methylene blue or under a phase-contrast microscope (Nikon TMS inverted microscope using Kodak Ektachrome 100 film). Quantification of morphological differentiation was done on the basis of the number of cells showing outgrowth more than twice the diameter of the cell body. Ten independent frames were considered for determinations of statistical significance. Considering the typical networking pattern observed after the RA treatment, the outgrowths were measured as the percentage of cells showing the second/third degree of collaterals.

Immunoprecipitation and Western blot. LA-N-5 cells were solubilized with 500 µl of lysis buffer (150 mM NaCl, 6 mM Na₂HPO₄, 4 mM NaH₂PO₄, 2 mM EDTA, 1% sodium deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], 1% aprotinin, 0.2 M sodium orthovanadate, and 0.1 M phenylarsineoxide). For IP of SRC, YES, and FYN, clarified lysates were assayed for total protein (Bio-Rad protein assay kit) using BSA as a standard. The clear lysates were immunoprecipitated by specific antibodies (1 µg protein) for 2 h after protein equilibration (2 to 4 mg protein). Immunoprecipitates were bound to pansorbin and resolved by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE). Membranes were immunoblotted with anti-human ß-actin to confirm equal loading. Individual bands were visualized by an enhanced chemiluminescence reagent combined with peroxidase-conjugated anti-rabbit or anti-mouse IgG using BCIP (50 mg/ml) and nitroblue tetrazolium (50 mg/ml). The relative density of the bands was plotted in arbitrary units using ImageJ, version 1.32j (NIH).

RAC1 pull-down assay. The glutathione S-transferase (GST) fusion protein corresponding to the human PAK-1 p21 binding domain (PBD) (residues 67 to 150) was expressed in E. coli. The final protein products were bound to glutathione agarose in a liquid suspension containing 300 µg of PAK-1 PBD in 333 µl of 50% agarose slurry of 20 mM phosphate-buffered saline (PBS) (pH 7.4) containing 50% glycerol. The pull-down assay was carried out at different time points following ATRA treatment in controls and CSK-overexpressing clones (21). In short, ATRA-treated cells were lysed with extraction buffer (25 mM HEPES [pH 7.5], 150 mM NaCl, 1% Igepal CA630, 10 mM MgCl₂, 1 mM EDTA, 10% glycerol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM NaF-1 mM sodium orthovanadate), and following centrifugation, 10 µl of PAK-1 PBD (1 µg/µl) was added per sample of lysate and incubated for 45 min at 4°C. For the positive control, lysates of each clone were treated with 10 mM EDTA and 100 µM GTP-S and incubated for 15 min at 30°C. Before adding PAK-1 PBD, the reaction was stopped by adding 60 mM MgCl₂ to the mixture. Agarose beads were resuspended in 30 µl Laemmli sample buffer to resolve protein by 15% SDS-PAGE. The membranes were probed with monoclonal RAC1 (1:1,000) antibody. The bands were quantified by densitometry (Eagle Eye II-Still Video system; Stratagene). The amount of total RAC1 protein in each lysate was quantitated as an additional loading control. We carried out an earlier time course of activation of RAC1 following ATRA administration. Cells were treated with ATRA (10^–5 M) in the dark at different time points (5 min, 15 min, 30 min, 1 h, 3 h, and 6 h), and activated RAC1 was pulled down from the cell lysates as mentioned above.

SRC, FYN, and YES kinase assays. The phosphotransferase activity of cell lysates or specific immunoprecipitates (SRC, FYN, or YES) was determined in vitro using a cell-free system, i.e., an SRC assay kit from Upstate Biotechnology (Lake Placid, NY), according to the manufacturer's instructions, as described previously (21). The in vitro kinase assay employs an exogenous SRC substrate. The data are expressed as changes (n-fold) in c-SRC, FYN, and c-YES kinase activities in LA-N-5 cells under different experimental conditions. The immunoprecipitates were resolved on SDS-PAGE to quantify c-SRC, FYN, and YES protein levels in IPs. Each experimental point was performed in triplicates. The time course of activation of c-SRC following ATRA administration was studied in LA-N-5 cells. Cells were treated either with ethanol or DMSO (vehicles for ATRA) or with ATRA (10^–5 M) in the dark for different time points (5 min, 15 min, 30 min, 1 h, 3 h, and 6 h), and the in vitro kinase activity of c-SRC was determined from the immunoprecipitated c-SRC as mentioned above. In separate experiments, in order to test the effect of RA-dependent binding of RAR to c-SRC on the activation of c-SRC in LA-N-5 cells, we immunoprecipitated RAR and c-SRC from LA-N-5 cells and performed an in vitro SRC kinase assay in the presence and absence of ATRA (see Fig. 7A). Endogenous c-SRC and RAR from the normalized clear lysates of LA-N-5 cells (growing in medium containing 10% FBS) were immunoprecipitated separately using the respective antibodies (rabbit polyclonal antibody for c-SRC and rabbit polyclonal antibody for RAR). Individual immunoprecipitants were then used for the SRC kinase assay. An in vitro kinase assay for SRC was carried out according to the manufacturer's protocol, with little modification. In short, the pellets obtained following the washings in buffer following an hour of incubation with secondary antibodies (pansorbin) were washed (one time) in pan-kinase buffer [0.1 M NaCl, 1% (vol/vol) aprotinin, 10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), pH 7.0]. The pellets were then resuspended in SRC kinase reaction buffer (100 mM Tris-HCl [pH 7.2], 125 mM MgCl₂, 25 mM MnCl₂, 2 mM EGTA, 0.25 mM sodium orthovanadate, and 2 mM dithiothreitol). The reaction mixture for the in vitro SRC kinase assay contained SRC kinase reaction buffer, SRC substrate peptide (150 to 375 µM/assay), immunoprecipitated c-SRC from LA-N-5 cells, immunoprecipitated RAR from LA-N-5 cells, and [-³²P]ATP. Freshly made ATRA (where mentioned) was added to the reaction mixture under light-protected conditions. Following 30 min of incubation in the dark at 30°C (with agitation), the reaction was stopped by pulse centrifugation at 10,000 rpm. The supernatant was used for the subsequent procedures according to the manufacturer's protocol.

View larger version (22K): [in this window] [in a new window]   FIG. 7. Effect of ATRA-dependent binding of RAR to c-SRC from LA-N-5 cells on SRC kinase activity. (A) In vitro c-SRC kinase activity from LA-N-5 cells. RAR and c-SRC were immunoprecipitated (i.p.) from LA-N-5 cell lysates using polyclonal antibodies. The immunoprecipitants were incubated with and without ATRA (10–5 M) under subdued-light conditions. Phosphotransferase activity towards an SRC-specific peptide was determined from the reaction mixture by an in vitro kinase assay. In short, endogenous c-SRC and RAR from the normalized clear lysates of LA-N-5 cells were immunoprecipitated separately using their respective antibodies (rabbit polyclonal antibody for c-SRC and rabbit polyclonal antibody for RAR). Individual immunoprecipitants were then used for the SRC kinase assay. The in vitro kinase assay for SRC was carried out according to the manufacturer's protocol with little modification, as described in Materials and Methods. In short, the reaction mixture representing lane 1 contained SRC kinase reaction buffer, SRC substrate peptide, immunoprecipitated c-SRC from LA-N-5 cells, and [-32P]ATP. The reaction mixture representing lane 2 contained SRC kinase reaction buffer, SRC substrate peptide, immunoprecipitated c-SRC from LA-N-5 cells, [-32P]ATP, and freshly prepared ATRA (10–5 M final concentration). The reaction mixture representing lane 3 contained SRC kinase reaction buffer, SRC substrate peptide, immunoprecipitated c-SRC from LA-N-5 cells, immunoprecipitated RAR from LA-N-5 cells, and [-32P]ATP. The reaction mixture representing lane 4 contained SRC kinase reaction buffer, SRC substrate peptide, immunoprecipitated c-SRC from LA-N-5 cells, immunoprecipitated RAR from LA-N-5 cells, [-32P]ATP, and freshly prepared ATRA (10–5 M final concentration). Bars represent kinase activity from three to four individual experiments. *, P < 0.05. Data show that kinase activity in the reaction mixture containing immunoprecipitated c-SRC and RAR from LA-N-5 cells was significantly higher in the presence of ATRA (bar corresponding to lane 4) than in the nontreated control (bar corresponding to lane 3). The kinase activities from immunoprecipitated c-SRC treated with and without ATRA (bars corresponding to lanes 1 and 2, respectively) served as negative controls. Representative immunoblots for c-SRC and RAR are shown in the bottom panels. Positive controls (recombinant proteins) for c-SRC and RAR are shown in lane 5. (B) Effects of wild-type CSK and PP1 on kinase activity of immunoprecipitated c-SRC from LA-N-5 cells. Endogenous c-SRC and RAR from normalized cell lysates of LA-N-5 cells were immunoprecipitated separately using their respective antibodies (rabbit polyclonal antibody for c-SRC and rabbit polyclonal antibody for RAR). Individual immunoprecipitants were then used for the SRC kinase assay. In short, the reaction mixture representing all the lanes (lanes 1 to 3) contained SRC kinase reaction buffer, SRC substrate peptide, immunoprecipitated c-SRC from LA-N-5 cells, immunoprecipitated RAR from LA-N-5 cells, [-32P]ATP, and freshly prepared ATRA (10–5 M final concentration). Immunoprecipitated c-SRC from control (LXSN) LA-N-5 cells (lane 1) and a clone (5P) of LA-N-5 cells overexpressing wild-type CSK (lane 2) were incubated in the presence of RAR with ATRA (10–5 M) under subdued-light conditions. Immunoprecipitated c-SRC in the presence of PP1 (4 µM) was also incubated as described above (lane 3). Phosphotransferase activity towards an SRC-specific peptide was determined from the reaction mixture by an in vitro kinase assay as described in Materials and Methods. Bars represent kinase activities from three to four individual experiments. *, P < 0.005. Data show that kinase activities of immunoprecipitated c-SRC from CSK-overexpressing LA-N-5 cells and, under conditions of PP1 treatment, were significantly inhibited (bars corresponding to lanes 2 and 3, respectively) compared to the nontreated vector control (bar corresponding to lane 1).

Confocal microscopy. The colocalization of c-SRC and RAR was studied from the pattern of distribution of exogenously expressed c-SRC and RAR in HEK293 cells. The transient expression of EGFP-tagged c-SRC and red fluorescent protein (RFP)-tagged RAR was carried out in HEK293 cells for these colocalization studies. In short, exponentially growing HEK293 cells were plated onto 12-mm glass coverslips (Fisher Scientific, Pittsburgh, PA) in six-well plates (four coverslips per well), and cells were allowed to attach overnight in medium containing 10% FBS. The following day, cells were either transiently transfected with EGFP-tagged c-SRC (0.8 µg) or RFP-tagged RAR (0.8 µg) or cotransfected with both EGFP-tagged c-SRC (0.4 µg) and RFP-tagged RAR (0.4 µg) using Lipofectamine 2000 reagent according to the manufacturer's protocol. Twenty-four hours after transfection, cotransfected cells were treated with either ATRA (10^–5 M) or DMSO (vehicle of ATRA) for 1 h under subdued-light conditions. Cells were fixed with warm PHEMO buffer (0.068 M PIPES, 0.025 M HEPES, 0.015 M EGTA, 0.003 M MgCl₂, 10% DMSO [pH 6.8]) containing 3.7% formaldehyde, 0.05% glutaraldehyde, and 0.5% Triton X-100 for 10 min at room temperature following a warm PBS wash. Coverslips were then washed three times in PBS for 5 min and mounted onto glass slides using Gel Mount mounting medium (Biomeda Corp., Foster City, CA). Cells were imaged using a Zeiss (Thornwood, NY) LSM 510 Meta confocal microscope with a 63x (1.4-numerical-aperture) or 100x (1.4-numerical-aperture) Plan-Apochromat oil objective. All images were acquired using Zeiss LSM 510 software and processed using Adobe Photoshop 7.0.

Interactions between RAR and c-SRC by SPR. A surface plasmon resonance (SPR)-based biosensor system, the BIAcore (Uppsala, Sweden) 3000 system, was used to measure the kinetic parameters for the interactions between soluble recombinant RAR protein (analyte) and the immobilized recombinant His-tagged SRC protein (ligand). The binding of RAR recombinant protein in the presence of ATRA to SRC was monitored in real time as described previously (71). Briefly, His-tagged SRC (9.23 nM) was covalently linked to the surface of a research-grade CM5 sensor chip via an amine-coupling reaction according to the manufacturer's instructions (BIAcore handbook), yielding a resonance signal of 200 resonance units (RU). One flow cell was intentionally left underivatized to allow for corrections for refractive index changes. The binding of RAR (1.85 nM) to immobilized SRC in the presence or absence of ATRA (20 µM) on the biosensor surface was determined by the change in the RU using the KINJECT function of the BIAcore control software (flow rate of 30 µl/min, with 3 min for association and 5 min for dissociation). A schematic representation of the sensor chip surface is shown in Fig. 6A (i, bottom). First, we tested the binding capacity of the individual components alone (see Fig. 6Ai). Different concentrations of ATRA were then titrated against the RAR concentration to determine if ATRA could induce the binding of RAR to c-SRC (see Fig. 6Aii). Finally, we examined the effect of free recombinant SRC protein on the binding of RAR to the immobilized SRC protein (see Fig. 6Aiii). We then tested the specificity of RAR binding to SRC in the presence of 9-cis-RA (20 µM), ATRA (20 µM), or 13-cis-RA (20 µM) (see Fig. 6Aiii). Each experiment was repeated at least twice to ensure reproducibility. The data analysis was performed using Bia-evaluation software supplied by the vendor.

View larger version (56K): [in this window] [in a new window]   FIG. 6. Ligand-dependent binding of RAR and c-SRC in vitro. (A) Real-time binding of RAR and c-SRC in the presence of ATRA (RA). The interactions between RAR and c-SRC were measured by SPR. (i) Interactions between immobilized c-SRC and individual analyte, respectively, are shown in sensorgram. A schematic representation of the sensor chip surface is represented below the sensorgram. Purified recombinant RAR (1.85 nM) and other analytes (as shown in the figure) were injected over the sensor chip coated with c-SRC as described in Materials and Methods. ATRA (RA), 9-cis-RA, 13-cis-RA, and RAR alone did not interact with SRC efficiently. (ii) When 20 µM of ATRA was mixed with 1.85 nM of RAR, a significant binding was observed. (iii) No significant binding between RAR and c-SRC was observed when ATRA was replaced by 9-cis-RA or 13-cis-RA. The specificity of the binding was detected by adding recombinant c-SRC (9.25 nM) in the analyte solution to compete for binding of RAR. (B) Binding of RAR and c-SRC in the presence of ATRA using Affi-Gel 15 in vitro. Affi-Gel beads coated with c-SRC (inset) were incubated in the presence of RAR with (lanes 7, 8, and 9) and without (lanes 4, 5, and 6) ATRA in the dark at 37°C. Following the reaction, samples were immunoblotted for RAR. Data show that the binding of RAR and c-SRC occurs in the presence of ATRA (lanes 7, 8, and 9) compared to untreated controls (lanes 4, 5, and 6). Densitometry evaluation showed a six- to eightfold increase (*, P < 0.0005) in the binding in the presence of ATRA (lanes 7, 8, and 9) compared to that in the absence of ATRA (lanes 4, 5, and 6), as shown in the RAR immunoblot. Uncoated Affi-Gel beads plus RAR with (lane 2) and without (lane1) blocking and c-SRC-coated beads with blocking (lane 3) served as negative controls. Recombinant (Recomb.) RAR was run as a positive control (lane 10). The coating of c-SRC on Affi-Gel beads was confirmed by running an immunoblot for c-SRC (inset) from the beads after coating (lane 11 of the inset) compared to the uncoated beads (lane 12 of the inset). No binding of RAR to unconjugated Affi-Gel beads was observed in the presence of ATRA. Recombinant c-SRC (lane 13 of the inset) was used as a positive control. (C) Binding of RAR to c-SRC in vivo. (i) Expression of FLAG-tagged RAR and HA-tagged c-SRC in HEK293 cells. HEK293 cells were either transiently cotransfected with FLAG-tagged RAR (0.2 µg or 0.4 µg) and HA-tagged c-SRC (0.2 µg or 0.4 µg) (lanes 2 and 3) or transfected separately with HA-tagged c-SRC (0.8 µg) (lane 4) and FLAG-tagged RAR (0.8 µg) (lane 5). Whole-cell extracts (250 µg) obtained 24 h after transfection were resolved by 10% SDS-PAGE and immunoblotted (IB) with anti-FLAG antibody (blot in top panel) and anti-HA antibody (blot in bottom panel). Lysates from mock-transfected (pcDNA3.1 for FLAG-tagged RAR and pCSA for HA-tagged c-SRC) HEK293 cells (lane 1) served as negative controls. Data show comparable amounts of expression of FLAG-tagged RAR and HA-tagged c-SRC following transfection (0.8 µg) and cotransfection (0.4 µg as shown in lane 2 and 0.8 µg as shown in lane 3) of the respective DNAs. (ii) RAR coimmunoprecipitates with c-SRC in cotransfected HEK293 cells. HEK293 cells were either transiently cotransfected with FLAG-tagged RAR (0.4 µg) and HA-tagged c-SRC (0.4 µg) (lanes 1, 3, and 8) or transfected separately with FLAG-tagged RAR (0.8 µg) (lanes 5, 10, and 12) and HA-tagged c-SRC (0.8 µg) (lanes 4 and 9). Whole-cell extracts (250 µg of protein) were incubated with anti-HA monoclonal antibody and then incubated with protein G-agarose beads. Preimmune control (C) for IP was performed by adding mouse IgG to cell lysates from cotransfected cells (lane 1). Immune complexes were resolved by 10% SDS-PAGE and immunoblotted with anti-FLAG antibody (blot in the top panel) and anti-HA antibody (blot in the bottom panel). Lysates from HEK293 cells (lane 6) and mock-transfected (0.4 µg of pcDNA3.1 and 0.4 µg of pCSA) HEK293 cells (lane 2) served as internal negative controls for IP. Expression levels of proteins (FLAG-tagged RAR and HA-tagged c-SRC) in the lysates (used for IP) were tested in the same gel (lanes 7 to 11). Lanes 7 and 11 represent lysates from mock-transfected HEK 293 cells and nontransfected HEK293 cells, respectively. Lysates from cells transfected with FLAG-tagged deletion mutations (amino acids 75 to 85 [75-85 RAR]) of RAR were included as internal controls (lane 12). (iii) c-SRC coimmunoprecipitates with RAR in cotransfected HEK293 cells. HEK293 cells were either transiently cotransfected with FLAG-tagged RAR (0.4 µg) and HA-tagged c-SRC (0.4 µg) (lanes 1, 3, 7, and 10) or transfected separately with FLAG-tagged RAR (0.8 µg) (lanes 4 and 8) and HA-tagged c-SRC (0.8 µg) (lanes 5, 9, and 11). Whole-cell extracts (250 µg of protein) were incubated with anti-FLAG polyclonal antibody and then incubated with pansorbin. Preimmune control (C) for IP was performed by adding rabbit IgG to cell lysates from cotransfected cells (lane 1). Immune complexes were resolved by 10% SDS-PAGE and immunoblotted with anti-HA monoclonal antibody (top blot) and anti-FLAG monoclonal antibody (bottom blot). Lysates from mock-transfected (0.4 µg of pcDNA3.1 and 0.4 µg of pCSA) HEK293 cells (lanes 2 and 6) served as internal negative controls. Expression levels of proteins (FLAG-tagged RAR and HA-tagged c-SRC) in the lysates (used for IP) were tested in the same gel (lanes 7 to 9). Lysates obtained from different batches of transfected cells expressing HA-tagged c-SRC and FLAG-tagged deletion mutations (amino acids 75 to 85 [75-85 RAR]) of RAR were included as internal controls (lanes 11 and 12, respectively). (iv) The proline-rich domain-deleted mutant (75-85) of RAR does not coimmunoprecipitate with c-SRC in cotransfected HEK293 cells. HEK293 cells were either transiently cotransfected with FLAG-tagged RAR (0.4 µg) and HA-tagged Y527-SRC (0.4 µg) (lanes 3 and 8 of the top panel); cotransfected with FLAG-tagged RAR (0.4 µg) and HA-tagged wild-type c-SRC (0.4 µg) (lane 4 of the top panel), with the FLAG-tagged 75-85 mutant of RAR (0.4 µg) and HA-tagged Y527-SRC (0.4 µg) (lane 5 of the top panel), or with the FLAG-tagged 75-85 mutant of RAR (0.4 µg) and HA-tagged wild-type (WT) c-SRC (0.4 µg) (lane 6 of the top panel); or transfected separately with HA-tagged Y527-SRC (0.8 µg) (lane 9 of the top panel), with FLAG-tagged wild-type RAR (0.8 µg) (lane 10 of the top panel), with HA-tagged wild-type c-SRC (0.8 µg) (lane 11 of the top panel), or with the FLAG-tagged 75-85 mutant of RAR (0.8 µg) (lane 12 of the top panel). For IP experiments, whole-cell lysates (250 µg of protein) from the transfected cells were first incubated with anti-HA monoclonal antibody and then transfected with protein G-agarose beads as mentioned in Materials and Methods. The preimmune control (C) experiment for IP was performed by adding mouse IgG to cell lysates from cotransfected cells (lane 1). Immune complexes were resolved by 10% SDS-PAGE and immunoblotted with anti-FLAG antibody (top blot) and anti-HA antibody (middle blot). The middle panel shows the expression of HA-tagged c-SRC and HA-tagged Y527-SRC (lanes 3, 4, 5, 6, 8, 9, and 11 of the middle panel) in both immunoprecipitants and lysates. Lysates from the mock-transfected (0.4 µg of pcDNA3.1 and 0.4 µg of pCSA) HEK293 cells (lane 2) served as internal negative controls for IP. Lane 7 represents lysate from mock-transfected HEK293 cells. Lysates from cells cotransfected with FLAG-tagged RAR and HA-tagged Y527-SRC were included as an internal control (lane 8 of the top panel). Expression of proteins (FLAG-tagged RAR, FLAG-tagged 75-85 mutant of RAR, HA-tagged c-SRC, and HA-tagged Y527-SRC) in cell lysates was tested in the same gel (lanes 9 to 12 of the top panel). The bottom blot shows the expression of FLAG-tagged RARs (wild-type protein as shown in lanes 3 and 4 as well as mutated protein as shown in lanes 5 and 6, respectively) in cell lysates that were used for the IP studies. (v) Colocalization of exogenous EGFP-tagged c-SRC with RFP-tagged RAR in the cytosol of HEK293 cells. HEK293 cells were transiently transfected together (photomicrographs in the bottom panel) or separately (photomicrographs in the top panel) with EGFP-tagged c-SRC and/or RFP-tagged RAR, respectively, as described in Materials and Methods. Cotransfected cells were treated with either ATRA (10–5 M) or vehicle for ATRA (DMSO) for 1 h under subdued-light conditions. Fixed cells were processed for confocal imaging. Photomicrographs in the top panel show the subcellular distribution of c-SRC (images a and c) and RAR (images d and f) in HEK293 cells that were transfected with EGFP-tagged c-SRC (images a, b, and c) and RFP-tagged RAR (images d, e, and f) along with their differential interference contrast (DIC) images (images b and e) and their merged confocal images (images c and f), respectively. Scale bar, 10 µm. Photomicrographs in the bottom panel show the colocalization of c-SRC and RAR in the cytosol of untreated (images a, b, and c) and ATRA-treated (images d, e, and f) HEK293 cells following cotransfection of EGFP-tagged c-SRC and RFP-tagged RAR. Merged images of EGFP-tagged c-SRC and RFP-tagged RAR from both untreated (images a and b are merged to form image c) and treated (images d and e are merged to form image f) groups show a clear change in color, indicating the colocalization of these two proteins in the cytosol of the cells. Arrowheads represent the characteristic "edge"-like c-SRC-rich adhesion structures along the plasma membrane that were observed in 100% of ATRA-treated cells. Scale bar, 10 µm. Results show (i) an exclusive cytoplasmic distribution of c-SRC, (ii) both nuclear and cytoplasmic distribution of RAR, and (iii) cytoplasmic colocalization of c-SRC and RAR in untreated and ATRA-treated HEK293 cells.

Binding of RAR to immobilized (linked to Affi-Gel 15) c-SRC.

Transient transfection and coimmunoprecipitation. HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The cells in 60-mm tissue culture dishes were either transiently cotransfected with FLAG-RAR (0.2 µg to 0.4 µg) and HA-c-SRC (0.2 µg-0.4 µg) plasmid DNAs or transfected separately with HA-c-SRC (0.8 µg) and FLAG-RAR (0.8 µg) plasmid DNAs. For coimmunoprecipitation experiments, cotransfections and transfections were done with 0.8 µg of plasmid DNA using Lipofectamine 2000 reagent according to the manufacturer's protocol. Empty vectors (0.8 µg) were used for mock transfections. Whole-cell extracts were prepared in lysis buffer (50 mM Tris-HCl [pH 8], 0.05% NP-40, 100 mM NaF, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.08 mM phenylmethylsulfonyl fluoride, 0.01 mg/ml leupeptin, 0.01 mg/ml aprotinin, and protease inhibitor cocktail tablet) after 24 h of transfection. Where mentioned, IP was performed on normalized lysates from the transfected cells by incubating them first with anti-HA antibody (monoclonal HA.11 antibody, clone 16B12) overnight at 4°C and then with protein G-agarose continuously for 1 h. Proteins with or without prior IP were resolved by 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and immunoprobed (immunoblotted) separately by anti-HA (anti-HA polyclonal antibody [1:1,000 dilution]) and anti-FLAG (anti-FLAG-M2 monoclonal antibody [1:1,000 dilution]) antibodies. FLAG-tagged proteins in normalized lysates from a parallel set of transfected cells were immunoprecipitated using anti-FLAG polyclonal antibody. Lysates were incubated first with anti-FLAG antibody (overnight at 4°C) and then with pansorbin (for 1 h). Resolved proteins were immunoblotted separately by anti-HA (anti-HA monoclonal antibody) and anti-FLAG (anti-FLAG monoclonal antibody) antibodies. Chemiluminescence was detected by standard enhanced chemiluminescence, Western blotting detection reagents, and an analysis system according to the protocol of Amersham Biosciences (see Fig. 6C). To strengthen our claim that RAR binds c-SRC in vivo, we generated a deletion mutant of the wild-type RAR protein (75-85) and tested the binding of this mutant to wild-type c-SRC by coimmunoprecipitation studies. HEK293 cells were transiently transfected with the FLAG-tagged 75-85 mutant of RAR and HA-tagged wild-type c-SRC plasmids. In a separate experiment, HA-tagged Y⁵²⁷-SRC was cotransfected with FLAG-tagged wild-type RAR plasmids. IPs of the normalized lysates from transfected cells were carried out using anti-HA antibody (monoclonal HA.11, clone 16B12) overnight at 4°C as mentioned above. Resolved proteins were immunoblotted separately by anti-FLAG (anti-FLAG-M2 monoclonal antibody [1:1,000 dilution]) and anti-HA (anti-HA polyclonal antibody [1:1,000 dilution]) antibodies. The binding of the FLAG-tagged 75-85 mutant of RAR and HA-tagged wild-type c-SRC was compared with the binding of FLAG-tagged wild-type RAR and HA-tagged wild-type c-SRC. Similarly, the binding of HA-tagged Y⁵²⁷-SRC and FLAG-tagged wild-type RAR was compared with the binding of HA-tagged Y⁵²⁷-SRC and the FLAG-tagged 75-85 mutant of RAR.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effects of ATRA on NB cells. ATRA treatment caused the characteristic growth inhibition and differentiation in all the NB cell lines tested. ATRA-induced differentiation in LA-N-5, LA-N-6, and SK-N-BE(2) cells showed the characteristic neurite outgrowth as shown in Fig. 1 and 2. Typically, cells extend axon-like processes by day 4 and morphologically resemble a tightly knit web (Fig. 2C). By day 7, cells formed a typical network of cellular extensions involving second/third-degree processes. This effect was found to be dose dependent from 10^–5 M to10^–7 M and was appreciable from days 5 to 7 of treatment, reaching a maximum at around days 10 to 11. Flow cytometry analyses in LA-N-5 cells showed a steady increase in the percentage of cells in G₀/G₁ phase from the 5th day (112% of the control) to the 11th day (128% of the control) of ATRA treatment (data not shown). This increase was associated with the concomitant induction of p27^kip1 expression and cell counts (data not shown). Interestingly, different NB cell lines showed the characteristics response to ATRA in terms of the degree of neurite outgrowth, and out of three cell lines tested, LA-N-5 cells were found to be the most sensitive to ATRA treatment (Fig. 1).

View larger version (16K): [in this window] [in a new window]   FIG. 1. Effect of pan-SFK inhibitor PP1 on ATRA-induced neurite outgrowth in NB cell lines. Neurite outgrowth was semiquantified from the morphological changes in LA-N-5, LA-N-6, and SK-N-BE(2) cells treated with ATRA (10–5 M) for 7 to 9 days. PP1 (4 µM) was added 1 h prior to ATRA administration. Each bar represents the percentage of cells (out of 10 randomly chosen fields) showing neurite outgrowth at the end of the treatment. Vehicle treatment served as the control. PP1 alone did not show any neurite outgrowth. *, P < 0.001 (n = 5). Data show that PP1 blocks ATRA-induced neurite outgrowth in NB cells.

View larger version (52K): [in this window] [in a new window]   FIG. 2. Overexpression of wild-type CSK and its effect on ATRA-induced neurite outgrowth in LA-N-5, LA-N-6, and SK-N-BE(2) cells. Overexpression (top panels) of wild-type CSK in LA-N-5, LA-N-6, and SK-N-BE(2) cells was determined by Western blot analysis. Bulk populations and individual clones of CSK-overexpressing cells were selected from parental LA-N-5 (A), LA-N-6 (B), and SK-N-BE(2) (C) cells infected with empty vectors (LXSN) and wild-type CSK. Clear lysates were resolved by 10% SDS-PAGE and probed with CSK antibody. Human ß-actin was run for the loading control. Bar diagrams in the top panels show the relative densities of protein bands in arbitrary units. Data show that all the clones of LA-N-5, LA-N-6, and SK-N-BE(2) cells have significantly higher levels of wild-type CSK than their respective vectors and wild-type controls. (A) Levels of expression of CSK in LA-N-5 clones 5P, 10P, and 15P (lanes 3, 4, and 5, respectively) are compared to the endogenous levels of CSK in the empty vector (LXSN)-infected cell line (lane 2) and the wild-type (WT) cell line (lane 1). (B) Levels of expression of CSK in LA-N-6 clones 8 and 10 (lanes 3 and 4, respectively) are compared to the endogenous levels of CSK in the empty vector (LXSN)-infected cell line (lane 2) and the wild-type cell line (lane 1). (C) Levels of expression of CSK in SK-N-BE(2) clones 1 and 2 (lanes 3 and 4, respectively) are compared to the endogenous levels of CSK in the empty vector (LXSN)-infected cell line (lane 2) and the wild-type cell line (lane 1). Morphological changes were observed in LA-N-5, LA-N-6, and SK-N-BE(2) cells treated with ATRA (10–5 M) for 7 to 9 days as mentioned in Materials and Methods. The neurite outgrowth response was examined morphologically and semiquantified (bottom panels). NB cell lines were plated and grown (3 x 106 cells) for 1 day, the media were removed, and differentiating media were added. The media were changed every 2 days. Differentiated cells were stained with methylene blue dye for morphological/semiquantification studies. Quantifications of neuritogenic responses to ATRA (10–5 M) for 7 to 9 days, as mentioned in Materials and Methods (bottom panels), are shown for LA-N-5 cells (parental cell line and empty vector control were compared with CSK-expressing clones 5P, 10P, and 15P) (A), LA-N-6 cells (parental cell line and empty vector control were compared with CSK-expressing clones 8 and 10) (B), and SK-N-BE(2) cells (parental cell line and empty vector control were compared with CSK-expressing clones 1 and 2) (C). Bars represent means ± standard deviations. *, P < 0.001 (n = 4). Representative photomicrographs of the neuritogenic responses to ATRA in SK-N-BE(2) cells (clone 1 is compared to vector control) are shown in C (right panel). The figure shows that the overexpression of wild-type CSK blocked ATRA-induced neurite outgrowth in LA-N-5, LA-N-6, and SK-N-BE(2) cells.

Stable overexpression of wild-type CSK in LA-N-5, LA-N-6, and SK-N-BE(2) cells. Recently, we reported the negative role of CSK in the TrkA-mediated neural differentiation of PC12 cells (21). Since CSK negatively regulated SFKs in the PC12 system, we overexpressed wild-type CSK in LA-N-5, LA-N-6, and SK-N-BE(2) cells (Fig. 2). Stable clones of LA-N-5 cells (clones 5P, 10P, and 15P as shown in Fig. 2, lanes 3, 4, and 5, respectively, compared to the wild type and vector control in lanes 1 and 2, respectively), LA-N-6 cells (clones 8 and 10 as shown in lanes 3 and 4, respectively, compared to the wild type and vector control in lanes 1 and 2, respectively), and SK-N-BE(2) cells (clones 1 and 2 as shown in lanes 3 and 4, respectively, compared to the wild type and vector control in lanes 1 and 2, respectively) overexpressed wild-type CSK as shown in the upper panels of Fig. 2A, B, and C, respectively. Data show a three- to fourfold (densitometry) increase in the expression of CSK in different clones (upper bar diagrams of Fig. 2A, B, and C) of LA-N-5, LA-N-6, and SK-N-BE(2) cells compared to the endogenous levels of their respective vector controls.

Effects of wild-type CSK overexpression on ATRA-induced neurite outgrowth in NB cells. In order to test the effect of the physiological inhibition of SFKs on ATRA-induced neurite outgrowth in NB, we treated the clones of LA-N-5, LA-N-6, and SK-N-BE(2) cells overexpressing CSK with ATRA and semiquantified their neuritogenic response. Figure 2A, B, and C show the effects of CSK on ATRA-induced neurite outgrowth in LA-N-5, LA-N-6, and SK-N-BE(2) cells, respectively. ATRA-induced neuritogenesis was significantly blocked by the overexpression of wild-type CSK compared to the empty vector controls (pLXSN) in all three NB cell lines tested. Although the neuritogenic response to ATRA varied between the cell lines, the effect of CSK was found to be uniformly inhibitory in the three different cell lines.

Effects of PP1 treatment and CSK overexpression on the kinase activities of SFKs in LA-N-5 cells. To determine the specificity of PP1 and wild-type CSK action in NB cells, we examined their effect on the kinase activities of the members of SFKs that are present in LA-N-5 cells. Figure 3A shows the kinase activities of three ubiquitously expressed members of SFKs in LA-N-5 cells under regular culture conditions as described in Materials and Methods. Treatment of PP1 (4 µM) blocked both c-SRC and FYN kinase activity in these cells compared to untreated controls (Fig. 3B). Similarly, the overexpression of CSK inhibited c-SRC and FYN kinase activity compared to vector controls (Fig. 3C). Our data demonstrate that the inhibitory effect of CSK on the kinase activity of c-SRC was more pronounced than that on the kinase activity of FYN in LA-N-5 cells. Anti-c-SRC (lanes 1 and 2) and anti-FYN (lanes 3 and 4) immunoblots corresponding to effects of PP1 and CSK were shown below the bars representing their respective kinase activities in Fig. 3B and C, respectively. Results indicate that treatment with a pharmacological inhibitor (PP1) and the overexpression of a physiological inhibitor of SFKs (CSK) reduced c-SRC and FYN kinase activities in LA-N-5 cells. Importantly, the inhibition of SFKs under these conditions corresponded with the blockade of ATRA-induced neurite outgrowth in LA-N-5 cells (Fig. 1 and 2A). In order to understand the role of SRC in retinoid signaling, we determined the time course of activation of c-SRC following ATRA administration in LA-N-5 cells. Results show that ATRA causes a transient activation of c-SRC (around a twofold increase in kinase activity compared to the untreated control) within 15 min of the treatment, which lasted for a total of 60 min (Fig. 3D).

View larger version (49K): [in this window] [in a new window]   FIG. 3. Effects of pan-SFK inhibitor PP1 or wild-type CSK overexpression on activity of the src family kinases c-SRC and FYN in LA-N-5 cells. Phosphotransferase activity towards an SRC-specific peptide was determined by IP of c-SRC, FYN, and c-YES from 100 µg of protein derived from LA-N-5 cells followed by an in vitro kinase assay as described in Materials and Methods. (A) Kinase activities of different members of SFKs in LA-N-5 cells. Bars represent changes in kinase activities from cpm values from three to four individual experiments. The changes in kinase activities were compared to the kinase activity of c-SRC (kinase activity of c-SRC represents "onefold"). *, P < 0.005. (B) Effect of PP1 treatment on c-SRC and FYN kinase activities in LA-N-5 cells. Kinase activities of c-SRC and FYN were determined from PP1 (4 µM for 1 h)-treated LA-N-5 cell lysates. Bars represent changes in kinase activities from cpm values from three to four individual experiments. **, P < 0.005; *, P < 0.05. Representative immunoblots for c-SRC and FYN are shown in the bottom panels. (C) Effect of overexpression of wild-type CSK on c-SRC and FYN kinase activities in LA-N-5 cells. Kinase activities of c-SRC and FYN were determined from the stable clone (5P) of LA-N-5 cells overexpressing wild-type CSK and compared with those of the vector control (LXSN). Bars represent changes in kinase activities from cpm values from three to four individual experiments. **, P < 0.0001; *, P < 0.005. Representative immunoblots for c-SRC and FYN are shown in the bottom panels. (D) Time course of activation of c-SRC in LA-N-5 cells following administration of ATRA. Wild-type LA-N-5 cells were treated with or without ATRA (10–5 M) under subdued-light conditions. Kinase activities of c-SRC were determined from the cell lysates following 5 min (lane 2), 15 min (lane 3), 30 min (lane 4), 60 min (lane 5), 180 min (lane 6), and 360 min (lane 7) of ATRA administration as described in Materials and Methods. Lane 1 represents the kinase activity from nontreated cells. Bars represent kinase activities from three to four individual experiments. *, P < 0.005. A representative immunoblot for c-SRC is shown in the bottom panel.

View larger version (46K): [in this window] [in a new window]   FIG. 4. Effects of CSK overexpression on ATRA (RA)-induced activation of RAC1 in LA-N-5 cells. (A) Activation of RAC1 in ATRA-stimulated LA-N-5 cells. Wild-type LA-N-5 cells were treated with or without ATRA (10–5 M) under subdued-light conditions. The conversion of GDP-RAC1 to GTP-RAC1 was determined at 24, 48, and 72 h of ATRA administration using GST fusion proteins representing the GTP-RAC1 binding CRIB domain of the PAK-1 kinase as described in Materials and Methods. The membranes were immunoblotted for RAC1. Total RAC1 was immunoblotted for loading controls (blot in the bottom panel). Lane 5 represents the positive control. Densitometry scanning analyses (bar diagrams in the top panel) of GTP-RAC1 with or without ATRA show that ATRA administration causes the activation of RAC1 in LA-N-5 cells compared to the 48-h nontreated control. (B) Overexpression of CSK abrogates ATRA-induced RAC1 activation in LA-N-5 cells. Stable clones of LA-N-5 cells overexpressing wild-type CSK (clones 5P, 10P, and 15P) were treated with ATRA. After 48 h, lysates were evaluated by pull-down assay for the detection of activated GTP-bound RAC1 as described in Materials and Methods. The activation of RAC1 after 48 h of ATRA treatment in the empty vector control (LXSN as in lane 2) was compared with that of wild-type CSK-overexpressing clones (clones 5P, 10P, and 15P in lanes 3, 4, and 5, respectively). Total RAC1 was immunoblotted for loading controls (blot in bottom panel). Densitometry analyses (bar diagrams in top panel) of the GTP-RAC1 blot show that CSK overexpression blocked ATRA-induced activation of RAC1 in LA-N-5 cells. Both lanes 1 and 6 are positive controls. Lane 1 represents the positive control for activated RAC1 using the lysates of LA-N-5 cells (lysates treated with GTP-S according to the manufacturer's protocol). Lane 6 represents the positive control for endogenous RAC1 protein in LA-N-5 cells (whole-cell lysates from LA-N-5 cells). For positive controls, lysates were treated with 100 µM GTP-S at 30°C for 15 min before the addition of PAK-1 PBD glutathione agarose conjugate. (C) Time course of activation of RAC1 following ATRA administration in LA-N-5 cells. Wild-type LA-N-5 cells were treated with or without ATRA (10–5 M) under subdued-light conditions. Conversion of GDP-RAC1 to GTP-RAC1 was determined at 5 min (lane 2), 15 min (lane 3), 30 min (lane 4), 60 min (lane 5), 180 min (lane 6), and 360 min (lane 7) after ATRA administration using GST fusion proteins representing the GTP-RAC1 binding CRIB domain of PAK-1 kinase as described in Materials and Methods. Membranes were immunoblotted for RAC1. Lane 1 represents nontreated cells. Lanes 2 to 7 represent ATRA-treated cells. Lane 8 represents the positive control (as mentioned above). Total RAC1 was immunoblotted for loading controls (blot in the bottom panel). Results show that the activation of RAC1 in LA-N-5 cells occurs within 15 min of administration of ATRA.

Coimmunolocalization of RAR and c-SRC in LA-N-5 cells.

View larger version (39K): [in this window] [in a new window]   FIG. 5. Immunolocalization of RAR and its colocalization with c-SRC in the cytoplasm of LA-N-5 cells. Cytoplasmic coimmunolocalization of RAR and c-SRC in LA-N-5 cells was determined by double immunofluorescence of RAR and c-SRC in LA-N-5 cells using mouse monoclonal antibody against c-SRC (1:50) and rabbit polyclonal antibody against RAR (1:50). Methanol-fixed LA-N-5 cells were stained with primary antibodies specific for RAR and c-SRC. Signals were visualized with secondary antibody conjugated to rhodamine for RAR and fluorescein isothiocyanate for c-SRC as described in Materials and Methods. Nuclei were counterstained with DAPI. Negative controls (CON and DAPI) were prepared by incubating the cells with secondary antibody only and secondary antibody plus DAPI, respectively. Merges of RAR with DAPI (RAR + DAPI) and c-SRC with DAPI (c-SRC + DAPI) were obtained using the Spot Advanced program. The superimposition of the images of RAR and DAPI and c-SRC and DAPI images (RAR c-SRC merge) shows a cytoplasmic coimmunolocalization (arrows) of the two proteins.

Real-time binding of RAR and c-SRC protein by SPR.

Ligand-dependent association of RAR with c-SRC linked to Affi-Gel 15. In order to study the binding interaction between RAR and c-SRC, we used Affi-Gel beads bound to c-SRC. Figure 6B shows the RAR immunoblot for the reaction mixtures containing RAR recombinant protein incubated with c-SRC linked to Affi-Gel 15 beads in the presence (lanes 7, 8, and 9) or absence (lanes 4, 5, and 6) of ATRA. Washed precipitates run on an SDS-PAGE gel were blotted for RAR with recombinant RAR as a positive control (lane 10). Beads alone with (Fig. 6B, lane 2) or without (lane 1) blocking were incubated with RAR as negative controls. Recombinant c-SRC-coated and blocked beads (lane 3) also served as negative controls. RAR binding to c-SRC-coated and blocked beads in the presence of ATRA was significantly higher (Fig. 6B, lanes 7, 8, and 9) than that in the absence of ATRA (Fig. 6B, lanes 4, 5, and 6). Densitometry evaluation showed a six- to eightfold increase (P < 0.0005) of binding in the presence of ATRA compared to that in the absence of ATRA as represented in the RAR immunoblot. Figure 6B (inset) shows a Western blot of beads conjugated in the presence of recombinant SRC (lane 11) and conjugated in the absence of SRC (lane 12) or recombinant c-SRC loaded onto the gel as a positive control (lane 13). As a control, no binding was observed between RAR and unconjugated Affi-Gel beads in the presence of ATRA. The combined results demonstrate a direct RA ligand-dependent induction for RAR binding to c-SRC.

In vivo binding of RAR with c-SRC. Next, we sought to determine if SRC can bind to RAR in vivo. Full-length human RAR (FLAG tagged) and c-SRC (HA tagged) were transiently expressed in HEK293 cells. Whole-cell lysates from HA-tagged c-SRC- and/or FLAG-tagged RAR-transfected cells were resolved by SDS-PAGE along with lysates from HA-tagged c-SRC- and FLAG-tagged RAR-cotransfected (with increasing DNA concentrations) cells and immunoblotted separately with anti-HA and anti-FLAG antibodies as shown in Fig. 6Ci. Comparable amounts of expression of both the proteins were observed following cotransfections of 0.4 µg and 0.8 µg of DNA. For coimmunoprecipitation studies, both RAR (FLAG tagged) and c-SRC (HA tagged) proteins were transiently coexpressed in HEK293 cells. To determine if SRC and RAR interact in vivo, we immunoprecipitated HA-tagged c-SRC or FLAG-tagged RAR separately as shown in Fig. 6C (panels ii and iii, respectively). FLAG-tagged RAR coimmunoprecipitated with HA-tagged c-SRC from lysates using an anti-HA antibody. Immunoblotting with anti-HA antibody showed that RAR (as detected by anti-FLAG antibody) (upper panel) was present in c-SRC IPs as shown in lane 3 (lower panel) of Fig. 6Cii. No RAR was detected in IPs of lysates following transfections of HA-tagged c-SRC and FLAG-tagged RAR alone, as in lanes 4 and 5, respectively, at the upper panel of Fig. 6Cii. Lane 4 of Fig. 6Cii (lower panel) showed that HA-c-SRC was immunoprecipitated following the transfection of HA-tagged c-SRC alone. No interacting proteins were detected in the preimmune samples (lane 1), the mock-transfected samples (lane 2), or HEK293 cell lysates (lane 6). Western blot analysis of whole-cell lysates from transfected cells was carried out simultaneously to confirm the expression of c-SRC and RAR in all transfections (full length and a deletion mutation of 75 to 85 amino acids) in the immunoprecipitated samples (lanes 7 to 12). Data show the expression of HA-tagged c-SRC (lower panel, lanes 8 and 9) and FLAG-tagged RAR (upper panel, lanes 8, 10, and 12) in the respective lysates. In order to further confirm the coimmunoprecipitation study, we performed reverse IP on the lysates from similar cotransfections; HA-tagged c-SRC was coimmunoprecipitated with FLAG-tagged RAR following IP by anti-FLAG polyclonal antibody as shown in Fig. 6Ciii. Immunoblotting with anti-FLAG antibody showed that c-SRC (as detected by anti-HA antibody) interacted with RAR as shown in lane 3 (upper panel) of Fig. 6Ciii. Similar to the results shown in Fig. 6Cii, no interactions were seen upon IP of lysates following transfections of FLAG-tagged RAR and HA-tagged c-SRC alone, as in lanes 4 and 5, respectively, of Fig. 6Ciii (upper panel). Lane 4 of Fig. 6Ciii (lower panel) showed that FLAG-tagged RAR was immunoprecipitated following the transfection of FLAG-tagged RAR alone. No binding was observed in the case of preimmune samples (lane 1), mock-transfected samples (lane 2), and lysates from mock-transfected HEK293 cells (lane 5). Western blot analysis of whole-cell lysates from transfected cells was carried out simultaneously to test the expression of c-SRC and RAR (full-length and deletion mutation of 75 to 85 amino acids) in the immunoprecipitated samples (lanes 7 to 12). The data demonstrate the expression of HA-tagged c-SRC (upper panel, lanes 7, 9, 10, and 11) and FLAG-tagged RAR (lower panel, lanes 7, 8, 10, and 12) in their respective lysates (Fig. 6Ciii) and clearly demonstrate an in vivo interaction of RAR and c-SRC. To further characterize the interaction between c-SRC and RAR, we have generated a deletion of 11 amino acids (75-85) within a proline-rich region of RAR and coexpressed this mutant in HEK293 cells with c-SRC. IP studies using lysates from HEK293 cells that were cotransfected with the FLAG-tagged 75-85 mutant of RAR and HA-tagged wild-type c-SRC demonstrated an abrogation of binding between these proteins (Fig. 6Civ). Additionally, we tested the binding of Y⁵²⁷-SRC with both wild-type and 75-85 RAR and compared the binding with wild-type SRC. Figure 6Civ shows that both wild-type c-SRC and Y⁵²⁷-SRC bind equally with wild-type RAR (top, lanes 3 and 4), while this binding was abrogated