MOLECULAR AND CELLULAR BIOLOGY, June 2007, p. 4179–4197
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 27, No. 11
CSK Controls Retinoic Acid Receptor (RAR) Signaling: a RAR–c-SRC
Signaling Axis Is Required for Neuritogenic Differentiation?
Nandini Dey,1Pradip K. De,1Mu Wang,2Hongying Zhang,1Erika A. Dobrota,3
Kent A. Robertson,3* and Donald L. Durden1*
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 300221; Department of
Pediatrics, Wells Center for Pediatrics Research, Riley Hospital for Children, Indiana University Medical Center,
Indianapolis, Indiana 462023; and 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
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 inhib-
ited RA-mediated neurite outgrowth, a result which correlated with the inhibition of the SFK c-SRC. Consis-
tent 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 colocal-
ization 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 coimmu-
noprecipitation 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.
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, embry-
onal 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) (?, ?, and ?) and retinoid X receptors
(?, ?, 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 inhi-
bition 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 malig-
nant cells include the inhibition of the AP-1 protein (18, 33),
the inhibition of c-Jun NH2-terminal kinase (38), the regula-
tion 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 po-
tential for nongenomic extranuclear cellular signaling down-
stream 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 undif-
ferentiated 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). More-
over, 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 in-
dicates 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) admin-
istration (28–30, 59, 66). It is generally held that RARs func-
tion as DNA binding transcription factors to induce NB dif-
ferentiation. 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,
* Corresponding author. Mailing address for Donald L. Durden:
Section of Pediatric Hematology/Oncology, Department of Pediatrics,
Aflac Cancer Center and Blood Disorders Services, Children’s Health-
care of Atlanta, Emory University School of Medicine, Atlanta, GA
30022. Phone: (404) 778-5069. Fax: (404) 727-4455. E-mail: don
firstname.lastname@example.org. Mailing address for Kent A. Robertson:
Department of Pediatrics, Wells Center for Pediatrics Research, Riley
Hospital for Children, Indiana University Medical Center, Indianapolis,
?Published ahead of print on 26 February 2007.
has not been explored to date. Herein, we have explored evi-
dence that another domain within RAR may functionally in-
teract with tyrosine kinases and play a critical role in neuronal
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 posttransla-
tional modification of proteins, e.g., phosphorylation, tubulin
acetylation, actin polymerization, etc. Therefore, we hypothe-
sized that there might be a more direct mechanism by which
the RARs may regulate and/or coordinate nuclear and cyto-
plasmic 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 con-
served C-terminal regulatory tyrosine (Y527in c-SRC) residue
(20, 57, 60) by a family of nonreceptor protein tyrosine kinases
comprised of C-terminal SRC kinase, CSK, and CSK-homol-
ogous 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
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
Antibodies and reagents. ATRA, 9-cis-RA, 13-cis-RA, and monoclonal anti-
body against human ?-actin were obtained from Sigma-Aldrich (St. Louis, MO).
A pan-SFK kinase inhibitor, PP1, was purchased from Biomole (Plymouth Meet-
ing, 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 (Buckingham-
shire, 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 chroma-
tography (as specified by Santa Cruz Biotechnology, Inc.). Commercially ob-
tained recombinant SRC (p60c-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 hydroxyap-
atite (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 ob-
tained from Amersham Biosciences (Buckinghamshire, England). For immuno-
fluorescence 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 frag-
ments in plasmid constructs were verified by restriction enzyme analysis and
automated standard DNA sequencing. The wild-type human RAR?1 (kindly
provided by Ron Evans) gene was amplified by PCR using two primers, 5?-GA
CCACCAATAAGGAGCGACTC-3? and 5?-CTAGCGGAATCTCAGGCTGG
GGACTTCAGGC-3? (with a 2? 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?-CAGCTCAG
TGGCTTGTAGACCACCATCTCCTCTGAGCTG-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?-CAGCTCAGAGGAGATGGTGG
TCTACAAGCCATGCTTCGTG-3? and 5?-CACGAAGCATGGCTTGTAGA
CCACCATCTCCTCTGAGCTG-3?. The wild-type FLAG-RAR? gene in the
pcDNA3.1 vector was used as the template. DNA sequencing ensured the suc-
cessful deletion of the domain at positions 75 to 85 (?75-85) of RAR?. A point
4180 DEY ET AL.MOL. CELL. BIOL.
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 instruc-
tions, and they were 5?-GACAGAGCCCCAGTTCCAGCCTG GAGAGAAC
C-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?-GTCATGCCATGGCGAGGTTCTCTCCAGGCT
G-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?-CGGGATCCCCCGGGGAAATAAGTTA
GCACAATCAT-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 supple-
mented 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?5to 10?6M 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 concentra-
tion of 4 ?M 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 ? 106cells per 10-cm dish. The fresh supernatants
from stable virus producer PG13 cells were filtered (0.22 ?m) 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 evalu-
ated by Western blot analysis. The clones (5P, 10P, 15P, 8, 10, 1, and 2) were
maintained in 300 to 500 ?g/ml of G418 in cultures, and expression was con-
firmed by Western blot analysis before every experiment (data not shown).
Neurite outgrowth assay. For the neurite outgrowth assay, cells were seeded
(3 ? 106cells) 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?5M) under amber-light conditions or left untreated in
control medium (25 ?l 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 mea-
sured as the percentage of cells showing the second/third degree of collaterals.
Immunoprecipitation and Western blot. LA-N-5 cells were solubilized with
500 ?l 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 phenylarsineox-
ide). 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 ?g 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 ?-actin to confirm
equal loading. Individual bands were visualized by an enhanced chemilumines-
cence 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
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 gluta-
thione agarose in a liquid suspension containing 300 ?g of PAK-1 PBD in 333 ?l
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 ?g/ml leupeptin, 10 ?g/ml aprotinin, and 1 mM NaF–1
mM sodium orthovanadate), and following centrifugation, 10 ?l of PAK-1 PBD
(1 ?g/?l) 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 ?M GTP-?S and incubated for 15 min at 30°C. Before adding PAK-1 PBD,
the reaction was stopped by adding 60 mM MgCl2to the mixture. Agarose beads
were resuspended in 30 ?l 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?5M) 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
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 immuno-
precipitates 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?5M) 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 sepa-
rate 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 manu-
facturer’s protocol, with little modification. In short, the pellets obtained follow-
ing the washings in buffer following an hour of incubation with secondary anti-
bodies (pansorbin) were washed (one time) in pan-kinase buffer [0.1 M NaCl,
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?5M) for 7 to 9 days. PP1 (4 ?M) 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
VOL. 27, 2007RETINOIC ACID RECEPTOR AND SRC IN NB DIFFERENTIATION 4181
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
?-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).
4182 DEY ET AL.MOL. CELL. BIOL.
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 ?M/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
Immunofluorescence studies. LA-N-5 cells were seeded onto glass coverslips
in 10-cm petri dishes and allowed to attach in culture medium containing 10%
FBS. Cells were fixed with chilled 100% methanol (10 min), permeabilized with
0.1% Triton X-100, and washed three times in PBS. Nonspecific binding was
blocked with 2% BSA in PBS for 30 min at 37°C. Staining was carried out using
mouse monoclonal anti-c-SRC (1:50) and rabbit polyclonal anti-RAR? (1:50)
antibodies. Primary antibodies were diluted in blocking buffer, and cells were
incubated for 1 h at 37°C. After washing three times in PBS, cells were incubated
with fluorescein goat anti-mouse IgG (secondary for mouse monoclonal anti-c-
SRC [1:1,000 dilution in blocking buffer]) and tetramethylrhodamine goat anti-
rabbit IgG (secondary for rabbit polyclonal anti-RAR? [1:1,000 dilution in block-
ing buffer]) antibodies in the dark for 45 min. Nuclei were counterstained with
DAPI. Cells were visualized under a Zeiss epifluorescence microscope, and
images were collected and merged using SPOT ADVANCE Fluorescence PC
software. The mean ratio of cytoplasmic to nuclear intensity and the correlation
between cytoplasmic intensity and nuclear intensity of RAR? in LA-N-5 cells
were determined using the MetaMorph Imaging system (Universal Imaging
Corp., Downingtown, PA).
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 fluo-
rescent 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 ?g) or RFP-tagged RAR? (0.8 ?g) or cotrans-
fected with both EGFP-tagged c-SRC (0.4 ?g) and RFP-tagged RAR? (0.4 ?g)
using Lipofectamine 2000 reagent according to the manufacturer’s protocol.
Twenty-four hours after transfection, cotransfected cells were treated with either
ATRA (10?5M) 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 63? (1.4-numer-
ical-aperture) or 100? (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 ?M) 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 ?l/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 ?M), ATRA (20 ?M), or
13-cis-RA (20 ?M) (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.
Binding of RAR? to immobilized (linked to Affi-Gel 15) c-SRC. Recombinant
c-SRC was linked to the activated affinity medium Affi-Gel 15 according to the
manufacturer’s instructions. Briefly, the linking was carried out by adding re-
combinant c-SRC protein to the beads (0.5-ml bed volume) prewashed in 10 mM
sodium acetate buffer (pH 4.5). After reaction for 2 h at room temperature, the
unreacted sites on beads were blocked using a solution containing 100 mM
Tris-HCl (pH 8.0) and 350 mM NaCl for 1 h. The beads were then tested for
SRC by immunoblot analysis (see Fig. 6B, lanes 11 to 13). Beads bound to SRC
were incubated with recombinant RAR? with or without ATRA in the dark for
1 h at 37°C. The SRC-conjugated beads were then resolved by SDS-PAGE and
immunoblotted for RAR?. Recombinant protein served as a positive control.
Beads alone with (see Fig. 6B, lane 2) or without (lane 1) blocking were incu-
bated with RAR? as negative controls. Recombinant c-SRC-coated and blocked
beads (see Fig. 6B, lane 3) also served as negative controls. The binding of RAR?
to unconjugated Affi-Gel beads in the presence of ATRA was tested as the
Transient transfection and coimmunoprecipitation. HEK293 cells were main-
tained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf
serum. The cells in 60-mm tissue culture dishes were either transiently cotrans-
fected with FLAG-RAR? (0.2 ?g to 0.4 ?g) and HA–c-SRC (0.2 ?g-0.4 ?g)
plasmid DNAs or transfected separately with HA–c-SRC (0.8 ?g) and FLAG-
RAR? (0.8 ?g) plasmid DNAs. For coimmunoprecipitation experiments, co-
transfections and transfections were done with 0.8 ?g of plasmid DNA using
Lipofectamine 2000 reagent according to the manufacturer’s protocol. Empty
vectors (0.8 ?g) 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 fluo-
ride, 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 mono-
clonal antibody [1:1,000 dilution]) antibodies. FLAG-tagged proteins in normal-
ized 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 anti
Morphological changes were observed in LA-N-5, LA-N-6, and SK-N-BE(2) cells treated with ATRA (10?5M) 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 ? 106cells) 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?5M) 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.
VOL. 27, 2007RETINOIC ACID RECEPTOR AND SRC IN NB DIFFERENTIATION 4183
body) and anti-FLAG (anti-FLAG monoclonal antibody) antibodies. Chemilu-
minescence 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 coimmu-
noprecipitation studies. HEK293 cells were transiently transfected with the
FLAG-tagged ?75-85 mutant of RAR? and HA-tagged wild-type c-SRC plas-
mids. 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
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 neu-
rite 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?5M to10?7M 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/G1phase 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 p27kip1expression 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
Effects of PP1 on ATRA-induced differentiation in NB cells.
In order to test the involvement of SFKs in retinoid-induced
NB differentiation, the pan-SRC inhibitor PP1 was used in this
study. ATRA-induced neurite outgrowth was semiquantified
following the treatment of PP1 in LA-N-5, LA-N-6, and SK-
N-BE(2) cells. At around days 7 to 9, 100% of the ATRA-
treated LA-N-5 cells were differentiated, compared to 10 to
15% in vehicle-treated cells. Interestingly, the pretreatment of
NB cell lines with PP1 completely abolished neurite outgrowth
(?5% of the control), as shown in Fig. 1. Although the per-
centages of neurite-bearing cells were less in LA-N-6 and SK-
N-BE(2) cells, the inhibitory effect of PP1 was consistent in all
NB cell lines.
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, respec-
tively, 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 out-
growth in LA-N-5, LA-N-6, and SK-N-BE(2) cells, respec-
tively. 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 de-
scribed in Materials and Methods. Treatment of PP1 (4 ?M)
blocked both c-SRC and FYN kinase activity in these cells
compared to untreated controls (Fig. 3B). Similarly, the over-
expression 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. Re-
sults 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 deter-
mined 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 in-
crease in kinase activity compared to the untreated control)
within 15 min of the treatment, which lasted for a total of 60
min (Fig. 3D).
Effect of CSK on the activation of RAC1 following ATRA
administration in LA-N-5 cells. Recent literature revealed a
4184DEY ET AL.MOL. CELL. BIOL.
major role of the small GTPase RAC1 in the regulation of
neuritogenesis, an effect mediated through its control over
cytoskeleton rearrangements in different cell types (5, 37). In
fact, RAC1 has been found to act downstream of receptor
protein tyrosine kinases in the neurite outgrowth response of
N1E-115 NB cells (25, 62). In order to substantiate the effect of
CSK on neurite outgrowth as shown in Fig. 2, we studied the
effects of CSK expression on the ATRA induction of RAC1
activation in LA-N-5 cells. LA-N-5 cells were treated for dif-
ferent times with ATRA, and GTP-RAC levels were quantified
using a GTP-RAC pull-down assay as described previously
(21). ATRA treatment induced a strong and time-dependent
increase in RAC1 activation in LA-N-5 cells (Fig. 4A). Similar
levels of activated RAC1 were observed 24 to 72 h following
ATRA treatment (Fig. 4A, lanes 2, 3, and 4). In order to find
the state of RAC1 activation at the terminal differentiation of
cells, we also measured the activation at 168 h (seventh day) of
ATRA treatment. Activation of RAC1 returned to the non-
stimulated level at 168 h (results not shown). Protein levels of
RAC1 in the lysates used to measure GTP-RAC1 were deter-
mined to be equal by immunoblot analysis. We then deter-
mined the effect of CSK overexpression on the capacity of
ATRA to induce the activation of RAC1 to its GTP-RAC1
form. The results shown in Fig. 4B demonstrate that CSK
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 ?g 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 ?M 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?5M) 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.
VOL. 27, 2007 RETINOIC ACID RECEPTOR AND SRC IN NB DIFFERENTIATION4185
overexpression completely abrogates the capacity of ATRA to
activate RAC1. From these data, we conclude that CSK reg-
ulates the RAR-induced activation of the small G protein
RAC1. This is correlated directly with the marked inhibition of
neurite outgrowth shown in Fig. 2. Furthermore, our results
show that in LA-N-5 cells, the activation of RAC1 occurs as
early as at 15 min of ATRA treatment (Fig. 4C). Convincingly,
the kinetics of RAC1 activation in LA-N-5 cells were compa-
rable to those of the activation of c-SRC under similar condi-
tions of ATRA stimulation (Fig. 3D).
Coimmunolocalization of RAR? and c-SRC in LA-N-5 cells.
Figure 5 shows immunofluorescence analyses of LA-N-5 cells
probed with specific antibodies directed against human RAR?
and c-SRC. Immunofluorescence of RAR? in LA-N-5 cells
shows a cytoplasmic staining for rhodamine corresponding to
the RAR? immunoreactivity. The mean ratio of cytoplasmic to
nuclear intensity for RAR? (expressed as relative pixel inten-
sity) was 2.2 ? 0.16 (standard error) (n ? 18 to 20) as mea-
sured using the MetaMorph Imaging system (Universal Imag-
ing Corp.). A correlation (R2? 0.7597) between nuclear
intensity and cytoplasmic intensity was also observed for
RAR? staining. For colocalization studies, LA-N-5 cells were
double immunostained with specific antibodies for RAR? and
c-SRC. A merge of the RAR? and c-SRC images using the
SPOT ADVANCE program showed coimmunolocalization, as
evidenced by the color change (Fig. 5). These results demon-
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?5M) 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 ?M 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?5M) 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.
4186 DEY ET AL.MOL. CELL. BIOL.
strate that both RAR? and c-SRC have distinct patterns of
staining, and an overlap of colors in the merged image is not
100%. It can be noted from this analysis that a fraction of
RAR? is present in the cytoplasm of LA-N-5 cells and is
colocalized with c-SRC.
Real-time binding of RAR? and c-SRC protein by SPR.
Boonyaratanakornkit et al. (10) found a direct interaction be-
tween the SH3 domain of SFKs and the proline-rich motif of
the nuclear steroid hormone receptor progesterone. A search
of the RAR? amino acid sequence revealed several proline-
rich motifs in the N terminus (residues 74 to 86). Our immu-
nofluorescence results demonstrated a “colocalization” be-
tween RAR? and SRC; hence, we sought to determine the
ability of purified recombinant RAR? to bind purified recom-
binant c-SRC by an SPR technique. This method would permit
real-time direct measurements of the association and dissoci-
ation kinetics of macromolecular interactions. Figure 6A rep-
resents (i) the basal binding of individual components to SRC,
(ii) the RA dose-dependent induction of association between
RAR? and SRC, and (iii) the capacity of free recombinant
SRC protein to compete for SRC-RAR?-RA binding. Figure 6
shows the representative BIAcore sensorgrams for the binding
between immobilized recombinant c-SRC and RAR? in the
presence or absence of its ligands (ATRA, 13-cis-RA, and
9-cis-RA). The RU signal change was close to 250 RU when
SRC bound to 1.85 nM RAR? in the presence of 20 ?M of
ATRA, compared to virtually no binding when 20 ?M 13-
cis-RA was used to replace ATRA. Lower concentrations of
ATRA (2 ?M to 0.002 ?M) did not cause any significant
change in the RU (Fig. 6Aii). A much lower binding affinity
(?25 RU) was observed when 20 ?M 9-cis-RA was used to
replace ATRA. Interestingly, when c-SRC (9.25 nM) was
mixed with 1.85 nM RAR? and 20 ?M ATRA, a more-than-
twofold decrease in binding affinity was observed (?80 RU),
suggesting that SRC in solution was competing with immobi-
lized SRC on the sensor chip surface to bind RAR (Fig. 6Aiii).
These results provide experimental evidence showing that c-
SRC is capable of directly binding to RAR? and that this
interaction is RA ligand dependent.
Ligand-dependent association of RAR? with c-SRC linked
to Affi-Gel 15. In order to study the binding interaction be-
tween RAR? and c-SRC, we used Affi-Gel beads bound to
c-SRC. Figure 6B shows the RAR? immunoblot for the reac-
tion mixtures containing RAR? recombinant protein incu-
bated 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. Re-
combinant 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
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.
VOL. 27, 2007RETINOIC ACID RECEPTOR AND SRC IN NB DIFFERENTIATION 4187
(inset) shows a Western blot of beads conjugated in the pres-
ence 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
In vivo binding of RAR? with c-SRC. Next, we sought to
determine if SRC can bind to RAR? in vivo. Full-length hu-
man 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 ?g and
0.8 ?g 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 us-
ing an anti-HA antibody. Immunoblotting with anti-HA anti-
body 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 pro-
teins 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 trans-
fected cells was carried out simultaneously to confirm the ex-
pression of c-SRC and RAR? in all transfections (full length
4188 DEY ET AL.MOL. CELL. BIOL.
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 ?M 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 ?g or 0.4 ?g) and HA-tagged c-SRC (0.2 ?g
or 0.4 ?g) (lanes 2 and 3) or transfected separately with HA-tagged c-SRC (0.8 ?g) (lane 4) and FLAG-tagged RAR? (0.8 ?g) (lane 5). Whole-cell
extracts (250 ?g) 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 ?g) and cotransfection (0.4 ?g as shown in lane 2 and 0.8 ?g 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 ?g) and HA-tagged c-SRC (0.4 ?g) (lanes 1, 3, and 8) or transfected separately with FLAG-tagged RAR? (0.8 ?g) (lanes
5, 10, and 12) and HA-tagged c-SRC (0.8 ?g) (lanes 4 and 9). Whole-cell extracts (250 ?g 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 ?g of pcDNA3.1 and 0.4
?g 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 ?g) and HA-tagged c-SRC (0.4 ?g) (lanes 1, 3, 7, and 10)
or transfected separately with FLAG-tagged RAR? (0.8 ?g) (lanes 4 and 8) and HA-tagged c-SRC (0.8 ?g) (lanes 5, 9, and 11). Whole-cell extracts
(250 ?g 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 ?g of pcDNA3.1 and 0.4 ?g 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 ?g) and HA-tagged Y527-SRC (0.4 ?g) (lanes 3 and 8 of the top panel); cotransfected with FLAG-tagged RAR? (0.4 ?g) and HA-tagged
wild-type c-SRC (0.4 ?g) (lane 4 of the top panel), with the FLAG-tagged ?75-85 mutant of RAR? (0.4 ?g) and HA-tagged Y527-SRC (0.4 ?g)
(lane 5 of the top panel), or with the FLAG-tagged ?75-85 mutant of RAR? (0.4 ?g) and HA-tagged wild-type (WT) c-SRC (0.4 ?g) (lane 6 of
the top panel); or transfected separately with HA-tagged Y527-SRC (0.8 ?g) (lane 9 of the top panel), with FLAG-tagged wild-type RAR? (0.8
?g) (lane 10 of the top panel), with HA-tagged wild-type c-SRC (0.8 ?g) (lane 11 of the top panel), or with the FLAG-tagged ?75-85 mutant of
RAR? (0.8 ?g) (lane 12 of the top panel). For IP experiments, whole-cell lysates (250 ?g 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 ?g of pcDNA3.1 and 0.4 ?g 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 RAR?s (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?5M) 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 ?m. 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 ?m. 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.
4190 DEY ET AL.MOL. CELL. BIOL.
and a deletion mutation of 75 to 85 amino acids) in the im-
munoprecipitated samples (lanes 7 to 12). Data show the ex-
pression 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 coimmuno-
precipitation study, we performed reverse IP on the lysates
from similar cotransfections; HA-tagged c-SRC was coimmu-
noprecipitated with FLAG-tagged RAR? following IP by anti-
FLAG polyclonal antibody as shown in Fig. 6Ciii. Immuno-
blotting 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 mu-
tation 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 character-
ize the interaction between c-SRC and RAR?, we have gen-
erated 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). Ad-
ditionally, 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 when wild-type RAR? was
replaced by the mutated RAR? (top, lanes 5 and 6). Control
experiments show that no binding was observed in the case of
preimmune samples (top panel, lane 1), mock-transfected sam-
ples (top panel, lane 2 [this lane represents IP out of lysates
from mock-transfected cells]), and whole-cell lysates from
mock-transfected HEK293 cells (top panel, lane 7). Western
blot analysis of whole-cell lysates from transfected cells was
carried out simultaneously to confirm the expression of c-SRC
and RAR? (full length and deletion mutant) in the immuno-
precipitated lysates (top panel, lanes 8 to 12). Data confirm the
expression of HA-tagged c-SRC (middle panel, lanes 3, 4, 5, 6,
8, 9, and 11) and FLAG-tagged RAR? (top panel, lanes 8, 10,
and 12) in the respective cell lysates. The immunoblot in the
bottom panel shows the expression of FLAG-tagged RAR?s
(wild-type protein as shown in lanes 3 and 4 as well as mutated
protein as shown in lanes 5 and 6, respectively) in the cell
lysates that were used in the IP studies. Data presented in Fig.
6Civ further confirm our observation that RAR? binds to c-
SRC in vivo.
Colocalization of exogenously expressed EGFP-tagged
c-SRC and RFP-tagged RAR? in HEK293 cells. In order to
confirm our result showing that endogenous c-SRC colocalizes
with RAR? (Fig. 5), we studied the colocalization of EGFP-
tagged c-SRC and RFP-tagged RAR? by confocal microscopy.
For this purpose, we first identified the subcellular distribution
of exogenously expressed c-SRC and RAR? in HEK293 cells
(Fig. 6Cv, top). In short, EGFP-tagged c-SRC and RFP-tagged
RAR? were transiently transfected (separately or together) in
HEK293 cells, and their subcellular distributions were exam-
ined. Confocal images from cells transfected with EGFP-
tagged c-SRC and RFP-tagged RAR? (separately or together)
showed a clear cytoplasmic distribution of c-SRC (images a
and c of Fig. 6Cv, top). RAR?, on the other hand, was local-
ized in both nuclear and cytoplasmic compartments of the cells
(images d and f of Fig. 6Cv, top). Figure 6Cv shows that the
pattern of subcellular distribution of c-SRC and RAR? as
identified by confocal microscopy was similar to the pattern of
distribution of endogenous c-SRC and RAR? as observed by
immunofluorescence staining of these proteins in LA-N-5 cells
(Fig. 5). When EGFP-tagged c-SRC was cotransfected with
RFP-tagged RAR?, a change in color (yellow/orange) of the
fluorescence was observed in the cytosol of the merged confo-
cal images (images c and f of Fig. 6Cv, bottom), indicating that
EGFP-tagged c-SRC was colocalized with RFP-tagged RAR?
in that compartment of the cells. A clear increase in the trend
of the merge between EGFP-tagged c-SRC and RFP-tagged
RAR? was observed following the administration of ATRA
(compare image c and image f of Fig. 6Cv, bottom). A similar
increase in the trend of the nuclear distribution of RFP-tagged
RAR? was observed following an hour of ATRA treatment in
these cotransfected cells. Interestingly, we observed a charac-
teristic morphological change in these cells following ATRA
administration. Confocal images (images d and f) of Fig. 6Cv
(bottom) show that following an hour of ATRA treatment,
cells exhibit characteristic protrusions of plasma membranes
resembling an “edge”-like focal adhesion structure (Fig. 6Cv,
bottom). These discrete “edge”-like adhesion structures of the
plasma membrane were observed in 100% of the ATRA-
treated cells (data not shown). Moreover, only c-SRC (not
RAR?) has been observed to be preferentially redistributed in
these structures, as evident from their green fluorescence, and
hence, no change of color in the merged images (with RFP-
tagged RAR?) was seen in these “edge”-like focal adhesion
Effect of RA-dependent binding of RAR? on the activation of
c-SRC in LA-N-5 cells. In order to determine if RA-dependent
binding to RAR? activated c-SRC in LA-N-5 cells, we immu-
noprecipitated RAR? and c-SRC from LA-N-5 cells and per-
formed an in vitro SRC kinase assay in the presence and
absence of ATRA (Fig. 7A). Lane 4 of Fig. 7A showed a
significantly higher (P ? 0.05) SRC kinase activity in reaction
mixtures containing both RAR? and c-SRC incubated with
ATRA than the control lanes (lanes 1, 2, and 3). To under-
stand the role of CSK in the ligand-dependent interaction
between SRC and RAR?, we studied the kinase activity of
SRC immunoprecipitated from PP1-treated cells or LA-N-5
cells overexpressing CSK (Fig. 7B). The kinase activity (counts
per minute [cpm]) of SRC immunoprecipitated from these
cells when incubated with RAR? in the presence of ATRA was
VOL. 27, 2007RETINOIC ACID RECEPTOR AND SRC IN NB DIFFERENTIATION4191
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 immunoprecipi-
tants were incubated with and without ATRA (10?5M) 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?5M 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?5M final concentration). Bars
4192DEY ET AL.MOL. CELL. BIOL.
markedly reduced compared to control untreated cells (Fig.
7B, lanes 2 and 3 and lane 1, respectively).
The results generated using a number of knockout mouse
models have confirmed a role for the nonreceptor protein
tyrosine kinases and their downstream substrates in neural
development. Neuronal developmental defects have been ob-
served in SRC, FYN, CSK, ABL, and DAB knockout mice (7,
26, 32). Since nuclear hormone receptors are known to partic-
ipate in neuronal development, we were prompted to examine
the role of the nonreceptor protein tyrosine kinase CSK in
nuclear hormone receptor signaling pathways. We focused on
the RAR signaling pathway in NB cells, partly because NB is a
malignancy currently treated with RA (49).
The results of our analysis demonstrate that RA-mediated
differentiation in NB involves a ligand-dependent binding of
RAR to the SFK (c-SRC), which leads to the enzymatic acti-
vation of this kinase. Our data provide evidence that c-SRC
interacts directly with the RAR and that the CSK-SFK signal-
ing axis regulates RA-induced neuritogenesis. These results
establish the existence of a nongenomic signaling pathway in-
volving SRC that is required for RAR-induced differentiation.
Our data provide important insights into how the RAR orches-
trates and transmits signals in both the nonnuclear and nuclear
cell compartments to orchestrate neurite outgrowth and pos-
sibly neuronal differentiation. The physiologic and biochemical
processes involved in neuronal differentiation include cytoskel-
etal changes, microtubule dynamics, cell cycle arrest, and the
induction of specific genes required for specialized neuronal
cell function. Our results, which implicate the tyrosine kinases
CSK and SRC in RAR signaling, may have implications for
strategies aimed at the pharmacologic control of neuronal dif-
In the present study, we have observed characteristic growth
inhibition, cell cycle arrest, and neuritogenic differentiation in
all three NB cell lines following ATRA exposure. In order to
determine the role of CSK and SFKs in ATRA-induced neu-
ritogenesis (neurite outgrowth), we overexpressed CSK in mul-
tiple NB cell lines. Interestingly, progesterone and estrogen
receptors have recently been implicated to signal through
members of the src family kinases (13, 17, 36, 50, 64). Boon-
yaratanakornkit et al. previously reported that the progester-
one receptor causes an activation of SRC following binding to
the SRC-SH3 domain through its proline-rich region (10).
Other members of the nuclear hormone family, including the
androgen, estrogen, and vitamin D receptors, were not found
to display this signaling paradigm. Upon sequence alignment
of the different RARs, we noted a group of conserved proline-
rich amino acid sequences within the coding regions of the
RAR?, RAR?, and RAR? receptors. Within the N-terminal
region of RAR?, a PxxP motif at amino acids 75 to 85 exists
(Fig. 8). This observation, together with our initial observation
that SFK inhibitors and CSK overexpression completely
blocked RA-induced differentiation of NB cells, led to our
efforts to determine if SFKs are somehow associated with
RAR signaling in neuronal cells.
To begin to explore whether a physical interaction occurs
between RAR? and SRC in NB cells, we utilized immunoflu-
orescence microscopy. The subcellular dynamics of RAR have
been examined previously by Kawata (35), and a transient
modulation of cytoplasmic and nuclear expression of RARs in
differentiating human NT2 cells was reported previously by
Borghi et al. (11). We were able to demonstrate the immuno-
localization of RAR? and c-SRC in LA-N-5 cells (Fig. 5).
Other investigations showed that 20% of RAR is present in the
cytoplasm of HL-60 cells and that RAR shuttles between cy-
toplasmic and nuclear compartments (48). In agreement with
that report, we observed a cytoplasmic staining for RAR? and
a correlation between cytoplasmic and nuclear intensity in LA-
N-5 cells (Fig. 5).
To confirm the interaction between SFK and RAR?, we
utilized several established biophysical methodologies com-
bined with a biochemical analysis of SFK activity. Our results
provide the first evidence that RAR? binds to c-SRC (Fig. 6).
Ligand-dependent real-time binding between RAR? and c-
SRC was observed by SPR analysis. Interestingly, only ATRA
and not 9-cis-RA/13-cis-RA was found to mediate the binding
of RAR? with c-SRC. Affi-Gel binding studies in vitro also
showed an ATRA-dependent binding between these two pro-
teins. Since RAR? has conserved proline-rich regions similar
to the progesterone receptors, we argue that the RAR? inter-
action may be mediated through the SH3 domain of SRC, as
reported previously by Boonyaratanakornkit et al. (10). We
observed that the ligand-mediated binding of RAR? to c-SRC
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 immunopre-
cipitants 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?5M 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?5M) under subdued-light conditions. Immunopre-
cipitated c-SRC in the presence of PP1 (4 ?M) 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).
VOL. 27, 2007 RETINOIC ACID RECEPTOR AND SRC IN NB DIFFERENTIATION4193
is direct and independent of RARE/DNA. Such an interaction
is possible only if the receptor undergoes a conformational
change following ligand binding in the absence of DNA. In line
with our argument, studies described previously by Leng et al.
showed that upon ligand binding, RAR acquires a specific
conformation involving its ligand binding domain, and this can
occur in the absence of DNA (40). Upon confirming the in
vitro interaction of RAR? with c-SRC by SPR and Affi-Gel
techniques, we went on to demonstrate the binding of RAR?
to c-SRC in vivo. Our result established an interaction between
RAR? and c-SRC in mammalian cells (Fig. 6C). The in vivo
IP results were consistent with our in vitro observation that
RAR? binds directly to c-SRC. This binding was observed in
the cases of both wild-type SRC and Y527F-mutated SRC
(Fig. 6Civ). From sequence analyses of RARs, we predicted
that this kind of direct binding involving the polyproline motif
in the N-terminal A/B domain of the receptor is likely to occur.
To test this idea, we deleted the proline-rich domain (from
amino acids 75 to 85 [?75-85 RAR?]) in the A/B domain of
RAR? (Fig. 6Civ). Coimmunoprecipitation experiments con-
firmed (Fig. 6Civ) that the deletion of the proline-rich domain
of RAR? abrogates the binding of the RAR? protein to c-
SRC. These results support a role for a direct binding of
RAR? to c-SRC in vivo and suggest a molecular basis for the
RAR?-SRC interaction. Since coimmunoprecipitation experi-
ments were performed in the absence of ATRA, the possibility
of the existence of a partial constitutive binding between these
proteins cannot be ruled out. However, as the cells were cul-
tured in medium containing10% FBS, and serum contains RA,
it is likely that the observed binding between these proteins is
FIG. 8. Nuclear and extranuclear RA signaling. ATRA binds to its cognate receptor (RAR). The interaction of the ligand-bound RAR with RARE
controls transcription and protein synthesis of its signature genes (classical genomic effect). The nongenomic mode of RA action involves the activation
of the SRC family of nonreceptor protein tyrosine kinases, SRC, following the ligand-dependent binding of RAR to the kinase. The inset shows a
schematic representation of the proline-rich motif in the functional domains of human RAR?1. A highly variable (A/B domain) amino-terminal domain,
a relatively conserved DNA binding domain (DBD or C domain), a hinge region (D domain), a C-terminal ligand binding domain (LBD or E domain),
and a small C-terminal domain (F domain) are schematically drawn (not to scale). The transcriptional activation regions (AF-1 and AF-2) are located
in the A/B and E domains, respectively. Numbers indicate amino acid positions along the sequences in relation to the domains. Proline-rich sequences
of the receptor in reference to its respective amino acid positions are indicated. The inset shows polyproline sequences in the A/B domains of RAR?1.
Interestingly, RAR?, RAR?, and RAR?2 have very similar proline-rich sequences in their A/B domains compared to RAR?1 (not shown in the
that leads to its interaction with c-SRC in the cytoplasm, leading to the activation of this kinase. The activation of SFKs activates a downstream signaling
cascade, which initiates the activation of RAC1 and leads to neurite outgrowth NB cells.
4194 DEY ET AL.MOL. CELL. BIOL.
mediated through RA present in FBS. Our experiments con-
firm the binding of SRC to the RAR? isoform. Considering the
conservation of proline-rich regions in RAR? and RAR?, it is
likely that a similar interaction may also occur in other forms
of RARs. Studies have been undertaken to characterize the
binding of SRC to different isoforms of RAR by mutational
In addition to the demonstration of the cytoplasmic colocal-
ization of endogenous c-SRC and RAR? proteins in LA-N-5
cells by immunofluorescence studies, we have tested the colo-
calization of these proteins using confocal microscopy. In
HEK293 cells, EGFP-tagged c-SRC is colocalized with RFP-
tagged RAR? in the cytoplasmic compartment of cells (Fig.
6Cv, bottom). This result (Fig. 6Cv) is in agreement with our
observation that endogenous RAR? and c-SRC proteins are
colocalized in the cytosol of LA-N-5 cells (Fig. 5). Data further
confirm our finding that both RAR? and c-SRC are colocal-
ized in the cytoplasm and favor the likelihood of an associa-
tion/binding between them. The identification of c-SRC-rich
“edge”-like adhesion structures of the plasma membrane in
ATRA-treated cells is a novel observation. Since c-SRC is
activated following ATRA treatment (Fig. 3D), and c-SRC has
been shown to be differentially localized in these structures, it
implies that the morphological changes occurring in the cells
following ATRA treatment are mediated through the intracy-
toplasmic relocalization of the activated c-SRC. The physio-
logical significance of the observed relocalization of c-SRC to
the periphery of the cell during ATRA stimulation remains an
Since RAR? associates directly with SRC, we next deter-
mined whether the RAR?-SRC interaction leads to the cata-
lytic activation of this tyrosine kinase. Since the activation of
SFKs occurs by “domain displacement” interactions involving
the binding of ligands to its SH2 and/or SH3 domains (9, 61),
we postulated that the binding of RAR? to c-SRC activated
the kinase in an ATRA-dependent way. Our results demon-
strate that c-SRC immunoprecipitated from LA-N-5 cell was
activated in the presence of RAR? plus ATRA. These results
indicate that the RA-RAR? binding to c-SRC leads to the
activation of the kinase (Fig. 7A). The mediation of cellular
functions involving a similar kind of an activation of SFKs
via interactions with its SH2/SH3 domains by other steroid
hormone receptors (progesterone, androgen, estrogen, and
vitamin D receptors) has been reported in literature (10, 13,
17, 36, 50).
To further explore one potential mechanism for how the
RAR?-SRC interaction induced by ATRA might contribute to
neurite outgrowth in LA-N-5 cells, we examined downstream
components of neurite outgrowth, the activation of Rho family
GTPases. The fact that SRC from LA-N-5 cells is activated
following its binding to RAR? in an RA-dependent way and
that the physiologic inhibition of SRC activity blocked ATRA-
induced neurite outgrowth in these cells prompted us to argue
that the activation of SRC following ATRA administration
presumably leads to a cascade of downstream signals culmi-
nating in the neurite outgrowth. Neurite outgrowth occurs
through the rearrangement in cytoskeletal dynamics of actin
(37, 51, 54), and the Rho family GTPases RAC1 and Cdc42 are
cytoskeleton switches for neuritogenesis (4, 5, 24, 42, 52, 69).
Recently, we reported that SFKs regulate the activation of
small GTPases in PC12 neuritogenesis (21). Others showed
previously that the inhibition of RAC1/Cdc42 abrogates neu-
rite outgrowth in various cell types (15, 37, 55). The overex-
pression of dominant negative RAC1-N17 and constitutively
active RAC1-V12 has been shown to block and induce ATRA-
induced neurite outgrowth, respectively, in SH-SY5Y cells
(58). Neuritogenesis in N1E-115 NB cells has been reported to
involve RAC1 (23, 25, 62). We propose that SRC may regulate
the ATRA-induced activation of RAC1 in our system. In
agreement with data reported previously by Alsayed et al., we
show that ATRA induces the activation of RAC1 in our NB
model (1). The fact that PP1 treatment or CSK overexpression
blocked the ATRA-induced activation of RAC1 vis-a `-vis neu-
rite outgrowth in LA-N-5 cells (Fig. 2 to 4) suggests that RAC1
may be involved in RAR-induced nonnuclear signals down-
stream of SRC in NB. A nongenomic mode of action of ATRA
in its neuritogenic response (in NB) was supported by the
demonstration of an early and transient activation of c-SRC
following the administration of ATRA in LA-N-5 cells (Fig.
3D). Furthermore, the kinetics of c-SRC activation by ATRA
match the pattern of an early activation of RAC1 following
ATRA treatment in LA-N-5 cells (Fig. 4C). The activation of
RAC1 within 15 to 30 min of treatment with ATRA suggests a
rapid and hence genome-independent mode of signaling in
retinoid function. Interestingly, the characteristic morpholog-
ical changes observed under a confocal microscope following
1 h of ATRA administration (Fig. 6Cv, bottom) strengthen our
argument for the role of c-SRC in the acute mode of signaling
mediated by the RAR in NB cells. Taken together, our study
identifies a direct binding of RAR? to c-SRC and provides the
first evidence for the biological significance of this RAR? sig-
naling pathway through the activation of SRC and its down-
stream effector RAC1 in the neuronal differentiation of NB
In conclusion, we propose a new paradigm for RAR signal-
ing in neuronal cells. In addition to its capacity to activate gene
expression, RAR engages in a dual function, the capacity to
activate SRC in the cytoplasm through a hormone-dependent
direct binding to SRC, a process required for neuritogenesis
(Fig. 8). In this manner, the RAR can coordinate and orches-
trate complex cytoplasmic, membrane, and nuclear events re-
quired for neuronal differentiation. Why would there be a
utility for the RAR to bind and activate cytoplasmic and nu-
clear effectors? Many signaling proteins, including membrane
proteins, coordinate downstream and upstream signals by vir-
tue of their multiple domains. Recent evidence suggests not
only that the epidermal growth factor receptor binds ligand at
the cell surface but also that a portion of its cytoplasmic do-
main is then cleaved to enter the nucleus to drive transcription
(1). Similarly, we envision that a multifunctional RAR may
exert its effects on cytoplasmic versus nuclear targets via dif-
ferent regions of the RAR protein (Fig. 8). We have now partly
mapped the regions of SRC and RAR? required for this in-
teraction in vivo. What is less clear at this time is to what extent
the cytoplasmic nonnuclear functions of RAR (SRC activa-
tion) can be separated from the transcriptional functions of the
retinoid receptor and how the coordination of these distinct
functions is mechanistically achieved. Preliminary data gener-
ated in CSK-overexpressing NB cells demonstrates that the
RA-RAR-induced cell cycle arrest and p27 induction re-
VOL. 27, 2007 RETINOIC ACID RECEPTOR AND SRC IN NB DIFFERENTIATION4195
sponses are intact, suggesting that these nonnuclear events are
not required for certain RAR functions. Many of the mecha-
nisms for the nonnuclear RAR function remain to be explored.
We will use our CSK-transduced NB cell lines to further ex-
plore these elements of RAR? structure and function (adapter
protein interactions, etc.). Finally, the recent evidence that
CSK and SRC regulate nongenomic androgen receptor signals
(74) suggests that this signaling axis may have an impact on
hormone-induced signals that relate to cellular transformation
in epithelial cells.
Wild-type human RAR?1 was kindly provided by Ron Evans (Salk
Institute, CA), and c-SRC was obtained from H. Fu (Emory Univer-
sity, GA). Mean ratios of cytoplasmic to nuclear intensity and corre-
lations between cytoplasmic intensity and nuclear intensity were de-
termined by Adam Marcus of the Confocal Microscope Facility,
Winship Cancer Center, Emory University, Atlanta, GA. We thank K.
Schafer-Hales (Cell Imaging and Microscopy Core, Winship Cancer
Institute) for her help with the confocal microscope and image pro-
We also acknowledge the support of the NIH for funding this work,
CA94233 to D.L.D. D.L.D. is supported by a Georgia Cancer Coali-
tion grant. The work is supported by the Aflac Cancer Center and
Blood Disorders Service.
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