From the Departments of Neurobiology,
Pathology, and Physical Medicine and Rehabilitation, Civitan
International Research Center, University of Alabama at Birmingham,
Birmingham, Alabama 35294-0021, the § Department of
Medicine, Boston University School of Medicine, Boston, Massachusetts
02118, and the ¶ Department of Pathology, University of Alabama at
Birmingham, Birmingham, Alabama 35294-0007
Received for publication, May 31, 2002, and in revised form, October 7, 2002
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ABSTRACT |
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We present evidence here that Erbin is a
negative regulator of the Ras-Raf-Erk signaling pathway. Expression of
Erbin decreases transcription of the AChR Extracellular signal-regulated kinases (Erk)1
are a subfamily of mitogen-activated
protein kinases (MAPK) that play important roles in a great array of
cell programs including proliferation, differentiation, and apoptosis
(1, 2). As exemplified by binding to growth factors such as EGF,
receptor tyrosine kinases are activated and undergo autophosphorylation
on tyrosine residues (3, 4). Phosphorylated tyrosine residues recruit
adaptor proteins to the plasma membrane by directly interacting with
modules including Src homology 2 (SH2) or phosphotyrosine binding
domain (PTB). Grb2, one of such adaptors, brings guanyl nucleotide
exchange factor (SOS) to the plasma membrane in proximity with Ras and expedites exchange of GDP for GTP on Ras (5). Activated Ras (GTP-bound)
then directly binds to Raf and allows the latter to be activated (1,
6). Active Raf triggers sequential activation of MEK, a MAPK kinase,
and Erk, leading to phosphorylation of various regulatory proteins
including nuclear transcription factors such as Elk-1 and Myc as well
as many cytoplasmic proteins (7, 8).
In the past, extensive efforts have been made to identify factors that
participate in regulation of the Ras-Raf-Erk pathway. Several
modulators have been identified that positively influence the pathway
at different levels. For example, the MEK partner 1 (MP1) was isolated
as a binding protein that interacts with both MEK1 and Erk1 to enhance
the efficiency of Erk phosphorylation by MEK (9). A second protein is
the kinase suppressor of Ras (KSR) that is believed to act as a
scaffold for Raf-1, MEK, and Erk (10). The Connector enhancer of KSR
(CNK) directly binds to Raf and is involved in activation of the
Raf/MEK/Erk pathway (11). Sur-8 is an interesting protein that contains
multiple leucine-rich repeats (LRRs) (12) and binds to both Ras and
Raf-1. Although Ras can directly associates with Raf upon activation, the presence of Sur-8 increases the interaction between Ras and Raf and
the activation of downstream signaling events (10). These non-enzymatic
factors are important regulators for normal cell proliferation and differentiation.
In addition, there are negative regulators of the Ras pathway in cells.
Sprouty, a Ras suppressor in Drosophila and its mammalian homologue Spred (Sprouty-related
EVH1 domain-containing protein) appear to serve
as negative feedback regulators of growth factor-mediated Erk pathway
(13, 14). The Ras effector RIN1 has been shown to inhibit Ras-induced
activation of Raf by competitively binding to active Ras (15).
Additionally, the Raf kinase inhibitory protein (RKIP), initially
isolated as a phosphatidylethanolamine-binding protein, binds directly
to the kinase domains of both Raf and MEK and inhibit MEK
phosphorylation (16). These negative regulators are important to ensure
that all programs are adequately executed through autonomous turn-on
and -off mechanisms. In addition, they may counterbalance overamplified
proliferative signals that are caused by Ras mutation frequently
occurring in human cancers. Such an inhibitory mechanism is key to
maintaining normal cell growth rate or function.
Erbin is a protein that was identified as a binding partner for ErbB2,
delta-catenin, and ARVCF (17-22). The 180-kDa protein contains two
identifiable domains: LRR and PDZ (17, 19). Because of the essential
role of ErbB2 in neuregulin (NRG)-induced synthesis of acetylcholine
receptors (AChR) (23-26), we investigated the effect of Erbin on AChR
subunit expression. Unexpectedly, Erbin was found to inhibit AChR
subunit transcription, an event that requires Erk activation (23-26),
suggesting that it may play a role in regulating Erk activation. This
study presents evidence that Ras-mediated Erk activation is indeed
negatively regulated by Erbin. We have explored the possible mechanism
by which Erbin inhibits Erk activation. Our results identify Erbin as a
novel suppressor of the Ras signaling.
Plasmid Construction--
The human Erbin N-terminal domain
(amino acids 1-391) consisting of 16 LRRs was generated by PCR
amplification using sense primer containing BamHI and
antisense primer containing XhoI. The resulting 1.2 kb-fragment was digested with BamHI and XhoI, and
subcloned in the BamHI-SalI sites of yeast vector
pGBT9 downstream of the Gal4 DNA binding domain
(Clontech). Myc-Erbin LRR, Myc-Erbin Cell Culture and Transfection--
HEK 293 cells and COS-1 cells
were cultured as described previously (29). The C2C12 cells were
maintained as undifferentiated myoblasts in Dulbecco's modified
Eagle's medium with high glucose supplemented with 20% fetal bovine
serum, and 0.5% chicken embryo extract. Fusing of myoblasts into
myotubes was induced by culturing myoblasts for 48 h in
differentiation medium DM (Dulbecco's modified Eagle's medium plus
4% horse serum). Mouse lung epithelial Mv1Lu cells and breast cancer
MCF-7 cells were maintained in Dulbecco's modified Eagle's medium
plus 10% fetal bovine serum. Rat pheochromocytoma-derived PC12 cells
were grown in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum and 5% horse serum. HEK293, COS-1, and C2C12
cells were transfected with the standard calcium phosphate technique
(29). PC12 cells and Mv1Lu cells were transfected with SuperFect
(Qiagen). Two days after transfection, cells were washed with
phosphate-buffered saline and lysed in the modified RIPA buffer
containing 20 mM sodium phosphate, pH 7.4, 50 mM sodium fluoride, 40 mM sodium pyrophosphate,
1% Triton X-100, 2 mM sodium vanadate, 10 mM
p-nitrophenyl phosphate, and protease inhibitors (25). Lysed
cells were incubated on ice for 20 min and centrifuged at 13,000 × g for 10 min at 4 °C. The clear supernatant was
designated as cell lysates.
Immunoprecipitation and Immunoblotting--
Cell lysates (~400
µg of protein) were incubated without or with indicated antibodies
1 h at 4 °C and subsequently with protein A- or protein
G-agarose beads overnight at 4 °C on a rotating platform. After
centrifugation, beads were washed five times with the modified RIPA
buffer. Bound proteins were eluted with the SDS sample buffer, resolved
by SDS-PAGE, and transferred onto nitrocellulose membranes (Schleicher
and Schuell). Nitrocellulose membranes were incubated at room
temperature for 1 h in the blocking buffer containing
Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% milk or
5% bovine serum albumin followed by incubation with indicated
antibodies in the blocking buffer. After washing three times for 5 min
each with TBS-T, membrane was incubated with horseradish
peroxidase-conjugated donkey anti-mouse, or anti-rabbit IgG (Amersham
Biosciences), or anti-rat IgG (Santa Cruz Biotechnology) followed by
washing. Immunoreactive bands were visualized with enhanced
chemiluminescence substrate (Pierce). In some experiments, the
nitrocellulose filter was incubated in a buffer containing 62.5 mM Tris-HCl, pH 6.7, 100 mM
Luciferase Assay--
C2C12 myoblasts were co-transfected with
or without Myc-Erbin, plus the Differentiation of PC12 Cells--
PC12 cells were cotransfected
pEGFP-C1 with empty vector pRK5-Myc, Myc-Erbin or its mutants, or
Erbin-siRNA duplex. 48 h after transfection, PC12 cells were
stimulated by 100 ng/ml or 20 ng/ml NGF for 2 days. Cells were examined
by fluorescence microscopy. Cells with processes 1.5 times longer than
the diameter of the cell body were considered to be differentiated.
Inhibition of Erbin Expression by RNA Interference
(RNAi)--
The target region of siRNA was 540 nucleotides downstream
of the start codon (31), which contained ~50% G/C content. The nucleotide sequence was 5'-UAG ACU GAC CCA GCU GGA A dTdT-3'
(nucleotides 866-884) (27). We searched the NCBI sequence bank against
this segment of DNA using the BLAST program, which revealed no match, suggesting of the specificity of target recognition by siRNA. The
21-nucleotide RNAs were chemically synthesized by Dharmacon Research
Inc. Synthetic oligonucleotides were deprotected and gel-purified. To
demonstrate the silencing effect of endogenous Erbin expression by
siRNA, cells in a 60-mm culture dish were co-transfected with empty
vector pEGFP-C1 and with siRNA duplex using SuperFect. Briefly, 2 µg
of pEGFP-C1 and 30 µl of 20 µM Erbin-siRNA duplex were
mixed with 300 µl of Opti-MEM (Invitrogen). After incubating 10 min
at room temperature, add Opti-MEM to obtain a final volume of 1 ml.
Cells were incubated with the mixture for 2-3 h at 37 °C and 5%
CO2 before the addition of 5 ml of growth medium. 72 h
after transfection, cells were resuspended in phosphate-buffered saline
buffer. GFP-positive cells were collected by fluorescence-activated cell sorting (FACS) analysis with the CellQuest software. Cells were
lysed in modified RIPA buffer, and lysates were subjected to
immunoblotting for expression of Erbin. In parallel experiments, GFP-positive PC12 cells were scored for differentiation.
Protein Assay--
Protein was assayed with Coomassie Protein
Assay Reagent (Pierce) using bovine serum albumin as a standard
(32).
Erbin Inhibits Erk Activation--
Previous studies from our
laboratory and others have demonstrated that NRG-induced AChR
expression requires ErbB2 tyrosine phosphorylation and activation of
the Ras-Raf-Erk signaling pathway (23-26,33). We speculated that
Erbin, interacting with ErbB2, may play a role in regulating NRG
signaling. To test this hypothesis, we examined the effect of Erbin on
the promoter activity of
To test this hypothesis, we characterized effects of Erbin on Erk1
activation in COS-1 cells. FLAG-Erk1 was activated in response to NRG
in cells cotransfected with ErbB4 (Fig. 1B) (29).
Coexpression of Erbin caused a decrease in phospho-Erk (Fig.
1B) and as well as Erk kinase activity (Fig. 1C),
indicating that Erbin negatively regulates the Ras-Raf-MEK-Erk pathway.
The inhibitory effect of Erbin featured the following: 1) It was
dose-dependent (Fig. 1D). 2) Erbin did not seem
to delay the peak Erk activation that usually occurred within 5 min of
stimulation (Fig. 1E, Ref. 25). 3) The inhibition was not
growth factor-specific. The expression of Erbin inhibited EGF- and
NGF-induced Erk activation (Fig. 1F and data not shown); and
4) it was Erk activation-specific, since expression of Erbin had no
apparent effect on NRG activation of Akt (Fig. 1G). Thus,
the results demonstrate that Erbin specifically inhibits Erk activation
with no effect on the PI 3-kinase pathway.
To identify the domain that inhibits the Erk activation, we examined
the effect of a series of Erbin mutants (Fig.
2A). The results revealed that
the inhibition of Erk did not require PDZ domain (Fig. 2B),
which is essential for interaction with ErbB2 (19, 27) or the region
between the LRR domain and the PDZ domain (Fig. 2B). In
contrast, deletion of the LRR domain disabled Erbin to inhibit Erk
activation (Fig. 2C). Furthermore, we demonstrated that the
LRR domain was sufficient to mediate the inhibitory effect (Fig.
2B). These results are in agreement with the differentiation assay (see Fig. 5) and point to an important role of Erbin in regulating the Ras-Raf-MEK-Erk pathway.
Erbin Inhibits Raf Activation--
Next we attempted to dissect
the position for Erbin action by walking upstream of Erk. Since Erbin
directly interacts with the cytoplasmic domain of ErbB2, it is possible
that Erbin interferes with the tyrosine kinase activation and/or
subsequently binding to adaptor proteins. To test these hypotheses, we
employed NeuT, an active form of ErbB2 (34). When expressed in COS-1
cells, NeuT was tyrosine-phosphorylated (Fig.
3A), resulting in an increase in the promoter activity of p3TP-Lux (Fig. 3B). When NeuT
was immunoprecipitated, association of Shc and Grb2 could be easily detected (Fig. 3A). Coexpression of Erbin had no effect on
either tyrosine phosphorylation of NeuT or its association with Shc and Grb2 (Fig. 3A). However, the NeuT-induced promoter activity
of 3TP was greatly inhibited by Erbin (Fig. 3B), suggesting
that the site of Erbin action is downstream of the adaptor proteins. Thus, we examined whether Erbin inhibits Erk activation by Raf or Ras.
Since expression of the active Raf can bypass the requirement of
upstream components for the MEK/Erk activation (7), Erbin would
attenuate Erk activation by active Raf, if Erbin acts downstream of
Raf. The results in Fig. 3, C and D showed that
coexpression of Erbin with active Raf did not have an effect on Erk
activation. In contrast, Erk activation by active Ras was evidently
inhibited (lanes 2 and 3, Fig. 3, C
and D). These results suggest that Erbin acts between Raf
and Ras.
Erbin Disrupts the Ras-Raf Interaction--
In considering the
mechanism of Erbin-induced inhibition of the Erk pathway, it is
reasonable to postulate that Erbin inhibits the interaction between
active Ras and Raf by competitive binding to either of them. To test
this, active Ras (FLAG-RasV12) was coexpressed with GST-Raf1 into
HEK293 cell. As shown in Fig.
4A, FLAG-RasV12 was found to
copurify with GST-Raf1. In a reciprocal experiment, Raf1 was also
detected in the immunoprecipitates of active Ras. Remarkably,
coexpression of Erbin decreased the interaction between active Ras and
Raf. In an alternative experiment, we employed the Ras pull-down assay
developed by Rooij and Bos, which takes advantage of high affinity of
GTP-Ras for the Ras-binding domain (RBD) of Raf-1 as compared with
GDP-Ras (35). The RBD (amino acids 50-150) was expressed as a GST
fusion protein and used to pull down GTP-Ras that had been activated in
cells cotransfected with or without Erbin. Basal Ras binding to RBD was
usually very low in control or Erbin-expressing cells. The effect of
Erbin, if any, on Ras-RBD binding was inconsistence. However, in cells transfected with FLAG-Ras, GST-Raf-RBD could pull-down activated Ras
(Fig. 4B, lane 2). Expression of Erbin inhibited the
interaction of Raf with growth factor-activated Ras, which was
accompanied by a decrease in phospho-Erk (Fig. 4B, lane 4).
These results suggest that Erbin inhibits the Erk pathway by
disrupting the interaction between active Ras and Raf.
Because our data did not support Erbin interaction with Raf1 (Fig.
4A), we attempted to determine whether Erbin associated with
Ras. In this experiment recombinant Erbin was immunoprecipitated from
HEK293 cells cotransfected with a constitutively active mutant of Ras
(V12) or a dominant negative mutant of Ras (N17). The resulting immunocomplex was probed with anti-FLAG antibody. As shown in Fig.
4C, only the active Ras (V12) was co-immunoprecipitated with Erbin, suggesting that transfected Erbin and active Ras may interact in
cells. We employed the yeast two-hybrid system to determine whether
Erbin and active Ras interact directly. Yeast Y190 cells were
cotransformed with pACT2-RasV12 and pGBT9-LRR, which contains the LRR
domain of Erbin. Cotransformation of Ras and Erbin did not result in a
significant change in Inhibition of NGF-induced PC12 Cell Differentiation by
Erbin--
To further study the physiological importance of the Ras
inhibition by Erbin, we examined the effect of its overexpression on
neuronal differentiation of rat pheochromocytoma (PC12) cells. By
chronic incubation with NGF, these cells differentiated and developed
sympathetic neuron-like phenotypes (37). The NGF-treated cells stopped
to divide and in turn developed long, sometimes branched processes. The
Ras-Raf-Erk pathway plays an essential role in NGF-induced
differentiation of PC12 cells (38-40). The inhibition of Erk
activation by Erbin suggests that it may alter NGF-induced
differentiation of PC12 cells. Expression of enhanced green fluorescent
protein (EGFP) and the pRK5-Myc empty vector (control) had no apparent
effect on differentiation whereas Erbin-transfected PC12 cells
exhibited altered morphology (Fig.
5A). The neurites became
shorter and were less branched. Quantitative analysis of Erbin's
effect was shown in Fig. 5B. Under the control condition, cells bearing neurites 1.5 times longer than the cell body accounted for 60 ± 5% of the total cell population, whereas the number of differentiated cells are significantly reduced by Erbin (Fig. 5B). Erbin contains three domains: an N-terminal region that
contains 16 LRRs, a C-terminal PDZ domain, and a middle region that
show no homology to known proteins (17-22). Ecptopic expression of the LRR domain showed similar effect on differentiation, suggesting that
the inhibitory activity was contained in this domain. In contrast,
cells transfected with
To confirm the inhibitory effect of Erbin on PC12 cell differentiation,
we examined the effect of suppressed Erbin expression in PC12 cells. To
this end, we employed the RNAi technique, which diminishes expression
of a specific gene in cells (41). Recently, RNAi has been shown to
specifically suppress the expression of endogenous and heterologous
genes in mammalian cell lines (31, 42). Thus, 21 nucleotide small
interfering RNA (siRNA) duplexes directed against Erbin (nucleotides
866-884, Ref. 27) were synthesized and transfected in PC12 cells to
suppress expression of endogenous Erbin. Two days after transfection,
cells expressing co-transfected EGFP were sorted out and analyzed for
Erbin expression by Western blot. As shown in Fig. 5C,
expression of Erbin in Erbin-siRNA-transfected cells was significantly
decreased in comparison with missense RNAi-transfected cells (control).
The suppressing effect by Erbin-siRNA appeared to be specific since it
had no effect on expression of endogenous Erk1. Moreover, expression of
cotransfected EGFP was unaffected (Fig. 5C). In agreement of
results with Erbin expression experiments, inhibition of Erbin
expression by Erbin-siRNA caused an increase in phospho-Erk in response
to NRG stimulation (Fig. 5D). Having demonstrated that
Erbin-siRNA inhibits expression of endogenous Erbin and increases
NRG-induced activation of Erk, we studied the effect of Erbin-siRNA on
PC12 cell differentiation. To capture the maximal effect of
Erbin-siRNA, a submaximal effective concentration of NGF was used, as
at this concentration, NGF caused differentiation of only 25 ± 2% PC12 cells (Fig. 5E, control). Remarkably, suppressing
Erbin expression by Erbin-siRNA enhanced NGF-mediated cell
differentiation (52 ± 2%), while missense RNA had no significant
effect (18 ± 2%). These results demonstrate that Erbin inhibits
NGF-induced differentiation of PC12 cells.
The current study showing that Erbin inhibits Erk activation together
with previous findings that it associates with ErbB2, p0071,
Erbin belongs to the LAP (LRR and
PDZ) protein family of PDZ domain-containing proteins
(17-22). In addition to Erbin, the family members include LET-413 in
Caenorhabditis elegans (27, 43); Scribble, a
Drosophila protein essential for epithelial integrity (44,
45); and Densin-180 (46) and Lano in mammals (47). Genetic studies in
non-vertebrates have demonstrated that LAP proteins play a role in cell
polarity and cell morphology of epithelial cells (43-45). Our results
raised the possibility that these previously thought scaffold proteins
may participate in regulation of cell signaling.
In summary, we provide evidence that Erbin is a negatively regulator of
the Ras signaling pathway. Since Erbin associates with activated, but
not inactive Ras, our results suggest a turn-off mechanism through
which Ras signaling to Raf is inhibited. Ras, upon activation by
extracellular signals, forms a complex with Erbin and thus becomes
unable to activate Raf.
-subunit gene, an
event that is mediated by Erk activation. Although it interacts with
the ErbB2 C terminus through the PDZ domain, Erbin has no effect on
ErbB2 tyrosine phosphorylation or binding to the adaptor proteins Shc
and Grb2. In contrast, expression of Erbin greatly impairs activation
of Erk, but not Akt, by ligands that activate receptor tyrosine
kinases. Moreover, Erbin inhibits the Erk activation by active Ras,
while it fails to do so in the presence of active Raf-1. Erbin
associates with active Ras, but not inactive Ras nor Raf. Consistently,
Erbin interferes with the interaction between Ras and Raf both in
vivo and in vitro. Finally, overexpression of Erbin
leads to inhibition of NGF-induced neuronal differentiation of PC12
cells, whereas down-regulation of endogenous Erbin by specific siRNA
exhibits an opposite effect. Collectively, our study has identified
Erbin as a novel suppressor of the Ras signaling by disrupting
the Ras-Raf interaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
965-1371 and Myc-Erbin
PDZ were generated by introducing a stop codon after the LRR domain following amino acids 965 or 1279 in pRK5-Myc-Erbin (19,
27). The N-terminal deletion mutant (pRK5-Erbin965) was described
previously (19). A fragment encoding the full-length Akt cDNA
generated by PCR using sense primer containing EcoRI and
antisense primer containing XhoI. The resulting 1.6-kb
fragment was digested with EcoRI and XhoI and
subcloned into EcoRI-XhoI sites of the mammalian
expression vector pCS2+MT (for the Myc tag at the N terminus). Wild
type-ErbB2 and constitutively active form of ErbB2 (NeuT) (generously
provided by Dr. M. C. Hung, University of Texas M. D. Anderson Cancer
Center) were subcloned downstream of the FLAG tag and an artificial
signal peptide in pCMV. pCMV-FLAG-Erk1 was generously provided by Dr.
Mike Weber (University of Virginia). FLAG-Ras, FLAG-RasV12,
FLAG-RasN17, and GST-Raf-BXB were described as previously (28).
-mercaptoethanol, and 2% SDS at 50 °C for 30 min, and washed
with 0.1% Tween 20 in 50 mM TBS at room temperature for
1 h, and reblotted with different antibodies. The following
antibodies were used: FLAG (M2, Sigma), Myc (9E10, Santa Cruz
Biotechnology), phospho-MAPK (Promega), phospho-Akt (Ser-473, New
England Biolab), H-Ras (238, Santa Cruz Biotechnology), and Erbin
(19).
-subunit promoter-luciferase
transgene that contains 416 nucleotides of the 5'-untranslated region
of the
-subunit gene (25) and a control plasmid pRL-SV40 (Promega).
24 h after transfection, myoblasts were incubated in DM to induce
myotube formation. Myotube formation was complete 48 h after
switch to DM. The C2C12 myotubes were stimulated with NRG at a final
concentration of 10 nM at 37 °C for 24 h. Mv1Lu
cells were transiently transfected with the promoter reporter construct
p3TP-Lux, which contains three AP-1 sites and the plasminogen activator
inhibitor-1 (PAI-1) promoter (30), and firefly luciferase gene.
pRL-SV40 (Promega) that express Renilla luciferase under the
control of SV40 promoter was cotransfected as a control to monitor the
transfection efficiency. 48 h after transfection, cells were lysed
and activities of the two different luciferases were assayed with
respective substrates with a dual luciferase assay kit (Promega).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
416-Luc, a transgene reporter that contains
416 nucleotides of the 5'-untranslated region of the
-subunit gene
(25). Expression of this transgene is up-regulated by NRG or active
forms of Ras and Raf and requires Erk activation (25, 33).
Unexpectedly, we found that Erbin inhibited expression of the
416-Luc transgene in control as well as NRG-stimulated muscle cells
(Fig. 1A), suggesting that
Erbin may regulate the Erk activation.
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Fig. 1.
Inhibition of NRG-induced transcription of
AChR -subunit gene and Erk activation by
Erbin. A, inhibition of the NRG-induced expression of an
AChR
-subunit reporter gene by Erbin. C2C12 cells were cotransfected
with empty vector pRK5-Myc or Myc-Erbin and
416-luc, which contains
the
-subunit 5'-flanking region driving the expression of firefly
luciferase. A Renilla luciferase plasmid pRL-SV40 under the
constitutive control of HSV promoter was cotransfected as a control to
monitor the transfection efficiency and sample handling. After
differentiation, transfectants were treated with or without 10 nM NRG for 24 h. Firefly luciferase activity was
normalized to Renilla luciferase activity. B,
Erbin inhibits Erk activation by NRG. COS-1 cells in 100-mm culture
dishes were transfected with FLAG-Erk1 (1 µg) either alone or with
Erbin (10 µg) and ErbB4 (5 µg). Transfected cells were
serum-starved for 6 h and then stimulated with 10 nM
NRG for 10 min. From the cell lysates, FLAG-Erk1 was immunoprecipitated
(IP) using a mouse anti-FLAG antibody (M2) coupled with Sepharose
beads. Resulting immunoprecipitates were then subjected to SDS-PAGE and
immunoblotting with anti-phospho-Erk and anti-FLAG antibodies. Cell
lysates were also subjected to immunoblotting (IB) with anti-Myc, and
anti-ErbB4 antibodies to detect expression of Erbin and ErbB4.
C, Erk kinase activity. Cells were transfected as in
B. Immunopurified FLAG-Erk1 was assayed using myelin basic
protein (MBP) as substrate in the presence of
[
-32P]ATP in vitro as described previously
(25). D, dose-dependent inhibition of Erk
activation by Erbin. COS-1 cells were co-transfected with FLAG-Erk1 and
ErbB4 and increasing concentrations of Myc-Erbin and then challenged
with 10 nM NRG for 10 min. Erk activation was assayed as in
B. Equal amount of FLAG-Erk1 was shown in a reblot of the
same membrane in a representative experiment. E, Erbin had
no effect on the Erk activation time course. F, inhibition
of EGF-mediated Erk activation by Erbin. COS-1 cells were transfected
with Flag-Erk1 (1 µg) with empty vector or Erbin (10 µg). Cells
were stimulated with 100 ng/ml EGF for 10 min. Phospho-Erk was
visualized with anti-phospho-MAPK antibody as described in
B. Equal amounts of FLAG-Erk1 and EGFR were shown in a
reblot. G, no effect of Erbin on Akt/protein kinase B kinase
activation. COS-1 cells co-transfected with ErbB4 (5 µg), Myc-Akt (1 µg) without or with Myc-Erbin (10 µg). 48 h after
transfection, cells were stimulated with 100 ng/ml EGF or 10 nM NRG for 10 min. Myc-Akt was immunoprecipitated with
anti-Myc antibody, and subjected to SDS-PAGE and immunoblotting for
phospho-Akt with a rabbit anti-phospho-Akt antibody.
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Fig. 2.
The LRR domain was required and sufficient to
inhibit Erk activation. A, schematic diagrams of Erbin
expression constructs. B, LRR was sufficient to inhibit Erk
activation. C, dependence of the inhibitory effect on the
LRR domain. COS-1 cells were transfected with FLAG-Erk1 alone, or
cotransfected with Myc-tagged LRR, 965-1371,
PDZ, Erbin in
A or 965 in B. Transfected cells were stimulated
NRG and assayed for Erk1 activation as described in Fig. 1. Lysates
were immunoblotted with anti-Myc antibody to demonstrate the expression
of Erbin and its mutants.
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Fig. 3.
Erbin acts above Raf. A,
Erbin did not alter NeuT tyrosine phosphorylation or binding to Shc or
Grb2. COS-1 cells transfected with FLAG-NeuT with or without Myc-Erbin
or Myc-Erbin PDZ. NeuT was immunoprecipitated with anti-FLAG
antibody. Resulting immunocomplexes were probed with antibodies against
phosphotyrosine, Shc, Grb2, or ErbB2. Expression of Erbin and its
mutant was detected by immunoblotting with anti-Myc antibody.
Non-immune, no antibody in immunoprecipitation.
B, Erbin inhibited NeuT-mediated gene expression. p3TP-Lux
was transfected alone or cotransfected with FLAG-NeuT without or with
Myc-Erbin in Mv1Lu cells. Cells were lysed, and luciferase activity
measured and normalized to cotransfected pRL-SV40. Data are shown as
means ± S.D. (n = 3). Asterisk,
p < 0.01, Student's t test. C,
inhibition of active Ras-, but not active Raf-mediated Erk activation.
COS-1 cells were cotransfected with FLAG-Erk1, constitutively active
Ras (V12) or Raf (BXB) without or with Myc-Erbin. Erk1 activation was
assayed as described in the legend to Fig. 1. Results of densitometric
analysis is shown on the right. Data are shown as means ± S.D. (n = 3). Asterisk, p < 0.01, Student's t test. D, Erk kinase
activity. Cells were transfected as in C. Immunopurified
FLAG-Erk1 was assayed using myelin basic protein (MBP) as
substrate in the presence of [
-32P]ATP in
vitro as described previously.
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Fig. 4.
Erbin blocks the interaction of Ras and
Raf. A, Erbin inhibited RasV12 binding to Raf. Cells
were transfected with Flag-RasV12, GST-Raf1, or Myc-Erbin. GST-Raf1 was
pull-downed from cell lysates with GSH-agarose beads. Bound proteins
were resolved by SDS-PAGE and then subjected to immunoblotting with
antibodies against FLAG, Myc, or Raf1. In reciprocal experiments,
FLAG-Ras was immunoprecipitated with anti-FLAG antibody. The
immunoprecipitates were resolved by SDS-PAGE and then immunoblotted
with anti-Raf1 or anti-FLAG antibodies. Lysates were also blotted with
anti-FLAG, anti-Myc, and anti-Raf1 antibodies to show the expression of
indicated proteins. B, Erbin inhibited NRG-activated Ras
binding to Raf. FLAG-Ras (wild type) and FLAG-Erk1 were cotransfected
without or with Myc-Erbin. After stimulation with NRG, cells were lysed
and cell lysates incubated with 5 µg of GST-Raf-RBD to purify
activated GTP-bound Ras. Bound proteins were resolved on SDS-PAGE and
subjected to immunoblotting with anti-FLAG antibody. Erk1 activation
was assayed as described in the legend to Fig. 1. Lysates were also
immunoblotted with anti-Myc or anti-FLAG antibodies to demonstrate
expression of Erbin and Ras, respectively. C, Erbin
interaction with active GTP-bound Ras. Cells were co-transfected with
Myc-Erbin with FLAG-RasV12 or FLAG-RasN17. Myc-Erbin was
immunoprecipitated from cell lysates with anti-Erbin antibodies.
Resulting immunoprecipitates were subjected to SDS-PAGE and then
immunoblotting with anti-FLAG or anti-Myc antibodies. Cell lysates were
also blotted with anti-FLAG antibody to show expression of Ras
constructs. D, interaction between endogenous Erbin and Ras.
MCF-7 cells (500 µg of protein) were incubated with anti-Ras
antibody, and anti-Myc antibody as a negative control. Resulting
immunocomplexes were subjected to immunoblotting with antibodies
against Erbin, Raf, or Ras. Input, 5% of proteins for
immunoprecipitation.
-galactosidase activity as compared with the
negative control, while the interaction between Ras and Raf was
evidently detected (36).2
This suggests that the two proteins may not interact directly. To
demonstrate the possible interaction of endogenous proteins, MCF-7 cell
lysates were incubated with monoclonal antibody against Ras and the
resulting immunocomplex was blotted for Erbin, Raf1, and Ras.
Immunoprecipitation brought down Raf1 (Fig. 4D). Of note was
the presence of Erbin in the complex, suggesting that endogenous Erbin
and Ras may interact or be in the same complex.
1-965 encoding C-terminal domain of Erbin
appeared to have normal differentiation.
View larger version (34K):
[in a new window]
Fig. 5.
Erbin inhibition of PC12 cell
differentiation. A, PC12 cells were cotransfected with
pEGFP-C1 and empty vector pRK5-Myc or wild-type Erbin, LRR domain, or
C-terminal domain of Erbin. Transfected cells were stimulated without
or with NGF (100 ng/ml) for 48 h. Representative fluorescent
images of cells were shown. B, quantitative analysis of data
in A (means ± S.D., n = 3).
Asterisk, p < 0.01, Student's t
test. C, inhibition of endogenous Erbin expression by RNAi.
Cells were cotransfected with pEGFP-C1 expression vector either with
RNAi against Erbin (10 µM), or with missense scramble RNA
(10 µM) as a control. Two days after transfection,
GFP-positive cells were sorted and lysed in RIPA buffer. Cell lysates
were subjected to SDS-PAGE and Western blotting with anti-Erbin, Erk1,
and GFP antibodies. D, increase in NRG-activated Erk
activity in Erbin-siRNA expressing cells. COS-1 cells were
co-transfected with FLAG-Erk1 and Erbin-siRNA, which were then
challenged with NRG for 10 min. Erk activation was assayed as in the
legend to Fig. 1. Equal amounts of FLAG-Erk1 was shown in a reblot of
the same membrane in a representative experiment. E,
increase in differentiated PC12 cells by Erbin-siRNA. PC12 cells
cultured on 12-well plates were cotransfected with pEGFP-C1 either
alone or with Erbin-siRNA (10 µM), or with missense RNA
(10 µM) as a control. Transfected cells were stimulated
without or with NGF (20 ng/ml) for 48 h. Data shown were
means ± S.D. (n = 3). Asterisk,
p < 0.01, Student's t test.
-catenin, and ARCVS (17-22) raises a possibility that Erbin may
participate in targeting signaling complex to specific subcellular compartment. The mechanism of Erbin inhibition remains unclear. Erbin
may compete with Raf in binding to active Ras. However, we have no
evidence at present that Erbin interacts directly with Ras.
Alternatively, Erbin may compete with Sur-8 for binding to Ras. Sur-8
is an LRR-containing protein that potentiates Ras signaling presumably
by enhancing the interaction between Ras and Raf-1 (10, 12). Sur-8
contains 18 LRRs, two more than Erbin and the LRRs of Sur-8 and Erbin
share only 30% amino acid identity (17, 19). The structure differences
may account for distinct roles of these proteins in regulating Ras
signaling. Erbin binding to Ras may dissociate the Sur-8/Ras/Raf
ternary complex, resulting in down-regulation of the Erk pathway.
Active mutations of the Ras gene render it to be the most frequent
oncogene found in human cancers and even more, many other oncogenes
exploit Ras and its downstream cohorts to execute their functions.
Further study on how Erbin inhibits Erk activation may not only
contribute to a better understanding of cell signaling, but also
identify targets in development of cancer diagnosis and therapy.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. M. Weber, M. C. Hung, and M. Slikowski for reagents.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant NS40480.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. E-mail:
lmei@ nrc.uab.edu.
Published, JBC Papers in Press, October 11, 2002, DOI 10.1074/jbc.M205413200
2 Y. Huang and L. Mei, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; RIPA, radioimmune precipitation assay buffer; GFP, green fluorescent protein; LRR, leucine-rich repeat; GST, glutathione S-transferase; AChR, acetylcholine receptor; NGF, nerve growth factor; NRG, neuregulin.
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