Background
ERK (extracellular-signal-regulated kinase)/MAPKs (mitogen-activated protein kinases) are cytoplasmic serine/threonine kinases that transduce signals from the surface to the interior of the cell [
1]. ERK1/2 is activated in response to multiple stimuli, including those that regulate cellular proliferation, differentiation, and survival [
1]. Once activated, ERK1/2 disperses throughout the cell and phosphorylates a broad spectrum of substrates localized in different subcellular compartments, including the nucleus, and the cytoplasm [
2]. The balance between the cytoplasmic and nuclear components of ERK1/2 signaling is critical for the biological outcomes resulting from ERK1/2 activation [
3,
4]. Dysregulation of ERK/MAPK signaling is closely correlated with multiple diseases, including cancer, autoimmunity, and Alzheimer’s disease [
5]. Increased ERK1/2 activity is found in majority of cancers and is a key event in tumor cell survival and proliferation [
6]. The RAS/RAF/MEK/ERK/MAPK pathway has been reported to be activated in over 88 % of gliomas [
7]. Both the RAS and RAF oncogenes are believed to promote initiation of human cancers by activating the ERK/MAPK signaling pathway [
8,
9]. The aberrant nuclear accumulation of activated ERKs leads to tumor progression [
10].
Leucine-rich repeat C4 protein (LRRC4), also known as netrin-G ligand-2 (NGL-2) [
11], is a member of the leucine-rich repeat (LRR) superfamily [
12]. It is predominantly localized to the postsynaptic side of excitatory synapses and is involved in early nervous system development and differentiation, especially synapse formation [
11,
13‐
15]. LRRC4 regulates the formation of excitatory synapses through the recruitment of pre- and postsynaptic proteins [
16], participates in the differentiation of neuron and glial cells, and promotes neurite outgrowth [
17].
LRRC4 also is a tumor suppressor gene, and it is decreased in World Health Organization (WHO) grades II and III gliomas and absent in glioblastoma (WHO, grade IV) [
18]. Promoter hypermethylation and miRNA dysregulation (miR-182, miR-381, and miR-185) have been identified as mechanisms underlying LRRC4 inactivation in glioma [
19‐
21]. Enforced expression of LRRC4 reduced the activity of the Ras/c-Raf/ERK/MAPK and PI-3 K/AKT signaling pathways and inhibited cell proliferation and invasion in glioblastoma cells [
22,
23].
Here, we demonstrated that amino acids 499-513 of the C-terminal of LRRC4 bind to ERK1/2 and constitute a reverse docking domain (D domain) with a consensus sequence: (R/K)
1-2-(X)
2-6-ØA-X-ØB (where ØA and ØB are Leu, Ile, or Val) [
24,
25]. LRRC4 abolished ERK1/2 activation and inhibited ERK1/2 nuclear translocation through a direct interaction with ERK1/2 via the D domain, which inhibited ERK1/2 binding to MEK. Our results provided a novel regulatory mechanism for ERK1/2 activation and identified LRRC4 as a key modulator in ERK1/2 nuclear translocation.
Discussion
The LRRC4 gene was first characterized from human chromosome 7q31-32 by our group [
12,
18,
33]. Our studies indicated that LRRC4 is specifically expressed in brain tissue [
12] and decreases in primary brain tumor biopsies, especially in gliomas (up to 87.5%) [
12,
18]. The absence of LRRC4 expression contributes to late events in the pathogenesis of malignant glioblastoma. Studies have shown that the low expression of LRRC4 is due to the loss of heterozygosity on chromosome 7q32, promoter hypermethylation, and miRNA dysregulation in U251 cells [
18,
34]. Ectopic LRRC4 expression inhibited glioblastoma cell proliferation and invasion in an ERK-dependent manner. Therefore, LRRC4 may act as upstream of ERK1/2 [
18]. In this study, we found that LRRC4 binds with ERK1/2 and anchors ERK1/2 in the cytoplasm in HEK293 cells. The ectopic expression of LRRC4 abrogated the MEK1/2-ERK1/2 interaction in U251 cells. LRRC4 competitively inhibited the binding of ERK1/2 with MEK1/2 and prevented the phosphorylation of ERK1/2 and nuclear translocation, which further suppressed ERK-mediated activation of the downstream transcripts to inhibit cell proliferation and invasion.
These data further supported our previous hypothesis [
18] that decreased LRRC4 accelerated the initiation and progression of glioblastoma [
18,
34]. Following exogenous signaling stimulation, such as EGF, bFGF, IGF, and PDGF [
35], the decreased LRRC4 also failed to block the ERK 1/2-MEK1/2 interaction, preventing MEK1/2 from sustaining activation for ERK1/2. Moreover, the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases [
36], which can further promote MEK/ERK/MAPK signal pathway activation. However, the MEK/ERK/MAPK pathway is one of the most frequently aberrantly activated signaling pathways in human cancers [
37], including over 88% of gliomas [
7].
Our studies also showed that LRRC4 binds to the CD domain of ERK1/2 via the D domain of the C-terminus. The D domain, also known as the kinase interaction motif (KIM), is a conserved amino acid sequence that has since been identified in nearly every MAPK regulatory protein, including MEKs, phosphatases, and substrates. The D domain is characterized by a consensus sequence: (R/K)1-2-(X)2-6-ØA-X-ØB (where ØA and ØB are Leu, Ile, or Val) [
25]. The CD domain is the docking domain in the C-terminal lobe of MAPKs that determines binding specificity with substrate proteins [
24,
38]. ERK1/2 and other MAPKs contain the CD domain, which includes aspartate residues 316 and 319 (labeled for ERK2) that are located on the opposite side of the TXY activation loop [
24,
39] and mediate interactions with the D domains [
39‐
41]. The D domain within Schnurri-3 mediated the interaction with ERK and inhibition of ERK activity and osteoblast differentiation [
42]. Human scribble (hScrib) interacts with ERK through two D domain docking sites and decreases activation of ERK [
43]. Ephrin-B3 (eB3), which contains a D domain, regulates synapse density by directly binding to ERK1/2 to inhibit postsynaptic Ras/MAPK signaling, and knockdown of eB3 resulted in a significant increase in the percentage of neurons with nuclear ERK1/2 localization [
44]. In this study, we found that LRRC4 competitively binds the CD domain of ERK1/2 via the D domain, anchors ERK1/2 in the cytoplasm and prevents the activation induced by MEK.
Moreover, LRRC4 suppressed EGF-induced ERK1/2 phosphorylation and the activation of downstream transcription factors, such as ELK1, FOXO3a, and CDC25a, by preventing ERK1/2 translocation into the nucleus. ELK1 is a member of the Ets family of transcription factors and of the ternary complex factor subfamily [
45,
46]. Activation of the ELK1 led to increased survival and proliferation following EGF stimulation in the U138 glioblastoma cells [
47]. FOXO3a belongs to the forkhead family of transcription factors, which are characterized by a distinct forkhead domain [
48,
49]. FOXO3a is an important regulator of proliferation and apoptosis in mantle cell lymphoma [
49]. FOXO3a functions as a growth factor and promotes the proliferation of serum-deprived hepatocellular carcinoma cells [
50]. CDC25a, a member of the CDC25 family of phosphatases, is required for progression from G1 to the S phase of the cell cycle [
51,
52]. Interfering with CDC25a suppresses the growth and invasion in tumor cells [
51,
53,
54]. Our results indicated that enforced LRRC4 expression prevents the activation of ERK downstream transcription factors to inhibit glioblastoma cell proliferation and invasion. The D domain is a critical domain for the LRRC4 anti-proliferation and anti-invasion activities.
Methods
Cells culture and reagents
The human glioblastoma cell line U251 and human embryonic kidney (HEK) 293 cells were maintained in DMEM medium with high glucose and sodium pyruvate and supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. Antibodies against ELK-1 (BM0191) and phospho-ELK-1 (BM1095) were purchased from Abzoom Biolabs, Inc. (Dallas, TX, USA). Antibodies against MEK1/2 (#9122) and phospho-ERK1/2 (#9101) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against ERK1 (sc-94), ERK2 (sc-154), and GAPDH (sc-32233) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against CDC25a (DP0870), phospho-CDC25a (DP0150), FOXO3a (DR1805), and phospho-FOXO3a (DP0315) were from UcallM Biotechnology Co., Ltd. (Wuxi, China). Antibodies against GFP (AG281), GST (AG768), and Histone H3 (AH433) were purchased from Beyotime Institute of Biotechnology (Jiangsu, China).
Cell transfection
Cell transfection was performed using Lipofectamine 2000 (Invitrogen–Life Technologies, Carlsbad, CA, USA) using the manufacturer’s instructions.
Confocal and image analysis
The cultured cells were plated on coverslips and transfected with plasmids. After transfection for 48 h, the cells were washed with PBS, fixed in 4% paraformaldehyde (PFA) at room temperature for 30 min, and incubated with 0.1% Triton X-100 in PBS for 10 min. Nuclear staining was performed with DAPI (Beyotime Institute of Biotechnology, Jiangsu, China). Coverslips were mounted and examined using a confocal laser scanning microscope (UltraView, Perkin Elmer, Cambridge, UK). Images were analyzed with ImageJ v1.440 (National Institutes of Health, Bethesda, MD).
Immunoprecipitation
Lysates in RIPA buffer were incubated with antibody (0.3–0.6 mg) overnight at 4 °C with gentle rotation. A total of 80 ml of protein A Sepharose CL-4B beads (for rabbit immunoglobulin G [IgG]) or protein G Sepharose CL-4B beads (for mouse IgG) was added to the tubes and rotated at 4 °C for 2 h. Beads were precipitated by centrifugation at 16,000×g for 30 s and washed three times with cold RIPA buffer containing 150 mM NaCl. The pellets were resuspended in 2× Laemmli buffer and incubated at 100 °C for 5 min. The supernatants were used for western blot analysis.
Western blotting
For total cell lysates, cells were lysed in lysis buffer that contained 25 mM Tris (pH 7.4), 2 mM NaVO4, 10 mM NaF, 10 mM Na4P2O7, 1 mM EGTA, 1 mM EDTA, and 1% NP-40. A protease inhibitor cocktail and PhosSTOP were added fresh to the lysis buffer before each experiment. Equivalent concentrations of protein (ranging 1–3 mg/ml) from each sample were placed in 1.5-ml tubes. Proteins were denatured in 1× Laemmli buffer by boiling at 100 °C for 5 min. The tubes were incubated at room temperature for 15 min before separation using SDS-PAGE. After resolving the proteins in SDS-PAGE, they were transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in Tris-buffered saline (TBS; pH 7.4) with the 10% blocking reagent provided with the BM Chemiluminescence Blotting Substrate (POD) assay system for 1 hr, followed by incubation with primary antibody in TBS-Tween (TBST; pH 7.4) with 5% blocking reagent at 4 °C overnight. After the incubation, the membrane was washed three times in TBST, followed by incubation with secondary antibody in TBST 10% blocking reagent for 1 h, and washed again in TBST (three times for 20 min). Immunoblots were developed using ChemicalDocTM XRS+ (Bio-Rad, Berkeley, CA, USA). The intensity of the protein bands was quantified using Quantity One software (Bio-Rad, Berkeley, CA, USA).
GST pull-down assay
Bacterial cells were lysed using the following buffer: 20 mmol/L Tris-Cl, 150 mmol/L NaCl, 2 mmol/L EDTA, 0.5% NP40, pH 7.5. To determine the interaction between the D domain (CD domain) and ERK1/2 (LRRC4), bacterial lysates containing GST-D domain were incubated with glutathione-Sepharose 4B beads at 4 °C for 1 h. The beads were washed and incubated with bacterial cell lysates containing ERK1/2 (LRRC4), allowing the interaction between GST-D domain (GST-CD domain) and ERK1/2 (LRRC4). After washing, the GST-D domain and the bound ERK1/2 were eluted from the beads and subjected to electrophoresis.
For nuclear protein extraction from cells in 6 cm plates, cells were removed from the dishes by scraping with 300 ml of cytoplasmic lysis buffer (10 mM HEPES [pH 7.5], 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 mM KCl, 10 mM NaF, 0.1 mM Na3VO4, protease inhibitor cocktail, and PhosSTOP). Following 15 min of incubation on ice, 25 ml of 10% NP-40 was added and vortexed for 10 s. The cells were centrifuged for 1 min at 16,000×g, and supernatants were collected to obtain the cytoplasmic fractions. The pellets were resuspended in 200 ml of nuclear lysis buffer (25 mM HEPES [pH 7.5], 500 mM NaCl, 10 mM NaF, 10% glycerol, 0 .2% NP-40, 5 mM MgCl2, and 10 mM dithiothreitol [DTT]). RIPA buffer was used instead of nuclear lysis buffer for immunoprecipitation experiments. The suspension was incubated on ice for 30 min. During this incubation, lysates were vortexed every 10 min. Finally, cells were centrifuged for 10 min at 16,000×g to obtain nuclear proteins. For nuclear extraction from liver tissues, 50 mg of liver tissue was cut in small pieces and washed once with ice-cold PBS. Nuclear proteins were isolated using a commercially available kit from Pierce according to the manufacturer’s instructions, with no modifications.
CCK8 assay
Cell viability was determined with CCK8 assays. Briefly, 2000 cells/well were seeded into 96-well plates and were treated by plasmid vector transient transfection, and the absorptions of the cells were measured using a CCK8 kit (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer’s instruction at different indicated time points. Data were derived from three separate experiments with four replicates each time.
Matrigel chamber invasion assay
Diluted matrigel (BD Biosciences) was added to the upper well of the Transwell chamber (Corning Inc., Corning, NY) and reconstituted for 1 h at 37 °C. The cells were starved overnight in serum-free medium and resuspended at a concentration of 2.5×105 cells/ml in serum-free medium containing 0.1% bovine serum albumin. Then, 0.2 ml cell suspension was added to the top of each well, and a 10 mg/ml fibronectin solution was added to the bottom well of the chamber as a chemoattractant. After 36 h, the cells that had not invaded were removed from the upper surface of the filters using a cotton swab. The cells that had invaded to the lower surface of the filter were fixed with methanol and stained with H&E, and 5 random fields (409) were counted. The data are expressed as the mean value of cells per field in triplicate in two independent experiments.
Statistical analysis
All experiments were performed three times, and the data were analyzed with GraphPad Prism 5 (La Jolla, CA, USA). Differences between the variables of the groups were tested using Student’s t test or one-way ANOVA, using the SPSS 15.0 program. A p-value of <0.05 was statistically significant.
Acknowledgments
We thank Wei Wang, Bo Xiang, and Li Wang for the excellent technical assistance.