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. 2015 Jul 9:6:7679.
doi: 10.1038/ncomms8679.

Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

Yue Zhou  1 , Naoki Yamada  1 , Tomohiro Tanaka  1 , Takashi Hori  2 , Satoru Yokoyama  3 , Yoshihiro Hayakawa  3 , Seiji Yano  4 , Junya Fukuoka  5 , Keiichi Koizumi  6 , Ikuo Saiki  3 , Hiroaki Sakurai  1
Affiliations

Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

Yue Zhou et al. Nat Commun. .

Abstract

Crosstalk between inflammatory signalling pathways and receptor tyrosine kinases has been revealed as an indicator of cancer malignant progression. In the present study, we focus on EphA2 receptor tyrosine kinase, which is overexpressed in many human cancers. It has been reported that ligand-independent phosphorylation of EphA2 at Ser-897 is induced by Akt. We show that inflammatory cytokines promote RSK-, not Akt-, dependent phosphorylation of EphA2 at Ser-897. In addition, the RSK-EphA2 signalling pathway controls cell migration and invasion of metastatic breast cancer cells. Moreover, Ser-897-phosphorylated EphA2 co-localizes with phosphorylated active form of RSK in various human tumour specimens, and this double positivity is related to poor survival in lung cancer patients, especially those with a smoking history. Taken together, these results indicate that the phosphorylation of EphA2 at Ser-897 is controlled by RSK and the RSK-EphA2 axis might contribute to cell motility and promote tumour malignant progression.

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Figures

Figure 1
Figure 1. Phosphorylation of EphA2 at Ser-897 is induced by TNF-α stimulation.
(a) Whole-cell lysates from HeLa cells treated with TNF-α (20 ng ml−1) for 10, 20 and 60 min were separated by Zn2+-Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and EGFR antibodies. (b) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min were separated by Zn2+-Phos-tag SDS–PAGE and immunoblotted with anti-EphA2, pS-EphA2 and pY-EphA2. (c) Whole-cell lysates from HeLa cells treated with ephrin-A1 (100 ng ml−1) for 10 min or TNF-α for 20 min were separated by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2 and α-tubulin antibodies. (d) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were electrophoresed and probed with primary antibodies against pS-EphA2, pY-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR and α-tubulin. (e) HeLa cells were stimulated with TNF-α for 20 and 60 min. After fixation and permeabilization, cells were immunofluorescently stained with pS-EphA2, EphA2 or EGFR (clone LA1). Scale bar, 20 μm. Shown are representative images from three independent experiments.
Figure 2
Figure 2. The phosphorylation of EphA2 at Ser-897 is induced by TAK1, but not by Akt.
(a,b) HeLa (a) or T98G (b left) cells were pre-treated with LY294002 (10 μM) or MK-2206 (10 μM) for 30 min and then stimulated with TNF-α for 20 min. T98G cells were starved using FCS-free medium for 24 h, treated with LY294002 for 30 min and then treated with 10% FCS for 10 min (b, right). (c) MDA-MB-231 and Panc-1 cells were treated with LY294002 for 30 min. (d) HeLa cells stably transfected shRNA expression vectors against luciferase and TAK1 were stimulated with TNF-α for 20 min. (e) HeLa cells were transfected with siRNAs against TAK1 or negative control. At 72 h post transfection, cells were treated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pAKT, pRSK, RSK1, RSK2, TAK1, β-actin and α-tubulin antibodies.
Figure 3
Figure 3. Phosphorylation of pS-EphA2 is induced by RSK.
(a) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin antibodies. (b,c) Whole-cell lysates from HeLa cells pre-treated with LY294002 (10 μM), SB203580 (10 μM), U0126 (5 μM) or BI-D1870 (10 μM) for 30 min and then stimulated with TNF-α for 20 min were separated by Zn2+-Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and pS-EphA2 antibodies (b), or by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR, pRSK, RSK1, RSK2 and α-tubulin antibodies (c). (d) HeLa cells were pre-treated with LY294002 or BI-D1870 for 30 min and then stimulated with NaCl (0.3 M), TPA (100 ng ml−1) or EGF (10 ng ml−1) for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT and α-tubulin. (e) T98G and U-87 MG cells starved in FCS-free medium for 24 h were treated with LY294002, MK-2206, U0126 and BI-D1870 for 30 min and then stimulated with 10% FCS for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT, pERK and α-tubulin.
Figure 4
Figure 4. EphA2 at Ser-897 is phosphorylated by RSK1/2.
(a,b) HEK293 cells were transfected with expression vectors for EphA2, RSK1 and its substitution mutants. At 24 h post transfection, whole-cell lysates were immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2, pRSK, RSK1 and α-tubulin antibodies. (c) HeLa cells were transfected with siRNAs against RSK1, RSK2 or negative control. At 72 h post transfection, cells were stimulated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin. (d) Recombinant human GST-EphA2 was incubated with recombinant human active GST-RSK1 or RSK2 in the absence or presence of BI-D1870 (0.1 μM) at 30 °C for 30 min. The reaction mixtures were analysed by immunoblotting with anti-pS-EphA2, EphA2, RSK1 and RSK2 antibodies.
Figure 5
Figure 5. The RSK–EphA2 axis controls cell motility.
(a) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min or untreated MDA-MB-231 cells were separated by Zn2+-Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 antibody. (b–f) MDA-MB-231 cells were pre-treated with BI-D1870 (10 μM) for 30 min and then scratched with a pipette tip. After 48 h of incubation, whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2 and β-actin (b) Migrated cells were counted manually under a microscope (c) Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. *P<0.05 by Student's t-test. At the same time, the migration border cells were immunofluorescently stained with anti-pS-EphA2 or EphA2 antibodies (d,e) and cells harbouring lamellipodia were counted manually under a microscope (f) Scale bar, 20 μm. Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. *P<0.05 by Student's t-test. (g,h) MDA-MB-231 cells were transfected with siRNA against EphA2 or negative control and EphA2 mutation-expression plasmids. The immunoblotting results from whole-cell lysates with anti-pS-EphA2, EphA2 and β-actin antibodies are shown in g and the results of scratch assay are shown in h. Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. *P<0.05 by analysis of variance followed by Tukey–Kramer HSD test.
Figure 6
Figure 6. Molecular-targeted agents inhibited pS-EphA2.
(a) Human melanoma cells (A2058, SK-MEL-28, A375, UACC62, UACC257 and SK-MEL-2), (b) DLD-1 colon cancer cells and (c) lung adenocarcinoma cells (PC-9, HCC827, HCC4006, NCI-H1650, H2228 and A549) were treated with vemurafenib (1 μM), BI-D1870 (10 μM), gefitinib (1 μM), crizotinib (10 μM) or U0126 (5 μM) for 30–60 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pY-EGFR, EGFR, β-actin and α-tubulin.
Figure 7
Figure 7. pS-EphA2 and pRSK are co-localized in cancer patients' specimens and the RSK–EphA2 axis is associated with the overall survival of lung cancer patients.
(a) A multi-cancer tissue microarray, including 1,010 cores from 13 organ cancer tissues, was adopted for immunohistochemical staining using primary antibodies against pS-EphA2 and pRSK. Typical staining images of lung cancer tissues, including adenocarcinoma (AD) and squamous cell carcinoma (SCC), at low- and high-power magnifications are shown. Scale bar, 20 μm. (b) Typical immunohistochemical staining of pS-EphA2 and pRSK in EGFR-mutated (exon 19 deletion) lung adenocarcinoma tissues are shown. Scale bar, 20 μm. (c–f) Postoperative overall Kaplan–Meier survival curves of all the lung cancer patients (c,d) or smoking patients (e,f) were compared according to pRSK negativity or positivity (c,e) or pS-EphA2/pRSK double positivity (d,f) P values were calculated by the log-rank tests.
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