Phosphorylation of epidermal growth factor receptor at serine 1047 by MAP kinase-activated protein kinase-2 in cultured lung epithelial cells treated with flagellin
Highlights
► Treatment with flagellin of A549 cells activated p38 MAP kinase and MAPKAPK-2. ► Flagellin treatment induced phosphorylation of EGFR at Ser1047. ► The phosphorylation was inhibited by p38 MAP kinase and MAPKAPK-2 inhibitors. ► TNFα treatment induced the phosphorylation of EGFR at both Ser1047 and Tyr1173. ► These results suggested that MAPKAPK-2 directly phosphorylated EGFR at Ser1047.
Section snippets
Materials
The following chemicals and reagents were obtained from the indicated sources: [γ-32P]ATP, PerkinElmer Japan Co. (Tokyo, Japan); Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS), Sigma Chemical Co. (St Louis, MO); fetal calf serum (FCS), HyClone (Logan, UT); DynaMarker Protein MultiColor, BioDynamics Laboratory Inc. (Tokyo, Japan); U0126, anti-MAPKAPK-2 antibody, anti-phospho-MAPKAPK-2 (Thr222) rabbit monoclonal antibody (9A7), anti-phospho-MAPKAPK-2 (Thr334) rabbit
Phosphorylation of EGFR at Ser1047 by flagellin
We first examined whether or not phosphorylation of EGFR was increased by the flagellin treatment in A549 cells (Fig. 1). The phosphorylation of EGFR at Ser1047 was increased approximately 3.2 and 14.6-fold by flagellin at 0.2 and 2 μg/ml, respectively, compared with the control (Fig. 1A). In contrast, no increase in phosphorylation at Tyr1173 was observed, suggesting that flagellin did not activate EGFR. When the protein level of EGFR was examined, no significant changes by flagellin were
Discussion
The experiments with MK2a inhibitor clearly indicated that MAPKAPK-2 was involved in the phosphorylation of EGFR at Ser1047 in A549 cells treated with flagellin and TNFα (Fig. 2, Fig. 6). We then confirmed that purified MAPKAPK-2 could phosphorylate EGFR at Ser1047 in vitro (Fig. 8). These results strongly suggested that MAPKAPK-2 directly phosphorylated EGFR at Ser1047. We found that the phosphorylation by TNFα was transient, but more pronounced than that by flagellin (Fig. 1, Fig. 2, Fig. 3,
Conflict of interest
The authors declare that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.
Acknowledgments
We thank Dr. H. Ichijo (University of Tokyo) and Dr. H. Nakanishi (Kumamoto University) for kindly providing the cDNAs for MAPKAPK-2 and EGFR, respectively. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (K.S. and H.Y.).
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