Ionizing radiation induces EphA2 S897 phosphorylation in a MEK/ERK/RSK-dependent manner

Abstract

Purpose: The EphA2 tyrosine kinase is frequently overexpressed in human tumors that are also treated with radiation. However, few studies have examined the effect of radiation on the EphA2 receptor itself. The purpose of this project was to investigate the impact of radiation on EphA2 to better understand mechanisms of radioresistance.

Materials and methods: Cell lines were exposed to X-rays and assayed for changes in EphA2 protein levels and phosphorylation over time by Western blotting. HEK293 cells stably expressing wild-type EphA2 or the S897A mutant were analyzed for cell survival from X-rays.

Results: Treatment of different cancer cell lines with 2 Gy of X-rays induced the phosphorylation of EphA2 on S897 but no changes were found in EphA2 total levels or its tyrosine phosphorylation. Radiation-induced S897 phosphorylation was unaffected by an AKT inhibitor but blocked by a MEK or RSK inhibitor. HEK293 cells expressing the EphA2 S897A mutant had a nearly 2-fold lower level of cell survival from X-rays than cells expressing wild-type EphA2.

Conclusions: These findings show that radiation induces S897 EphA2 phosphorylation, an event associated with increased cell survival. Therefore, targeting pathways that mediate EphA2 S897 phosphorylation may be a beneficial strategy to reduce radioresistance.

Keywords:

• Radiosensitivity
• signal transduction
• cellular radiobiology

Acknowledgements

We thank Dr Elena Pasquale for wild-type and S897A mutant EphA2 plasmids and Dr Sean Palecek for NCI-H1650 cells. We also thank Pamela Ross, Dr Andreas Kyriacou and Dr John Parameritis for cell irradiation and Dr Peck Sun-Lin for technical advice.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Notes on contributors

Paul Graves received his Ph.D. from Indiana University and completed post-doctoral training at Washington University. He is chief of radiobiology at New York-Presbyterian Brooklyn Methodist Hospital and is engaged in teaching and research. Email: [email protected]

Shaun Din received his M.D. from St. George’s University. He spent two years as a post-doctoral fellow at Memorial Sloan-Kettering Cancer Center. He is now a third-year resident in radiation oncology at New York-Presbyterian Brooklyn Methodist Hospital. Email: [email protected]

Mark Ashamalla is a radiation oncology resident at New York-Presbyterian Brooklyn Methodist Hospital. He received his M.D. from Jefferson Medical College and is engaged in patient care and laboratory research. Email: [email protected]

Hani Ashamalla is chairman of the radiation oncology department at New York-Presbyterian Brooklyn Methodist Hospital. He received his M.D. degree from Ain Shams University Faculty of Medicine. Email: [email protected]

Thomas Gilbert is a research technician at the UNC Department of Pharmacology. He holds a B.S. degree in Cellular and Molecular Biology from the University of North Carolina at Asheville. Email: [email protected]

Lee Graves received his Ph.D. from the University of Illinois and is a professor in the department of Pharmacology at the University of North Carolina. He is actively engaged in teaching and research. Email: [email protected]

Additional information

Funding

This research was supported by funding from New York-Presbyterian Brooklyn Methodist Hospital and by a grant from the University Cancer Research Fund (UCRF) [grant number P30 CA016086] to Lee Graves.