Research Highlights
June 2006
Researchers Profile Cells to Determine Radiation Response
In research supported by the U.S. Department of Energy (DOE) Low Dose Radiation program, Pacific Northwest National Laboratory (PNNL) researchers have successfully profiled human skin cells to explore differences in low- and high-dose irradiation responses at the protein phosphorylation level. They combined several techniques to analyze the human skin fibroblast (HSF) cell phosphoproteome, and their results provide an initial view of phosphorylation events in response to low- and high-dose radiation and a basis for determining potential functions for site-specific protein phosphorylation. The research appeared in the May 2006 issue of the Journal of Proteome Research.
Human cellular response to high doses of radiation includes up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well-documented biological effects, but the underlying regulatory mechanisms are still poorly understood.
To profile the phosphoproteome of normal HSF cells and determine how irradiation affects the phosphoproteome profiles, the researchers combined protein extraction, peptide methylation, phosphopeptide enrichment with immobilized metal affinity chromatography, nanoflow reversed-phase HPLC electrospray ionization, and tandem mass spectrometry to analyze the HSF cell phosphoproteome.
They identified 692 phosphorylation sites that are assigned to 347 proteins. In some cases, a clear cellular function was associated with the identified phosphorylation sites, providing possible insights into the role of phosphorylation following radiation treatment. However, many of the sites identified in this study have not been characterized previously; therefore, identifying these novel phosphorylation events may shed light on the regulatory mechanisms involved in important cellular functions.
Significantly, the researchers found that the phosphorylation profile for proteins identified from low-dose irradiated HSF cells was different from the profile obtained for proteins irradiated at the high-dose level. This suggests that both low- and high-doses of radiation might affect distinct signaling pathways. These dose-dependent radiation-responses may form a basis for identifying relevant molecular markers that can be used in the future to better evaluate human health risks at low doses of irradiation and to develop low-dose radiation counter measurements. Further improvements to the present measurements are anticipated.
The research team includes Feng Yang, David Stenoien, Eric Strittmatter, Mary Lipton, Matt Monroe, Carrie Nicora, Marina Gritsenko, Keqi Tang, Ruihua Fang, Josh Adkins, Dave Camp, and Dick Smith, all PNNL; and Junhua Wang, Lianghao Ding, and David Chen, University of Texas Southwestern Medical Center.
Survey of phosphorylation motifs to determine if distinct signaling pathways are activated by low- and high-dose irradiation,. Primary sequences surrounding the identified phosphorylation sites in each phosphopeptide were queried against distinct kinase motifs and revealed potential kinase activities (copyright © 2006 American Chemical Society).
Reference
Yang F, DL Stenoien, EF Strittmatter, J Wang, L Ding, MS Lipton, ME Monroe, CD Nicora, MA Gritsenko, K Tang, R Fang, JN Adkins, DG Camp, II, DJ Chen, and RD Smith. 2006. "Phosphoproteome Profiling of Human Skin Fibroblast Cells in Response to Low- and High-Dose Irradiation." J Proteome Res 5(5):1252-1260.