Mass spectrometry-based quantification of the cellular response to ultraviolet radiation in HeLa cells

<div><p>Ultraviolet (UV) irradiation is a common form of DNA damage that can cause pyrimidine dimers between DNA, which can cause gene mutations, even double-strand breaks and threaten genome stability. If DNA repair systems default their roles at this stage, the organism can be damaged and result in disease, especially cancer. To better understand the cellular response to this form of damage, we applied highly sensitive mass spectrometry to perform comparative proteomics of phosphorylation in HeLa cells. A total of 4367 phosphorylation sites in 2100 proteins were identified, many of which had not been reported previously. Comprehensive bioinformatics analysis revealed that these proteins were involved in many important biological processes, including signaling, localization and cell cycle regulation. The nuclear pore complex, which is very important for RNA transport, was changed significantly at phosphorylation level, indicating its important role in response to UV-induced cellular stress. Protein–protein interaction network analysis and DNA repair pathways crosstalk were also examined in this study. Proteins involved in base excision repair, nucleotide repair and mismatch repair changed their phosphorylation pattern in response to UV treatment, indicating the complexity of cellular events and the coordination of these pathways. These systematic analyses provided new clues of protein phosphorylation in response to specific DNA damage, which is very important for further investigation. And give macroscopic view on an overall phosphorylation situation under UV radiation.</p></div

Network of phosphoproteins derived from data and the expanding view of phosphorylation level changes for parts of representative proteins.

<p>The different colors represent different ratios from -2 to +2. The highlight part are mismatch repair related protein—MSH6 network picture, DNA replication related protein—POLA1 and POLE network and nuclear pore complex protein—Nup153, Nup50, Nup188 and Nup214 network part.</p

Influenced phosphoproteins inrelated to DNA repair pathway under UV treatment.

<p>Diamond represents mismatch repair proteins, ellipse represents base excision repair proteins and rectangle stands for nucleotide excision repair proteins. The different colors represent different ratios of phosphorylation level from -2 to +2.</p

Quantitative overview of phosphorylated peptides and proteins in <i>HeLa</i> cells following UV treatment.

<p>(A) Number of phosphorylated proteins and proteins’sites quantified in HeLa cells in response to UV treatment. (B) Distribution of identified phosphorylated peptides at Serine, Threonine and Tyrosine sites. (C) Distribution of phosphorylated and non-phosphorylated amino acids in secondary structure. (D) Comparison of phosphorylated peptides identified in this study (I) and Phospho.ELM database (II) (<a href="http://phospho.elm.eu.org/" target="_blank">http://phospho.elm.eu.org/</a>).Validation of phosphorylation sites in RPA1 (S38) and RFC3 (T76) by western blot. Plasmids with 3xFLAG-S38A or 3xFLAG-T76A mutation were transfected to HeLa cells and precipitated by M2 beads. The phosphorylation level between wildtype and mutant proteins were evaluated by western blotting using specific antibody.</p

Phosphorylation-specific motifs using the Motif-X algorithm.

<p>(A) Pro-directed motif centered on Thr and Ser with a strong preference for additional Pro residues C-terminal to the phosphate. (B) Double-phosphorylation motifs found in our study.</p