Abstract
To investigate the presence of, and the potential biological roles of, protein tyrosine phosphorylation in the glioblastoma pathogenesis, two-dimensional gel electrophoresis- (2DGE-) based Western blotting coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis was used to detect and identify the phosphotyrosine immunoreaction-positive proteins in a glioblastoma tissue. MS/MS and Mascot analyses were used to determine the phosphotyrosine sites of each phosphopeptide. Protein domain and motif analysis and systems pathway analysis were used to determine the protein domains/motifs that contained phosphotyrosine residue and signal pathway networks to clarify the potential biological functions of protein tyrosine phosphorylation. A total of 24 phosphotyrosine-containing proteins were identified. Each phosphotyrosine-containing protein contained at least one tyrosine kinase phosphorylation motif and a certain structural and functional domains. Those phosphotyrosine-containing proteins were involved in the multiple signal pathway systems such as oxidative stress, stress response, and cell migration. Those data show 2DGE-based Western blotting, MS/MS, and bioinformatics are a set of effective approaches to detect and identify glioblastoma tyrosine-phosphorylated proteome and to effectively rationalize the biological roles of tyrosine phosphorylation in the glioblastoma biological systems. It provides novel insights regarding tyrosine phosphorylation and its potential role in the molecular mechanism of a glioblastoma.
Highlights
Tyrosine phosphorylation that is an addition of phosphogroup (–HPO3 to –OH or –H3PO4 to –NH2) to the tyrosine residue is a type of protein posttranslational modification that plays key roles in the signal transduction and participates in many physiological and pathological processes such as growth, proliferation, differentiation, aging, cancer, and inflammatory diseases [1,2,3]
Tyrosine phosphorylation and dephosphorylation are a reversibly dynamic mechanism that is regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) [4]
518 human protein kinase genes [8] including 90 known tyrosine kinases that include 58 receptor tyrosine kinases (RTKs) [9, 10] and 107 tyrosine phosphatases [11] have been discovered for potential targets of anticancer drugs, most tyrosine kinases are regulated negatively and only activated under certain conditions [8], and interestingly tyrosine kinases accounting for 0.3% of genome contribute to a large proportion (30%) of 100 known dominant oncogenes [10, 12]
Summary
Tyrosine phosphorylation that is an addition of phosphogroup (–HPO3 to –OH or –H3PO4 to –NH2) to the tyrosine residue is a type of protein posttranslational modification that plays key roles in the signal transduction and participates in many physiological and pathological processes such as growth, proliferation, differentiation, aging, cancer, and inflammatory diseases [1,2,3]. Tyrosine phosphorylation and dephosphorylation are a reversibly dynamic mechanism that is regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) [4]. Tyrosine kinase phosphorylation generally occurs within a consensus pattern/motif [R/K]-x(2)-[D/E]-x(3)-Y or [R/K]-x(3)-[D/E]x(2)-Y (Y = the phosphorylation site) [5,6,7]. Characterization of altered modification and functional activities of phosphotyrosine-containing proteins in different types of cancers has helped in the discovery of specific tyrosine kinase inhibitors to treat a cancer [9, 14]. It emphasizes the scientific importance of investigating phosphotyrosine-containing proteins in a cancer
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