Identification of Brk cellular substrates by phosphoproteomic and signaling approaches
Date Issued
2009
Date
2009
Author(s)
Li, Fang-Yen
Abstract
Breast tumor kinase (Brk), a non-receptor tyrosine kinase, is frequently overexpressed in breast cancers. Previous studies have shown that Brk acts downstream of epidermal growth factor to promote cell proliferation and migration. These biological functions of Brk are thought to be resulted from its phosphorylation of cellular proteins, but only a limited number of substrates have been discovered to date. In the thesis, we took use a systematic proteomic approach to identify putative Brk substrates by comparing the tyrosine phosphorylated proteins in Brk-overexpresing cells and control cells. Using immunoaffinity purification of tyrosine-phosphorylated proteins followed by enrichment of phosphopeptides, 127 phosphoproteins are observed only in Brk-overexpresing cells. Most of them belong to RNA-binding proteins involving in various RNA metabolic processes, such as splicing, transcription, and RNA processing. In addition, several of them are known to elicit effects on regulating actin cytoskeleton and/or cell migration. Importantly, several known Brk substrates, including Sam68, SLM-2, and PSF, are also recovered. We next validate the Brk-induced tyrosine phosphorylation of two identified proteins, GIT2 and ARHGEF6. Importantly, Brk not only promotes their phosphorylation in vivo but also physically interacts with both proteins. Moreover, we show that the tyrosine residues Y286 and Y592 of GIT2 are both involved in Brk-induced phosphorylation. Finally, we have extended our analysis to EPS8, another putative Brk substrate identified previously in our laboratory. Using in vitro kinase assay, we demonstrate EPS8 as a direct substrate of Brk and identify five Brk-targeting tyrosine residues. Together, our analysis has uncovered several Brk downstream targets, which would provide a basis for future analysis of Brk’s biological functions.
Subjects
phosphoproteomics
mass spectrometry
SDGs
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