Analysis of CSF-1R and SRC-induced tyrosine phosphorylation in breast epithelial cells

Activation of the receptor tyrosine kinase (RTK), colony stimulating factor 1 receptor (CSF-1R), is associated with breast cancer progression, but the mechanism by which CSF-1 signaling mediates this is not well defined. While the downstream targets of CSF-1R are poorly characterized in epithelial cells, it is known that CSF-1R activation leads to increased tyrosine phosphorylation and activation of the nonreceptor tyrosine kinase SRC. Previous work has determined that CSF-1R activation transforms mammary epithelial cells and disrupts cell-cell adhesion through the re-localization of E-cadherin in a SRC-dependent manner.;This dissertation describes the analysis of CSF-1R-induced changes in tyrosine phosphorylation and the identification of specific SRC-regulated proteins to define key substrates of CSF-1R and SRC in breast cancer progression and the regulation of cell adhesion. Using global quantitative phosphoproteomic analysis, we detected 98 proteins that displayed increased tyrosine phosphorylation in cells expressing an activated mutant of CSF-1R. These proteins included known targets of RTK and CSF-1R and proteins linked with metastasis. Nineteen proteins previously unlinked to the CSF-1 pathway were identified and may yield novel insights into CSF-1 signaling.;We identified 19 proteins in the CSF-1R-induced phosphoprotein set whose phosphorylation was dependent on SRC recruitment to CSF-1R. Motif analysis of the sequences surrounding the phosphorylated tyrosine indicated that many are direct SRC targets. Phosphorylation of five proteins, including p120-catenin (p120) and Hrs, significantly correlated with SRC activation in human tumors. p120 and Hrs are known to regulate E-cadherin stability, and knockdown of either p120 or Hrs by small interfering RNAs mimicked CSF-1R-induced, SRC-dependent changes in adhesion. The similarity between the effect of p120 and Hrs loss and CSF-1R-SRC kinase activity on cell adhesion suggests that p120 and Hrs phosphorylation may inhibit their function. Further studies will be required to define the importance of these SRC-dependent phosphorylation sites on p120 and Hrs.;In summary, our analyses of CSF-1R-induced tyrosine phosphorylation identified novel targets of this RTK and defined a subset of proteins that are candidate targets of SRC activated by CSF-1R. Future studies of these proteins may provide a better understanding of how unregulated tyrosine phosphorylation promotes tumor progression.