Growth factors and their cognate receptor tyrosine kinases (RTK) are key regulators of tumor cell initiation and progression 
. Growth factor binding and subsequent RTK auto-phosphorylation lead to the activation of pathways that regulate cell proliferation, survival, growth, adhesion and motility. Inappropriate RTK activation can drive tumor cell growth, survival, invasion and metastasis. RTKs like epidermal growth factor receptor (EGFR) and epidermal growth factor receptor 2 (Her2/ERBB2) are overexpressed or activated in a variety of human cancers 
. In non-small cell lung cancers (NSCLC), activating EGFR
mutations are found in 10–15% of Caucasian and 30–40% Asian patients 
. ERBB2, as a further example, is upregulated by gene amplification in 15–30% of invasive mammary ductal cancers 
. RTKs are known to activate several downstream tyrosine kinases, including members of the SRC, ABL, and JAK kinase families. These cytosolic kinases make a significant contribution to the dramatic increase in tyrosine phosphorylation induced by RTKs. It has been difficult to define the precise subset of proteins targeted by any individual tyrosine kinase that is a component of these kinase signaling cascades due to the overlap in substrates phosphorylated by activated RTKs and non-receptor tyrosine kinases. Previous studies have utilized activated mutant variants of non-receptor tyrosine kinases like SRC to identify downstream substrates of this kinase subfamily; however, these overexpressed, constitutively active mutants likely display promiscuous activities that do not necessarily reflect the substrates of the endogenous protein when activated by an upstream RTK.
SRC and other SRC family kinases (SFKs) are activated downstream of many different RTKs 
. SRC activity is critical for several phenotypic events induced by RTK activation including DNA synthesis, cytoskeletal reorganization and disruption of cell-cell adhesion 
. In human tumors, RTK activation of SFKs may contribute to tumor progression and lead to more aggressive tumor phenotypes 
. Dominant negative SRC mutants, pharmacological inhibition of SRC kinase activity, and SRC-specific docking site RTK mutants have been used to address the specific role of SRC in RTK signal transduction 
. Studies in breast cancer models using these methods have demonstrated that inhibition of SRC kinase activity suppresses phenotypic effects induced by the overexpression or activation of RTKs like EGFR and ERBB2, e.g. anchorage-independent growth, motility and survival 
. Therefore, RTK-induced SRC activity drives aspects of RTK signaling important in tumor progression, and the identification of RTK-induced SRC substrates will offer further insight into the role of SRC in tumorigenesis.
We previously demonstrated 
that Src activation regulates a subset of phenotypic alterations induced by the colony stimulating factor 1 receptor tyrosine kinase (CSF-1R), which has been implicated in the progression of multiple types of carcinoma including breast cancer 
. SRC activity was found to be critical for CSF-1R-induced disruption of cell-cell adhesion of MCF-10A cells, immortalized, non-transformed mammary epithelial cells 
. These alterations are associated with loss of plasma membrane association of E-cadherin, a key regulator of epithelial intercellular adhesion. The CSF-1R tyrosine phosphorylation site that binds to SRC and other SFKs, Y561, was found to be critical for disruption of cell-cell adhesion 
. Expression of a dominant-negative mutant of SRC (c-SRC K295R) or pharmacological inhibition of SRC kinase activity in the presence of CSF-1R activation preserves cell-cell adhesion, providing additional evidence that SRC activity and phosphorylation of SRC substrates are involved in the disruption observed 
Few studies have examined the specific set of SRC substrates phosphorylated after RTK activation 
. A recent mass spectrometry (MS) analysis by Amanchy and colleagues identified 43 candidate SRC substrates downstream of platelet-derived growth factor (PDGF) receptor in fibroblasts using SU6656, a pharmacological inhibitor of SRC kinase activity 
. While this work identified candidate substrates of endogenous SRC, all cellular SRC family kinases were inhibited by this inhibitor, so this analysis did not distinguish substrates that are directly downstream of PDGFR activation from those regulated by adhesion receptors and other cellular processes. Several MS analyses have identified proteins that display increased tyrosine phosphorylation in cells overexpressing wild-type SRC or an activated mutant variant 
. However, the relevance of these targets in human tumors is not clear, as SRC is not frequently mutated or overexpressed in breast cancers, and it is more likely activated in breast carcinomas by an upstream activated RTK or another receptor known to couple to SRC 
Using quantitative MS analysis, we examined CSF-1R-induced tyrosine phosphorylation using quantitative PhosphoScan® analysis. PhosphoScan® analysis is an MS approach that utilizes immunoaffinity purification of tyrosine phosphorylated peptides prior to MS analysis to enrich their representation in the sample 
. We profiled site-specific changes in tyrosine phosphorylation induced by activated CSF-1R and activated CSF-1R defective for the recruitment of SFKs in MCF-10A cells. To do this, we utilized a previously described constitutively active variant of CSF-1R (CA-CSF-1R) and CA-CSF-1R with a tyrosine to phenylalanine substitution at Y561 (CA-CSF-1R Y561F), the recruitment site for SRC and SFKs 
. This phosphotyrosine proteomic MS analysis of parental MCF-10As and cells expressing the two CSF-1R variants allowed us to characterize CSF-1R-induced tyrosine phosphorylation events and to distinguish Y561-independent and Y561-dependent phosphorylation events stimulated by the activated CSF-1R. The Y561-dependent substrates revealed a set of putative SRC substrates and subsequent bioinformatics analyses of these targets in human tumor datasets identified potential biomarkers of SRC activation in human tumors.