The Src Homology 2 (SH2) and Src Homology 3 (SH3) domain-containing protein Crk is the prototypical member of a class of adaptor proteins that play essential roles in signaling downstream of tyrosine kinases. By promoting the assembly of protein complexes mediated by the SH2 and SH3 domains, evidence accumulated over the past two decades has elucidated a canonical pathway for Crk signaling whereby the SH2 domain binds tyrosine phosphorylated proteins and the N-terminal SH3 domain (SH3N) binds guanine-nucleotide exchange factors that activate Rac1, Rap1, and Ras. The clinical significance of Crk in human cancer has been enumerated in recent years, as Crk is frequently over-expressed in several different cancers, including breast, ovarian, gastric, lung, glioblastoma, and sarcomas and siRNA-mediated knockdown of Crk reverses the malignant and metastatic features of these cancers. These observations have led to a new urgency to understand the mechanisms by which Crk promotes malignant transformation in the hope that new information can be exploited to develop therapeutics, particularly for tumors with a predisposition towards invasion and metastasis. In this application we have identified a new signaling paradigm for Crk by the identification of two previously uncharacterized tyrosine phosphorylation sites located within the carboxyl-terminal SH3 (SH3C) domain, an atypical SH3 domain that has no clear biological function. Tyr251 is located in the highly conserved RT-loop of the Crk SH3C while Tyr239 is located at the boundary of the linker and SH3C and comprises a region of Crk implicated in the negative regulation and auto-clamping of the SH3C to the SH3N. The central hypothesis to be tested is that in addition to its conventional role as an adaptor protein, Crk has an unorthodox, non-canonical role in signaling by virtue of being phosphorylated on Tyr239 and Tyr251 in the SH3C, phosphorylation which will define new binding sites for proteins with SH2 or PTB domains, and hence engage novel phosphorylation dependent signaling pathways. Since both Tyr251 and Tyr239 show elevation in tyrosine phosphorylation upon EGF stimulation, and the Y251F Crk mutant has a diminished ability to promote cell migration and invasion towards EGF compared to WT Crk, we hypothesize that mechanistic studies will be immediately relevant to invasive triple negative breast cancer cells that have elevated EGFR. These studies may have important implications in the development of therapeutic strategies to profile and target Crk-expressing tumors, but also provide rationale for exploring this biology in a wide spectrum of human cancers. PUBLIC HEALTH RELEVANCE: Src Homology-2 (SH2) and Src Homology-3 (SH3) containing proteins are among the most versatile proteins in eukaryotic cells, and critically important for signaling in human cancers. The research outlined in this proposal defines a new function for the Crk oncogene product. In recent years, an emerging body of evidence suggests that Crk proteins are overexpressed in human tumors and the expression levels correlate with aggressive and malignant behavior of cancer cells. These properties of Crk proteins make them potential cancer prognosis markers and therapeutic targets.