SHEP1 is a signaling protein that contains a guanine nucleotide exchange factor-like domain, which binds Ras family GTPases and also forms a stable complex with the scaffolding protein Crk-associated substrate (Cas). SHEP1 and Cas have several common functions, such as increasing c-Jun N-terminal kinase activity, promoting T cell activation, and regulating the actin cytoskeleton. However, it is unclear whether a physical association between SHEP1 and Cas is required for these activities. We reported previously that SHEP1 is tyrosine-phosphorylated downstream of the EphB2 receptor; in this study, we further demonstrate that activated EphB2 inhibits SHEP1 association with Cas. To investigate whether phosphorylation negatively regulates the SHEP1-Cas complex, we have identified by mass spectrometry several SHEP1 tyrosine phosphorylation sites downstream of EphB2; of particular interest among them is tyrosine 635 in the Cas association/exchange factor domain. Mutation of this tyrosine to glutamic acid, but not to phenylalanine, disrupts Cas binding to SHEP1 without inhibiting Ras GTPase binding. The glutamic acid mutation also makes SHEP1 unable to promote Cas-Crk association, membrane ruffling, and cell migration toward epidermal growth factor (EGF), implying that these activities of SHEP1 depend upon a physical interaction with Cas. Association with Cas also seems to be necessary for EGF-induced SHEP1 tyrosine phosphorylation, which is mediated by a Src family kinase. It is noteworthy that EGF stimulation does not cause dissociation of SHEP1 from Cas. These data show that SHEP1 regulates membrane ruffling and cell migration and that binding to Cas is probably critical for these functions. Furthermore, the SHEP1-Cas complex may have different roles downstream of EphB2 and the EGF receptor.