A murine embryonic cDNA library was screened for potential substrates of the Src family kinase, Lyn, using a phosphorylation-screening strategy. One cDNA that we identified encodes Dok-related protein (DokR), a protein with homology to p62(dok) (Dok), and members of the insulin receptor substrate-1 family of proteins. Analysis of murine tissue extracts with DokR-specific antisera revealed that DokR protein is expressed at highest levels in lymphoid tissues. Co-expression of a FLAG epitope-tagged form of DokR (FLAG-DokR) with Lyn in embryonic kidney 293T cells resulted in constitutive phosphorylation of FLAG-DokR on tyrosine residues and consequential physical association with RasGTPase-activating protein (GAP) and the Nck adaptor protein. Stimulation of BaF/3 hematopoietic cells co-expressing the epidermal growth factor (EGF) receptor tyrosine kinase and FLAG-DokR with EGF also induced phosphorylation of FLAG-DokR and promoted its association with GAP. Immunoprecipitation experiments using DokR-specific antibodies revealed an interaction between endogenous DokR and a 150-kDa protein that is tyrosine-phosphorylated in EGF-stimulated BaF/3 cells. The molecular basis of the interactions involving DokR with GAP and Nck was investigated using a novel glutathione S-transferase fusion protein binding assay and/or site-directed mutagenesis. Tandem SH2-binding sites containing Tyr-276 and Tyr-304 were shown to mediate binding of DokR to GAP, whereas Tyr-351 mediated the binding of DokR to Nck. These results suggest that DokR participates in numerous signaling pathways.