Independent SH2-binding sites mediate interaction of Dok-related protein with RasGTPase-activating protein and Nck.
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.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Binding Sites, Carrier Proteins, Cell Lineage, DNA, Complementary, GTPase-Activating Proteins, Gene Library, Hematopoietic Stem Cells, Insulin Receptor Substrate Proteins, Intracellular Signaling Peptides and Proteins, Lymphoid Tissue, Male, Membrane Proteins, Mice, Molecular Sequence Data, Oncogene Proteins, Phosphoproteins, Phosphorylation, Protein Binding, Proteins, Sequence Homology, Amino Acid, Substrate Specificity, Tissue Distribution, src Homology Domains, src-Family Kinases
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Binding Sites, Carrier Proteins, Cell Lineage, DNA, Complementary, GTPase-Activating Proteins, Gene Library, Hematopoietic Stem Cells, Insulin Receptor Substrate Proteins, Intracellular Signaling Peptides and Proteins, Lymphoid Tissue, Male, Membrane Proteins, Mice, Molecular Sequence Data, Oncogene Proteins, Phosphoproteins, Phosphorylation, Protein Binding, Proteins, Sequence Homology, Amino Acid, Substrate Specificity, Tissue Distribution, src Homology Domains, src-Family Kinases
J. Biol. Chem.
Date: Aug. 06, 1999
PubMed ID: 10428862
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