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.
Lock
P
P
Ludwig Institute for Cancer Research and the Cooperative Research Center for Cellular Growth Factors, P. O. Box 2008, Royal Melbourne Hospital, Parkville 3050, Australia. Peter.Lock@Ludwig.edu.au
Casagranda
F
F
Dunn
A R
AR
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D019070:Cell Lineage; D018076:DNA, Complementary / Q000235:genetics; D020690:GTPase-Activating Proteins; D015723:Gene Library; D006412:Hematopoietic Stem Cells; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008221:Lymphoid Tissue; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018076:DNA, Complementary; C498444:Dok2 protein, mouse; C106534:FRS2 protein, human; D020690:GTPase-Activating Proteins; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526298:Irs2 protein, mouse; D008565:Membrane Proteins; C078332:Nck protein; D015513:Oncogene Proteins; D010750:Phosphoproteins; D011506:Proteins; D019061:src-Family Kinases