Kinetics of p56lck and p60src Src homology 2 domain binding to tyrosine-phosphorylated peptides determined by a competition assay or surface plasmon resonance.
Src homology 2 (SH2) domains are phosphotyrosine-binding modules found within various signal-transducing proteins. We have determined by 125I competition assay and surface plasmon resonance that the SH2 domains of Src and Lck bind to a variety of phosphopeptides with similar affinity and specificity. Both bound with highest affinity [Kd(app) approximately 3.7 nM; ka = 2.4 x 10(5) M-1 x s-1; kd = 1.2 x 10(-3) s-1] a phosphopeptide having a Tyr(P)-Glu-Glu-Ile motif found in the hamster polyomavirus middle-sized tumor antigen. Intermediate affinity (5- to 40-fold lower) was observed with phosphopeptides corresponding to the regulatory domains of Src and Lck, containing Tyr527 and Tyr505, respectively. Lowest affinity (80- to 300-fold lower) was observed with phosphopeptides corresponding to phosphorylated tyrosines of GTPase-activating protein, insulin receptor substrate 1, and SH2 domain-containing protein-tyrosine-phosphatase 1.
Payne
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Shoelson
S E
SE
Gish
G D
GD
Pawson
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T
Walsh
C T
CT
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D016391:Genes, src; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism*; D010748:Phosphopeptides / Q000378:metabolism*; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives
D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D015688:Oncogene Protein pp60(v-src)