Using a phage display library to identify basic residues in A-Raf required to mediate binding to the Src homology 2 domains of the p85 subunit of phosphatidylinositol 3'-kinase.
Src homology 2 (SH2) domains are found in a variety of cytoplasmic proteins involved in mediating signals from cell surface receptors to various intracellular pathways. They fold as modular units and are capable of recognizing and binding to short linear peptide sequences containing a phosphorylated tyrosine residue. Here we show that each of the SH2 domains of the p85 subunit of phosphatidylinositol 3-kinase selects phage displayed peptide sequences containing the core (L/I)-A-(R/K)-I-R. The serine/threonine kinase A-Raf, containing the sequence LQRIRS, is associated with the p85 protein in both quiescent and growth factor stimulated cells. This suggests that p85 and A-Raf exist in a protein complex in cells and that complex formation does not require growth factor stimulation. We also show that p85 and A-Raf can bind directly to each other in vitro and that this interaction is mediated in part by the p85 SH2 domains. Further, the p85 SH2 domains require at least one of four distinct basic-X-basic sequence motifs within A-Raf for binding. This is the first description of a phosphotyrosine-independent SH2 domain interaction that requires basic residues on the SH2 ligand.
King
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Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
Fang
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Y
Mahon
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Anderson
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DH
D016475:3T3 Cells; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D006801:Humans; D051379:Mice; D009154:Mutation; D019151:Peptide Library; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D055672:Static Electricity; D018909:src Homology Domains
D019151:Peptide Library; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D019869:Phosphatidylinositol 3-Kinases; D019908:Proto-Oncogene Proteins c-raf