Identification of residues in the beta platelet-derived growth factor receptor that confer specificity for binding to phospholipase C-gamma 1.
The SH2 domains of cytoplasmic signaling proteins bind to autophosphorylated growth factor receptors by direct recognition of specific phosphotyrosine-containing sites. To identify the phosphotyrosine involved in association of phospholipase C (PLC)-gamma 1 with the beta platelet-derived growth factor receptor (PDGFR), and to investigate which contiguous residues confer specificity for PLC-gamma 1, phosphotyrosine-containing glutathione S-transferase (GST) fusion proteins possessing different regions of the beta-PDGFR were incubated with lysates of Rat-2 cells that overexpress PLC-gamma 1. The phosphorylated C-terminal tail of the PDGFR bound PLC-gamma 1, but did not associate with phosphatidylinositol (PI) 3'-kinase or GTPase-activating protein (GAP). High-affinity binding of PLC-gamma 1 was dependent on phosphorylation of Tyr-1021. Creation of a new phosphorylation site by replacing Asp-1018 with tyrosine did not restore binding of PLC-gamma 1 in the absence of Tyr-1021, indicating that the location of the phosphorylated tyrosine is important for PLC-gamma 1 binding. Substitution of the proline at the +3 position relative to Tyr-1021 with methionine (Y1021IIP-->Y1021IIM) in the phosphorylated PDGFR tail did not alter PLC-gamma 1 association, but conferred binding activity towards PI 3'-kinase, indicating that this residue is critical in discriminating between PLC-gamma 1 and PI 3'-kinase. Progressive conversion of the three residues C-terminal to Tyr-1021 to the consensus for PI 3'-kinase binding (YMDM) allowed PI 3'-kinase association, but did not block PLC-gamma 1 binding, suggesting that additional residues other than the three residues immediately following the phosphotyrosine may contribute to the association of PLC-gamma 1 with the PDGFR. These results indicate that phosphorylation at Tyr-1021 in the tail of the PDGFR creates a specific binding site for PLC-gamma 1. Proline at the +3 position relative to Tyr-1021 is crucial in conferring specificity for binding to PLC-gamma 1.
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Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Gish
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Shoelson
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Pawson
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010770:Phosphotransferases / Q000378:metabolism; D019000:Phosphotyrosine; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases