Interaction of the Flt-1 tyrosine kinase receptor with the p85 subunit of phosphatidylinositol 3-kinase. Mapping of a novel site involved in binding.
We have examined the interactions of the p85 regulatory subunit of phosphatidylinositol 3-kinase with the endothelium-specific Flt-1 receptor tyrosine kinase using the yeast two-hybrid system. We find that both the amino- and carboxyl-terminal SH2 domains of p85 bind to Flt-1. We have performed site-directed mutagenesis on the carboxyl-terminal tail of the Flt-1 receptor in order to identify the site(s) that is responsible for the p85 interactions. A single tyrosine to phenylalanine change at position 1213 inhibits the binding of both p85 SH2 domains. Phosphopeptide mapping of the wild type and mutant protein expressed in insect cells verifies that this amino acid is a target for autophosphorylation. The amino acids following this tyrosine are VNA and thus define a novel binding site for p85.
Cunningham
S A
SA
Department of Pharmacology, Texas Biotechnology Corporation, Houston 77030, USA.
Waxham
M N
MN
Arrate
P M
PM
Brock
T A
TA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015246:Deoxyribonuclease EcoRI; D004730:Endothelium, Vascular / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D018411:Spodoptera; D014162:Transfection; D014471:Umbilical Veins; D040281:Vascular Endothelial Growth Factor Receptor-1
D015246:Deoxyribonuclease EcoRI; D046911:Macromolecular Substances; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011518:Proto-Oncogene Proteins; D020794:Receptor Protein-Tyrosine Kinases; D017978:Receptors, Growth Factor; D011993:Recombinant Fusion Proteins; D040281:Vascular Endothelial Growth Factor Receptor-1