A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine.

AUTOR(ES)

Escobedo, J A

RESUMO

Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.

Documentos Relacionados

• Platelet-derived growth factor β receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3′ kinase signaling
• Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells.
• Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors.
• Tyrosine mutations within the alpha platelet-derived growth factor receptor kinase insert domain abrogate receptor-associated phosphatidylinositol-3 kinase activity without affecting mitogenic or chemotactic signal transduction.
• Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.