Abstract
Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein-tyrosine phosphatase SHP-2, and phospholipase C-gamma1 (PLC-gamma1), to specific phosphotyrosine residues. The roles of these various effectors in PDGF-induced generation of H(2)O(2) have now been investigated in HepG2 cells expressing various PDGF receptor mutants. These mutants included a kinase-deficient receptor and receptors in which various combinations of the tyrosine residues required for the binding of PI3K (Tyr(740) and Tyr(751)), GAP (Tyr(771)), SHP-2 (Tyr(1009)), or PLC-gamma1 (Tyr(1021)) were mutated to Phe. PDGF failed to increase H(2)O(2) production in cells expressing either the kinase-deficient mutant or a receptor in which the two Tyr residues required for the binding of PI3K were replaced by Phe. In contrast, PDGF-induced H(2)O(2) production in cells expressing a receptor in which the binding sites for GAP, SHP-2, and PLC-gamma1 were all mutated was slightly greater than that in cells expressing the wild-type receptor. Only the PI3K binding site was alone sufficient for PDGF-induced H(2)O(2) production. The effect of PDGF on H(2)O(2) generation was blocked by the PI3K inhibitors LY294002 and wortmannin or by overexpression of a dominant negative mutant of Rac1. These results suggest that a product of PI3K is required for PDGF-induced production of H(2)O(2) in nonphagocytic cells, and that Rac1 mediates signaling between the PI3K product and the putative NADPH oxidase.
Highlights
Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein-tyrosine phosphatase Src homology 2 domain-containing protein-tyrosine phosphatase-2 (SHP-2), and phospholipase C-␥1 (PLC-␥1), to specific phosphotyrosine residues
These results suggest that a product of PI3K is required for PDGF-induced production of H2O2 in nonphagocytic cells, and that Rac1 mediates signaling between the PI3K product and the putative NADPH oxidase
The kinase(Ϫ) mutant was generated by mutating Lys634 to Arg; the F740/751 mutant by replacing the residues that constitute the PI3K binding site (Tyr740 and Tyr751) with Phe; the F771 mutant by replacing the GAP binding site (Tyr771) with Phe; the F1021 mutant by replacing the PLC-␥1 binding site (Tyr1021) with Phe; the F740/751/771 mutant by replacing Tyr740, Tyr751, and Tyr 771 with Phe; and the F5 mutant by replacing the five tyrosine residues Tyr740, Tyr751, Tyr 771, Tyr1009, and Tyr1021 with Phe (Fig. 1)
Summary
Vol 275, No 14, Issue of April 7, pp. 10527–10531, 2000 Printed in U.S.A. Platelet-derived Growth Factor-induced H2O2 Production Requires the Activation of Phosphatidylinositol 3-Kinase*. Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein-tyrosine phosphatase SHP-2, and phospholipase C-␥1 (PLC-␥1), to specific phosphotyrosine residues. The roles of these various effectors in PDGF-induced generation of H2O2 have been investigated in HepG2 cells expressing various PDGF receptor mutants. Experiments with a dominant negative mutant of Rac (N17Rac1) suggest that Rac participates in the PDGFR-induced production of H2O2 in HepG2 cells
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