Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.
Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis.
Kazlauskas
A
A
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Durden
D L
DL
Cooper
J A
JA
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis; D004285:Dogs; D004848:Epithelium / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006801:Humans; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis; D011506:Proteins / Q000032:analysis; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D017479:Receptors, Platelet-Derived Growth Factor; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000032:analysis; D014443:Tyrosine / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D011956:Receptors, Cell Surface; D020703:ras GTPase-Activating Proteins; D014443:Tyrosine; D004247:DNA; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases