title: Tyrosine phosphorylation of ras GTPase-activating protein does not require association with the epidermal growth factor receptor.
abstract: The importance of the carboxyl-terminal domain of the epidermal growth factor (EGF) receptor and its five autophosphorylation sites in the in vivo interaction and tyrosine phosphorylation of the ras GTPase-activating protein (rasGAP) has been investigated, using NIH 3T3 cells transfected with mutant EGF receptors. Phosphorylation of rasGAP by EGF receptor mutants, in which one to four autophosphorylation sites (Tyr-1173, -1148, -1086, and -1068) were mutated to phenylalanine, was reduced by 50-60% compared to the wild-type receptor. Elimination of these four autophosphorylation sites by truncation of 123 carboxyl-terminal residues of the EGF receptor paralleled results obtained with point mutants. Substantial inhibition (about 90%) of rasGAP tyrosine phosphorylation by the EGF receptor occurred only when the remaining autophosphorylation site (Tyr-992) was mutated, in the context of this truncated receptor or in the full-length receptor mutated at all four other autophosphorylation sites. However, a point mutation of only Tyr-992 in the full-length receptor suppressed tyrosine phosphorylation of rasGAP only by 50%. In contrast, an EGF receptor lacking the last 214 amino acid residues (Dc214), which encompasses all five autophosphorylation sites, phosphorylated rasGAP to the same extent as the wild-type receptor. However, this truncated receptor was significantly impaired in its capacity to phosphorylate phospholipase C-gamma 1. Interestingly, while EGF receptor autophosphorylation sites are required for EGF-induced rasGAP association with the receptor, maximal phosphorylation of rasGAP by the truncated receptor Dc214 occurred without detectable formation of receptor-rasGAP complexes. Furthermore, the capacity of mutated EGF receptors to bring about focal transformation was correlated with their capacity to phosphorylate rasGAP.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
authors.forename: C
authors.initials: C
authors.lastname: Soler
authors.forename: L
authors.initials: L
authors.lastname: Beguinot
authors.forename: A
authors.initials: A
authors.lastname: Sorkin
authors.forename: G
authors.initials: G
authors.lastname: Carpenter
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D004277:DNA, Single-Stranded; D066246:ErbB Receptors / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins
chemical_list: D004277:DNA, Single-Stranded; D020690:GTPase-Activating Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D014443:Tyrosine; D066246:ErbB Receptors; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases