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.
Highlights
epidermal growthfactor (EGF) receptor paralleled results obtained with point mu- GAP),and phosphotyrosine phostants
Fig. 1depicts the EGF receptor constructs lunes 6 and 8),was tested, EGFproduced a 2-fold higher extent used in this study and their EGF receptor expression level. of tyrosine phosphorylation of this substrate thaPnDGF
To determine whether the daotbatained with truncationmutants could be extrapolated to the full-length EGF receptor, we examined receptor mutants with single or multiple substitution(s) of tyrosine autophosphorylation sites to phenylalanine (Table 11).Compared to the wild-type receptor, tyrosine phos
Summary
Materials-EGF was isolated frommouse submaxillary gland according to the method of Savage and Cohen [50] and iodinated as described by Carpenter and Cohen [51]. 1*51-Labeledrabbit anti-mouse. To examine tyrosine phosphorylation of total cellular proteins in response to EGF, an aliquot of cell lysates (50-100 pg) was mixed with 2 x Laemmli buffer,electrophoresedon 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and blotted with phosphotyrosine antibodies. Receptor Association Studies-NIH 3T3 cellsexpressing wild-type or mutant EGF receptors were grown, treated with EGF (100 ng/ml) or PDGF-BB (50 ng/ml) for1h at 4 "C, and lysed as indicated above.EGF receptor precipitation was performed using mouse monoclonalantibodies against the extracellular domain of the EGF receptor and protein G-Sepharose 4B beads. RusGAP was immunoprecipitated and tyrosine phosphorylation analyzed by immunoblot were performed, the total number of EGF receptors occupied by EGF was determined in parallel cultures incubated with lZ6I-EGF(100ng/ ml).
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