C. Soler et al., TYROSINE PHOSPHORYLATION OF RAS GTPASE-ACTIVATING PROTEIN DOES NOT REQUIRE ASSOCIATION WITH THE EPIDERMAL GROWTH-FACTOR RECEPTOR, The Journal of biological chemistry, 268(29), 1993, pp. 22010-22019
The importance of the carboxyl-terminal domain of the epidermal growth
factor (EGF) receptor and its five autophosphorylation sites in the i
n vivo interaction and tyrosine phosphorylation of the ras GTPase-acti
vating protein (rasGAP) has been investigated, using NIH 3T3 cells tra
nsfected with mutant EGF receptors. Phosphorylation of rasGAP by EGF r
eceptor mutants, in which one to four autophosphorylation sites (Tyr-1
173, -1148, -1086, and -1068) were mutated to phenylalanine, was reduc
ed 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 mu
tants. Substantial inhibition (about 90%) of rasGAP tyrosine phosphory
lation by the EGF receptor occurred only when the remaining autophosph
orylation site (Tyr-1992) was mutated, in the context of this truncate
d receptor or in the full-length receptor mutated at all four other au
tophosphorylation 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 emcompasses all five autophosphorylation
sites, phosphorylated rasGAP to the same extent as the wild-type recep
tor. However, this truncated receptor was significantly impaired in it
s capacity to phosphorylate phospholipase C-gamma1. Interestingly, whi
le EGF receptor autophosphorylation sites are required for EGF-induced
rasGAP association with the receptor, maximal phosphorylation of rasG
AP by the truncated receptor Dc214 occurred without detectable formati
on of receptor.rasGAP complexes. Furthermore, the capacity of mutated
EGF receptors to bring about focal transformation was correlated with
their capacity to phosphorylate rasGAP.