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Identification of sites on epidermal growth factor receptors which are phosphorylated by pp60src in vitro
J D Wright1, C W Reuter, M J Weber
1Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Abstract:
The Epidermal Growth Factor Receptor (EGF-R) becomes constitutively tyrosine phosphorylated on two novel sites in v-Src transformed cells, and these phosphorylations are associated with enhanced signaling activity [1]. To determine whether Src could directly phosphorylate these sites, we have examined the ability of the Src kinase to phosphorylate both wild-type and kinase-defective EGF-Rs in vitro. Although purified Src could phosphorylate EGF-Rs, the pattern of phosphorylation sites was not identical to what was previously found in vivo [1]: Src in vitro directly phosphorylated EGF-Rs on one autophosphorylation site (Tyr 1173) which was not a site of re-induced in vivo phosphorylation, suggesting the in vivo inaccessibility of this site. One Src-specific in vitro phosphorylation site (Tyr 03) appeared to correspond to one of the in vivo Src-induced sites (sPY2), but the other Src-specific in vivo site (sPY1) was not significantly phosphorylated in vitro, raising the possibility of a Src-induced tyrosine kinase cascade. The ability of Src to phosphorylate the EGF-R is consistent with the suggestion that the receptor can function as a kinase substrate independent of its intrinsic enzymatic activity, as implied by recent studies on signaling by kinase-defective EGF-Rs.
Insights
The v-Src kinase directly phosphorylates the Epidermal Growth Factor Receptor (EGF-R) at specific sites. However, the in vitro phosphorylation pattern differs from in vivo findings, suggesting complex signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- Epidermal Growth Factor Receptor (EGF-R) signaling is crucial for cell growth and is often dysregulated in cancer.
- Constitutive tyrosine phosphorylation of EGF-R occurs in v-Src transformed cells, leading to enhanced signaling.
- The precise mechanisms by which v-Src influences EGF-R phosphorylation remain incompletely understood.
Purpose of the Study:
- To investigate whether the v-Src kinase directly phosphorylates specific sites on the EGF-R.
- To compare in vitro phosphorylation patterns by v-Src with previously observed in vivo phosphorylation sites.
Main Methods:
- In vitro kinase assays using purified v-Src and wild-type or kinase-defective EGF-Rs.
- Analysis of EGF-R tyrosine phosphorylation sites using biochemical methods.
Main Results:
- Purified v-Src directly phosphorylated EGF-R at Tyr 1173 in vitro, a site not previously identified as re-phosphorylated in vivo.
- One v-Src-specific in vitro phosphorylation site (Tyr 03) corresponded to an in vivo site (sPY2), but another in vivo site (sPY1) was not significantly phosphorylated in vitro.
- These findings suggest potential inaccessibility of certain sites in vivo and the possibility of a v-Src-induced tyrosine kinase cascade.
Conclusions:
- v-Src can directly phosphorylate EGF-R, acting as a kinase substrate.
- The distinct in vitro and in vivo phosphorylation patterns indicate complex regulation and potential involvement of other kinases in the signaling cascade.
- EGF-R can function as a kinase substrate independently of its own intrinsic enzymatic activity.