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Activated SRC oncogene phosphorylates R-ras and suppresses integrin activity
June X Zou1, Yanqiu Liu, Elena B Pasquale
1Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA.
Abstract:
One of the prominent effects of the Src kinase is to reduce cell adhesion. The small GTPase, R-Ras, affects cell adhesion by maintaining integrin activity, and the ability of R-Ras to do so can be regulated by phosphorylation of a tyrosine residue located in its effector domain by an Eph receptor kinase (Zou, J. X., Wang, B., Kalo, M. S., Zisch, A. H., Pasquale, E. B., and Ruoslahti, E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13813-13818). Here we show that Src regulates cell adhesion through R-Ras and integrins. Reduced substrate attachment of 293T cells transfected with the cDNA for an activated form of Src (v-Src) was accompanied by phosphorylation of endogenous R-Ras. v-Src also phosphorylated R-Ras in vitro. An activated form of Src similar to one that has been found in human cancers, Src527, shared with v-Src the ability to phosphorylate R-Ras. Stronger R-Ras phosphorylation was seen in Madin-Darby canine kidney cells cells transformed with temperature-sensitive v-Src at the permissive temperature than at the non-permissive temperature, and R-Ras and Src co-immunoprecipitated at the permissive temperature. Mutation analysis showed that the Src phosphorylation site in R-Ras was tyrosine 66, the position critical to the ability of R-Ras to support integrin activity. Finally, activated R-Ras in which tyrosine 66 is mutated to phenylalanine rendered cells partially resistant to the effects of Src on cell adhesion. Regulation of cell adhesion by Src through R-Ras may be at least partially responsible for the reduced adhesion and the resulting increased invasiveness of Src-transformed cells.
Insights
Src kinase reduces cell adhesion by phosphorylating R-Ras, a key regulator of integrin activity. This molecular mechanism explains how Src-transformed cells exhibit decreased adhesion and increased invasiveness.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Src kinase is known to decrease cell adhesion.
- R-Ras, a small GTPase, maintains cell adhesion via integrin activity.
- Eph receptor kinases can regulate R-Ras activity through phosphorylation.
Purpose of the Study:
- To investigate the mechanism by which Src regulates cell adhesion.
- To determine if Src-mediated regulation of cell adhesion involves R-Ras and integrins.
Main Methods:
- Transfection of 293T cells with activated Src (v-Src).
- In vitro kinase assays using v-Src and R-Ras.
- Co-immunoprecipitation studies with R-Ras and Src.
- Site-directed mutagenesis of R-Ras phosphorylation site (tyrosine 66).
Main Results:
- Activated Src (v-Src and Src527) phosphorylates R-Ras.
- Src and R-Ras co-immunoprecipitated, indicating a complex formation.
- Phosphorylation of R-Ras by Src occurs at tyrosine 66, critical for integrin activity.
- Mutating tyrosine 66 of R-Ras confers partial resistance to Src-induced cell adhesion reduction.
Conclusions:
- Src kinase directly phosphorylates R-Ras at tyrosine 66.
- This phosphorylation event by Src impairs R-Ras function in maintaining integrin activity.
- Src-mediated regulation of R-Ras contributes to reduced cell adhesion and increased invasiveness in Src-transformed cells.