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Identification of a novel RalGDS-related protein as a candidate effector for Ras and Rap1
S N Peterson1, L Trabalzini, T R Brtva
1Division of Cell Biology, Glaxo Wellcome Inc., Research Triangle Park, North Carolina 27709, USA.
Abstract:
Although Ras and Rap1 share interaction with common candidate effector proteins, Rap1 lacks the transforming activity exhibited by Ras proteins. It has been speculated that Rap antagonizes Ras transformation through the formation of nonproductive complexes with critical Ras effector targets. To understand further the distinct biological functions of these two closely related proteins, we searched for Rap1b-binding proteins by yeast two-hybrid screening. We identified multiple clones that encode the COOH-terminal sequences of a protein that shares sequence identity with RalGDS and RGL, which we have designated RGL2. A 158-amino acid COOH-terminal fragment of RGL2 (RGL2 C-158) bound to Ras superfamily proteins which shared identical effector domain sequences with Rap1 (Ha-Ras, R-Ras, and TC21). RGL2 C-158 binding was impaired by effector domain mutations in Rap1b and Ha-Ras. Furthermore, RGL2 C-158 bound exclusively to the GTP-, but not the GDP-bound form of Ha-Ras. Finally, coexpression of RGL2 C-158 impaired oncogenic Ras activation of transcription from a Ras-responsive promoter element and focus-forming activity in NIH 3T3 cells. We conclude that RGL2 may be an effector for Ras and/or Rap proteins.
Insights
Researchers identified RGL2 as a potential effector protein for Ras and Rap1. This protein may antagonize Ras-mediated cell transformation by forming nonproductive complexes with effector targets.
Area of Science:
- Molecular biology
- Cell signaling
- Cancer research
Background:
- Ras and Rap1 proteins share effector interactions but differ in transforming activity.
- Rap1 is hypothesized to antagonize Ras transformation via nonproductive complexes.
- Understanding these distinct functions requires identifying specific binding partners.
Purpose of the Study:
- To identify Rap1b-binding proteins using yeast two-hybrid screening.
- To characterize the interaction of identified proteins with Ras superfamily members.
- To investigate the functional role of RGL2 in Ras signaling.
Main Methods:
- Yeast two-hybrid screening to identify Rap1b-binding proteins.
- Biochemical assays to analyze RGL2 C-158 binding to Ras proteins.
- Cell-based assays to assess RGL2's effect on Ras-driven transcription and transformation.
Main Results:
- Identified RGL2, a protein related to RalGDS and RGL, as a Rap1b-binding protein.
- Demonstrated that RGL2 C-158 binds to Ras superfamily proteins with shared effector domains.
- Showed RGL2 C-158 binding is dependent on effector domain integrity and GTP-bound Ras.
- Observed that RGL2 C-158 inhibits oncogenic Ras-induced transcription and focus formation.
Conclusions:
- RGL2 may function as an effector for Ras and/or Rap proteins.
- RGL2's interaction with Ras may contribute to the antagonism of Ras transformation.
- Further research is needed to elucidate the precise role of RGL2 in cellular signaling pathways.