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R-Ras regulates migration through an interaction with filamin A in melanoma cells
Joanna E Gawecka1, Genevieve S Griffiths, Barbro Ek-Rylander
1Natural Products and Cancer Biology, Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
Background:
Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins.
Methods And Findings:
We identified Filamin A (FLNa) as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin beta1, beta2 and beta7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaDelta3) abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaDelta3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly.
Conclusions:
These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration.
Insights
Researchers found that R-Ras binds to Filamin A (FLNa) repeat 3, enhancing melanoma cell migration and potentially promoting metastasis. This interaction is crucial for R-Ras to regulate cell movement and matrix assembly.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Tumor microenvironment changes in cell adhesion and migration are critical for metastasis.
- R-Ras, a small GTPase, regulates cell adhesion and migration, but its mechanisms are not fully understood.
- Identifying R-Ras binding proteins is key to elucidating its role in integrin-mediated processes.
Purpose of the Study:
- To identify novel proteins that bind to R-Ras using a yeast two-hybrid approach.
- To investigate the functional consequences of R-Ras and its binding partners on cell migration and integrin activity.
Main Methods:
- Yeast two-hybrid screening to identify R-Ras interacting proteins.
- Co-immunoprecipitation and pull-down assays to confirm R-Ras and Filamin A (FLNa) interaction.
- Analysis of FLNa deletion mutants (FLNaDelta3) to map the interaction site.
- Cell migration assays, integrin activation studies, and fibronectin matrix assembly measurements.
- siRNA knockdown of R-Ras to assess its role in FLNa-dependent processes.
Main Results:
- Filamin A (FLNa) was identified as an R-Ras interacting protein.
- R-Ras specifically binds to repeat 3 of FLNa, confirmed by interaction and co-localization studies.
- Co-expression of R-Ras and FLNa significantly increased melanoma cell migration and fibronectin matrix assembly.
- The R-Ras/FLNa interaction enhanced integrin activation but did not alter cell adhesion.
- R-Ras knockdown impaired FLNa-dependent fibronectin matrix assembly.
Conclusions:
- R-Ras functionally associates with FLNa, regulating integrin-dependent cell migration.
- The R-Ras-FLNa complex promotes melanoma cell migration, suggesting a cooperative role in metastasis.
- This interaction highlights a novel pathway for regulating cell motility in the tumor microenvironment.
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