R-Ras regulates migration through an interaction with filamin A in melanoma cells.
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.


RESULTS
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.
Gawecka
Joanna E
JE
Natural Products and Cancer Biology, Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
Griffiths
Genevieve S
GS
Ek-Rylander
Barbro
B
Ramos
Joe W
JW
Matter
Michelle L
ML
D001483:Base Sequence; D015153:Blotting, Western; D002465:Cell Movement / Q000502:physiology*; D003285:Contractile Proteins / Q000502:physiology*; D017931:DNA Primers; D064448:Filamins; D006801:Humans; D008545:Melanoma / Q000473:pathology*; D008840:Microfilament Proteins / Q000502:physiology*; D008856:Microscopy, Fluorescence; D018631:ras Proteins / Q000502:physiology*
D003285:Contractile Proteins; D017931:DNA Primers; D064448:Filamins; D008840:Microfilament Proteins; C085929:RRAS protein, human; D018631:ras Proteins