The leucine-rich region of Flightless I interacts with R-ras to regulate cell extension formation

Abstract

Flightless I (FliI) is a calcium-dependent, actin severing and capping protein that localizes to cell matrix adhesions, contributes to the generation of cell extensions, and colocalizes with Ras. Currently, the mechanism by which FliI interacts with Ras to enable assembly of actin-based cell protrusions is not defined. R-Ras, but not K-ras, H-ras, or N-ras, associated with the leucine-rich region (LRR) of FliI. Mutations of the proline-rich region of R-ras (P202A, P203A) prevented this association. Knockdown of Ras GTPase-activating SH3 domain-binding protein (G3BP1) or Rasgap120 by small interfering RNA inhibited the formation of cell extensions and prevented interaction of R-ras and G3BP1 in FliI wild-type (WT) cells. Pull-down assays using G3BP1 fusion proteins showed a strong association of R-ras with the C-terminus of G3BP1 (amino acids 236–466), which also required the LRR of FliI. In cells that expressed the truncated N-terminus or C-terminus of G3BP1, the formation of cell extensions was blocked. Endogenous Rasgap120 interacted with the N-terminus of G3BP1 (amino acids 1–230). We conclude that in cells plated on collagen FliI-LRR interacts with R-ras to promote cell extension formation and that FliI is required for the interaction of Rasgap120 with G3BP1 to regulate R-ras activity and growth of cell extensions.

Flightless anchors IQGAP1 and R-ras to mediate cell extension formation and matrix remodeling

“…FliI colocalized with IQGAP1 in immunostaining and bound in co-immunoprecipitation studies; pull-down experiments showed specific associations between IQGAP1 and the FliI-LRR but not the FliI-GLD domain. These findings are consistent with the multifunctional nature of FliI ( Liu and Yin, 1998 ; Arora et al., 2018 ), a member of the gelsolin family of proteins. Whereas the actin capping activity of FliI regulates actin remodeling through its GLD, its LRR region acts as an adaptor to integrate the functions of proteins involved in signaling.…”

“…The molecules that regulate the formation of the cell extensions required for the remodeling of collagen are not defined. FliI, by virtue of its multidomain structure ( Liu and Yin, 1998 ), performs two quite separate functions ( Arora et al., 2018 ). The GLDs are involved in actin cytoskeletal rearrangements while the LRR region may regulate signal transduction through its association with R-ras.…”

“…While the regulation of filopodia by cdc42 is well established ( Ohta et al., 1999 ; Dimchev et al., 2017 ), the involvement of other GTPases and their role in the formation of longer cell extensions are not well defined. One member of the Ras family of GTPases, R-ras, mediates a diverse range of cellular processes involving cytoskeletal rearrangements such as the generation of cell extensions, adhesion, migration and phagocytosis ( Keely et al., 1999 ; Self et al., 2001 ; Kwong et al., 2003 ; Arora et al., 2018 ). Small GTPases like R-ras exist in inactive GDP-bound and active GTP-bound states that are regulated by guanine nucleotide exchange factors, which promote conversion from a GDP-bound to a GTP-bound state, and by GTPase-activating proteins, which accelerate the hydrolysis of GTP to GDP.…”

“…FliI is enriched at nascent cell–matrix adhesions in fibroblasts ( Mohammad et al., 2012 ) and colocalizes with Ras ( Davy et al., 2001 ). More recent data show that R-ras associates with FliI through the N-terminal LRR domain of FliI, an interaction that enables the generation of plasma membrane extensions in fibroblasts by an unknown mechanism ( Arora et al., 2018 ). Here we considered that FliI acts as an adaptor protein to bind IQGAP1; FliI and IQGAP1 then co-ordinate with cdc42 and R-ras to control the formation of cell extensions.…”

R‐Ras regulates vascular permeability, but not overall healing in skin wounds

“…Whereas all other members of the Ras family cause malignant transformation, R‐Ras has very little or no transforming activity . Furthermore, the opposing functions of R‐Ras and other Ras members extend to cell adhesion, differentiation and signalling . These opposing functions suggest that the balance between R‐Ras and other Ras members acts as a switch that controls proliferation and migration versus quiescence in cells .…”