Human Mena Associates with Rac1 Small GTPase in Glioblastoma Cell Lines

Morihiro Higashi,Motoo Kitagawa,Michiyuki Matsuda,Akihiro Toyoda,Chieko Ishikawa,Hidetada Kawana,Jianyong Yu,Kazuo Kurokawa,Kenichi Harigaya,Joshua Z Rappoport

doi:10.1371/journal.pone.0004765

Morihiro Higashi, Motoo Kitagawa + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0004765

Copy DOI

Journal: PloS one	Publication Date: Mar 11, 2009
Citations: 61	License type: CC BY 4.0

Affiliation: Chiba University, Kyoto University

Abstract

Mammarian enabled (Mena), a member of the Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) family of proteins, has been implicated in cell motility through regulation of the actin cytoskeleton assembly, including lamellipodial protrusion. Rac1, a member of the Rho family GTPases, also plays a pivotal role in the formation of lamellipodia. Here we report that human Mena (hMena) colocalizes with Rac1 in lamellipodia, and using an unmixing assisted acceptor depletion fluorescence resonance energy transfer (u-adFRET) analysis that hMena associates with Rac1 in vivo in the glioblastoma cell line U251MG. Depletion of hMena by siRNA causes cells to be highly spread with the formation of lamellipodia. This cellular phenotype is canceled by introduction of a dominant negative form of Rac1. A Rac activity assay and FRET analysis showed that hMena knock-down cells increased the activation of Rac1 at the lamellipodia. These results suggest that hMena possesses properties which help to regulate the formation of lamellipodia through the modulation of the activity of Rac1.

Highlights

The Ena/VASP family of proteins comprised of Mammarian enabled (Mena), a mammalian ortholog of Drosophila Ena, VASP and Ena/VASPlike (EVL) play an important role in linking signaling pathways to the remodeling of the actin cytoskeletal structure including the formation of lamellipodia and filopodia which leads to cell motility [1,2,3]
The merged images indicated that human Mena (hMena) and Rac colocalized at the lamellipodia (Figure 1C, Movie S1)
Our study demonstrated that hMena protein interacts with Rac1 protein at lamellipodia and their association may to suppress the activation of Rac1

Summary

Introduction

The Ena/VASP family of proteins comprised of Mena, a mammalian ortholog of Drosophila Ena, VASP and Ena/VASPlike (EVL) play an important role in linking signaling pathways to the remodeling of the actin cytoskeletal structure including the formation of lamellipodia and filopodia which leads to cell motility [1,2,3]. The proline-rich region binds to the actin binding protein, profilin, and the EVH2 domain is required for multimerization and direct F-actin binding in vitro [9]. The one major link between Ena/VASP and actin dynamics had been thought to be via profilin. Both profilin and Ena/VASP localize to the leading edges of lamellipodia, and Ena/VASP is thought to recruit profilin-actin complexes to the sites of actin assembly [10]. It was reported that VASP-deficient murine fibroblasts showed prolonged activity of Rac and p21activated kinase (PAK) [13]

Methods

Results

Conclusion