A novel role for Lsc/p115 RhoGEF and LARG in regulating RhoA activity downstream of adhesion to fibronectin

TY - JOUR

T1 - A novel role for Lsc/p115 RhoGEF and LARG in regulating RhoA activity downstream of adhesion to fibronectin

AU - Dubash, Adi D

AU - Wennerberg, Krister

AU - García-Mata, Rafael

AU - Menold, Marisa M

AU - Arthur, William T

AU - Burridge, Keith

PY - 2007/11/15

Y1 - 2007/11/15

N2 - Adhesion of cells to extracellular matrix proteins such as fibronectin initiates signaling cascades that affect cell morphology, migration and survival. Some of these signaling pathways involve the Rho family of GTPases, such as Cdc42, Rac1 and RhoA, which play a key role in regulating the organization of the cytoskeleton. Although significant advances have been made in understanding how Rho proteins control cytoskeletal architecture, less is known about the signals controlling activation of the GTPases themselves. The focus of this study was to determine which guanine nucleotide exchange factor(s) are responsible for activation of RhoA downstream of adhesion to fibronectin. Using an affinity pulldown assay for activated exchange factors, we show that the RhoA-specific exchange factors Lsc/p115 RhoGEF and LARG are activated when cells are plated onto fibronectin, but not other exchange factors such as Ect2 or Dbl. Knockdown of Lsc and LARG together significantly decreases RhoA activation and formation of stress fibers and focal adhesions downstream of fibronectin adhesion. Similarly, overexpression of a catalytically inactive mutant of Lsc/p115 RhoGEF inhibits RhoA activity and formation of stress fibers and focal adhesions on fibronectin. These data establish a previously uncharacterized role for the exchange factors Lsc/p115 RhoGEF and LARG in linking fibronectin signals to downstream RhoA activation.

AB - Adhesion of cells to extracellular matrix proteins such as fibronectin initiates signaling cascades that affect cell morphology, migration and survival. Some of these signaling pathways involve the Rho family of GTPases, such as Cdc42, Rac1 and RhoA, which play a key role in regulating the organization of the cytoskeleton. Although significant advances have been made in understanding how Rho proteins control cytoskeletal architecture, less is known about the signals controlling activation of the GTPases themselves. The focus of this study was to determine which guanine nucleotide exchange factor(s) are responsible for activation of RhoA downstream of adhesion to fibronectin. Using an affinity pulldown assay for activated exchange factors, we show that the RhoA-specific exchange factors Lsc/p115 RhoGEF and LARG are activated when cells are plated onto fibronectin, but not other exchange factors such as Ect2 or Dbl. Knockdown of Lsc and LARG together significantly decreases RhoA activation and formation of stress fibers and focal adhesions downstream of fibronectin adhesion. Similarly, overexpression of a catalytically inactive mutant of Lsc/p115 RhoGEF inhibits RhoA activity and formation of stress fibers and focal adhesions on fibronectin. These data establish a previously uncharacterized role for the exchange factors Lsc/p115 RhoGEF and LARG in linking fibronectin signals to downstream RhoA activation.

KW - Animals

KW - Cell Adhesion

KW - Fibroblasts/cytology

KW - Fibronectins/metabolism

KW - Focal Adhesions/metabolism

KW - Guanine Nucleotide Exchange Factors/chemistry

KW - Humans

KW - Mice

KW - Mutant Proteins/metabolism

KW - NIH 3T3 Cells

KW - Protein Transport

KW - Rats

KW - Receptors, G-Protein-Coupled/metabolism

KW - Rho Guanine Nucleotide Exchange Factors

KW - Stress Fibers/metabolism

KW - rhoA GTP-Binding Protein/metabolism

U2 - 10.1242/jcs.003806

DO - 10.1242/jcs.003806

M3 - Journal article

C2 - 17971419

SN - 0021-9533

VL - 120

SP - 3989

EP - 3998

JO - Journal of Cell Science

JF - Journal of Cell Science

IS - Pt 22

ER -