βPak-interacting Exchange Factor-mediated Rac1 Activation Requires smgGDS Guanine Nucleotide Exchange Factor in Basic Fibroblast Growth Factor-induced Neurite Outgrowth

Eun-Young Shin,Chan-Soo Lee,Tae Goo Cho,Young Gyu Kim,Sukgil Song,Yong-Sung Juhnn,Sang Chul Park,Ed Manser,Eung-Gook Kim

doi:10.1074/jbc.m602399200

Abstract

Neuritogenesis requires active actin cytoskeleton rearrangement in which Rho GTPases play a pivotal role. In a previous study (Shin, E. Y., Woo, K. N., Lee, C. S., Koo, S. H., Kim, Y. G., Kim, W. J., Bae, C. D., Chang, S. I., and Kim, E. G. (2004) J. Biol. Chem. 279, 1994-2004), we demonstrated that betaPak-interacting exchange factor (betaPIX) guanine nucleotide exchange factor (GEF) mediates basic fibroblast growth factor (bFGF)-stimulated Rac1 activation through phosphorylation of Ser-525 and Thr-526 at the GIT-binding domain (GBD). However, the mechanism by which this phosphorylation event regulates the Rac1-GEF activity remained elusive. We show here that betaPIX binds to Rac1 via the GBD and also activates the GTPase via an associated GEF, smgGDS, in a phosphorylation-dependent manner. Notably, the Rac1-GEF activity of betaPIX persisted for an extended period of time following bFGF stimulation, unlike other Rho GEFs containing the Dbl homology domain. We demonstrate that C-PIX, containing proline-rich, GBD, and leucine zipper domains can interact with Rac1 via the GBD in vitro and in vivo and also mediated bFGF-stimulated Rac1 activation, as determined by a modified GEF assay and fluorescence resonance energy transfer analysis. However, nonphosphorylatable C-PIX (S525A/T526A) failed to generate Rac1-GTP. Finally, betaPIX is shown to form a trimeric complex with smgGDS and Rac1; down-regulation of smgGDS expression by short interfering RNA causing significant inhibition of betaPIX-mediated Rac1 activation and neurite outgrowth. These results provide evidence for a new and unexpected mechanism whereby betaPIX can regulate Rac1 activity.

Highlights

Rho GTPases regulate cytoskeletal dynamics and play a pivotal role in a wide range of biological responses, including cell polarity, cell adhesion and migration, and neuritogenesis [1,2,3]
Chem. 279, 1994 –2004), we demonstrated that ␤p21-activated kinase (Pak)-interacting exchange factor (␤PIX) guanine nucleotide exchange factor (GEF) mediates basic fibroblast growth factor-stimulated Rac1 activation through phosphorylation of Ser-525 and Thr-526 at the GIT-binding domain (GBD)
We demonstrate that C-terminal half of ␤PIX (C-PIX), containing proline-rich, GB domain (GBD), and leucine zipper domains can interact with Rac1 via the GBD in vitro and in vivo and mediated basic fibroblast growth factor (bFGF)-stimulated Rac1 activation, as determined by a modified Guanine nucleotide exchange factor (GEF) assay and fluorescence resonance energy transfer analysis

Summary

Introduction

Rho GTPases regulate cytoskeletal dynamics and play a pivotal role in a wide range of biological responses, including cell polarity, cell adhesion and migration, and neuritogenesis [1,2,3]. 279, 1994 –2004), we demonstrated that ␤Pak-interacting exchange factor (␤PIX) guanine nucleotide exchange factor (GEF) mediates basic fibroblast growth factor (bFGF)-stimulated Rac1 activation through phosphorylation of Ser-525 and Thr-526 at the GIT-binding domain (GBD). We show here that ␤PIX binds to Rac1 via the GBD and activates the GTPase via an associated GEF, smgGDS, in a phosphorylation-dependent manner.

Results

Conclusion