betaPak-interacting exchange factor-mediated Rac1 activation requires smgGDS guanine nucleotide exchange factor in basic fibroblast growth factor-induced neurite outgrowth

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...

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Published in:The Journal of biological chemistry 2006-11, Vol.281 (47), p.35954-35964
Main Authors: Shin, Eun-Young, Lee, Chan-Soo, Cho, Tae Goo, Kim, Young Gyu, Song, Sukgil, Juhnn, Yong-Sung, Park, Sang Chul, Manser, Ed, Kim, Eung-Gook
Format: Article
Language:English
Subjects:
Animals
Cell Cycle Proteins - chemistry
Cell Cycle Proteins - physiology
Cell Proliferation
Enzyme Activation
Fibroblast Growth Factor 2 - chemistry
Green Fluorescent Proteins - chemistry
Guanine Nucleotide Exchange Factors - chemistry
Guanine Nucleotide Exchange Factors - physiology
Humans
Models, Biological
Neurites - metabolism
p21-Activated Kinases
PC12 Cells
Protein-Serine-Threonine Kinases - chemistry
Protein-Serine-Threonine Kinases - physiology
rac1 GTP-Binding Protein - metabolism
Rats
Recombinant Proteins - chemistry
Rho Guanine Nucleotide Exchange Factors
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Summary: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.
ISSN:0021-9258