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 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.
Shin
Eun-Young
EY
Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju 361-763, Korea.
Lee
Chan-Soo
CS
Cho
Tae Goo
TG
Kim
Young Gyu
YG
Song
Sukgil
S
Juhnn
Yong-Sung
YS
Park
Sang Chul
SC
Manser
Ed
E
Kim
Eung-Gook
EG
D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000502:physiology*; D049109:Cell Proliferation; D004789:Enzyme Activation; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D008954:Models, Biological; D016501:Neurites / Q000378:metabolism*; D016716:PC12 Cells; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000502:physiology*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D064067:Rho Guanine Nucleotide Exchange Factors; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D011994:Recombinant Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; C515969:smgGDS protein, rat; D016222:Fibroblast Growth Factor 2; D049452:Green Fluorescent Proteins; C515911:PAK3 protein, human; C515913:Pak3 protein, rat; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein