Basic fibroblast growth factor stimulates activation of Rac1 through a p85 betaPIX phosphorylation-dependent pathway.
In a previous study (Shin, E. Y., Shin, K. S., Lee, C. S., Woo, K. N., Quan, S. H., Soung, N. K., Kim, Y. G., Cha, C. I., Kim, S. R., Park, D., Bokoch, G. M., and Kim, E. G. (2002) J. Biol. Chem. 277, 44417-44430) we reported that phosphorylation of p85 betaPIX, a guanine nucleotide exchange factor (GEF) for Rac1/Cdc42, is a signal for translocation of the PIX complex to neuronal growth cones and is associated with basic fibroblast growth factor (bFGF)-induced neurite outgrowth. However, the issue of whether p85 betaPIX phosphorylation affects GEF activity on Rac1/Cdc42 is yet to be explored. Here we show that Rac1 activation occurs in a p85 betaPIX phosphorylation-dependent manner. A GST-PBD binding assay reveals that Rac1 is activated in a dose- and time-dependent manner in PC12 cells in response to bFGF. Inhibition of ERK or PAK2, the kinases upstream of p85 betaPIX in the bFGF signaling, prevents Rac1 activation, suggesting that phosphorylation of p85 betaPIX functions upstream of Rac1 activation. To directly address this issue, transfection studies with wild-type and mutant p85 betaPIX (S525A/T526A, a non-phosphorylatable form) were performed. Expression of mutant PIX markedly inhibits both bFGF- and nerve growth factor (NGF)-induced activation of Rac1, indicating that phosphorylation of p85 betaPIX is responsible for activation of this G protein. Both wild-type and mutant p85 betaPIX displaying negative GEF activity (L238R/L239S) are similarly recruited to growth cones, suggesting that Rac1 activation is not essential for translocation of the PIX complex (PAK2-p85 betaPIX-Rac1). However, expression of mutant p85 betaPIX (L238R/L239S) results in retraction of the pre-existing neurites. Our results provide evidence that bFGF- and NGF-induced phosphorylation of p85 betaPIX mediates Rac1 activation, which in turn regulates cytoskeletal reorganization at growth cones, but not translocation of the PIX complex.
Shin
Eun-Young
EY
Department of Biochemistry, College of Medicine, Medical Research Institute and Biotechnology Research Institute, College of Natural Sciences, Chungbuk National University, San 48, Gaesin-dong, Heungduk-ku, Cheongju 361-763, Korea..
Woo
Kyung-Nam
KN
Lee
Chan-Soo
CS
Koo
Seong-Hoe
SH
Kim
Young Gyu
YG
Kim
Won-Jai
WJ
Bae
Chang-Dae
CD
Chang
Soo-Ik
SI
Kim
Eung-Gook
EG
D018797:Cell Cycle Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D019255:NADPH Oxidases / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology; D010766:Phosphorylation; D021381:Protein Transport; D064067:Rho Guanine Nucleotide Exchange Factors; D013997:Time Factors; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D016222:Fibroblast Growth Factor 2; D020932:Nerve Growth Factor; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein