Guanine nucleotide exchange factors (GEFs) have been implicated in growth factor-induced neuronal differentiation through the activation of small GTPases. Although phosphorylation of these GEFs is considered an activation mechanism, little is known about the upstream of PAK-interacting exchange factor (PIX), a member of the Dbl family of GEFs. We report here that phosphorylation of p85 PIX/Cool/p85SPR is mediated via the Ras/ERK/PAK2 pathway. To understand the role of p85 PIX in basic fibroblast growth factor (bFGF)-induced neurite outgrowth, we established PC12 cell lines that overexpress the fibroblast growth factor receptor-1 in a tetracycline-inducible manner. Treatment with bFGF induces the phosphorylation of p85 PIX, as determined by metabolic labeling and mobility shift upon gel electrophoresis. Interestingly, phosphorylation of p85 PIX is inhibited by PD98059, a specific MEK inhibitor, suggesting the involvement of the ERK cascade. PAK2, a major PAK isoform in PC12 cells as well as a binding partner of p85 PIX, also functions upstream of p85 PIX phosphorylation. Surprisingly, PAK2 directly binds to ERK, and its activation is dependent on ERK. p85 PIX specifically localizes to the lamellipodia at neuronal growth cones in response to bFGF. A mutant form of p85 PIX (S525A/T526A), in which the major phosphorylation sites are replaced by alanine, shows significant defect in targeting. Moreover, expression of the mutant p85 PIX efficiently blocks PC12 cell neurite outgrowth. Our study defines a novel signaling pathway for bFGF-induced neurite outgrowth that involves activation of the PAK2-p85 PIX complex via the ERK cascade and subsequent translocation of this complex.
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 PIX, 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 PIX phosphorylation affects GEF activity on Rac1/Cdc42 is yet to be explored. Here we show that Rac1 activation occurs in a p85 PIX phosphorylationdependent 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 PIX in the bFGF signaling, prevents Rac1 activation, suggesting that phosphorylation of p85 PIX functions upstream of Rac1 activation. To directly address this issue, transfection studies with wild-type and mutant p85 PIX (S525A/ T526A, a non-phosphorylatable form) were performed. Expression of mutant PIX markedly inhibits both bFGFand nerve growth factor (NGF)-induced activation of Rac1, indicating that phosphorylation of p85 PIX is responsible for activation of this G protein. Both wildtype and mutant p85 PIX 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 PIX-Rac1). However, expression of mutant p85 PIX (L238R/ L239S) results in retraction of the pre-existing neurites. Our results provide evidence that bFGF-and NGF-induced phosphorylation of p85 PIX mediates Rac1 activation, which in turn regulates cytoskeletal reorganization at growth cones, but not translocation of the PIX complex.
Abstractp21-activated kinase (PAK) targeting to the plasma membrane is essential for PC12 cell neurite outgrowth. Phospholipase C-γ γ γ γ1 (PLC-γ γ γ γ1) can mediate the PAK translocation in response to growth factors, since PLC-γ γ γ γ1 binds to both tyrosine-phosphorylated receptor tyrosine kinases and PAK through its SH2 and SH3 domain, respectively. In the present study, we examined a potential role for PLC-γ γ γ γ1 in the basic fibroblast growth factor (bFGF)-induced PAK translocation using stable PC12 cell lines that overexpress in a tetracycline-inducible manner either the wild-type FGFR-1 or the Y766F FGFR-1 mutant. Phosphatidylinositol hydrolysis was increased 6.5-fold in response to bFGF in the wild type cells but negligible in the mutant cells. The recombinant GST-PLC-γ γ γ γ1 SH3 was able to bind to PAK1 but not GST alone. However, examination of PLC-γ γ γ γ1 as an adaptor for translocation of PAK1 in cells showed that both cells transfected with pEGFP-PAK1 was able to differentiate for 24 h, as visualized by laser confocal microscopy. Translocation of PAK1 to growth cones occurs at similar levels in both wild and mutant cells. These results suggest that a protein(s) other than PLC-γ γ γ γ1 is functionally relevant for PAK targeting.
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