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.

The activity of Rho GTPases in migrating cells is regulated by binding of myosin II to GEFs.

Bioinformatic and functional data link integrin-mediated cell adhesion to cellular senescence; however, the significance of and molecular mechanisms behind these connections are unknown. We now report that the focal adhesion–localized βPAK-interacting exchange factor (βPIX)–G protein–coupled receptor kinase interacting protein (GIT) complex controls cellular senescence in vitro and in vivo. βPIX and GIT levels decline with age. βPIX knockdown induces cellular senescence, which was prevented by reexpression. Loss of βPIX induced calpain cleavage of the endocytic adapter amphiphysin 1 to suppress clathrin-mediated endocytosis (CME); direct competition of GIT1/2 for the calpain-binding site on paxillin mediates this effect. Decreased CME and thus integrin endocytosis induced abnormal integrin signaling, with elevated reactive oxygen species production. Blocking integrin signaling inhibited senescence in human fibroblasts and mouse lungs in vivo. These results reveal a central role for integrin signaling in cellular senescence, potentially identifying a new therapeutic direction.

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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.