Phosphorylation of p85 beta PIX, a Rac/Cdc42-specific guanine nucleotide exchange factor, via the Ras/ERK/PAK2 pathway is required for basic fibroblast growth factor-induced neurite outgrowth.
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 betaPIX/Cool/p85SPR is mediated via the Ras/ERK/PAK2 pathway. To understand the role of p85 betaPIX 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 betaPIX, as determined by metabolic labeling and mobility shift upon gel electrophoresis. Interestingly, phosphorylation of p85 betaPIX 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 betaPIX, also functions upstream of p85 betaPIX phosphorylation. Surprisingly, PAK2 directly binds to ERK, and its activation is dependent on ERK. p85 betaPIX specifically localizes to the lamellipodia at neuronal growth cones in response to bFGF. A mutant form of p85 betaPIX (S525A/T526A), in which the major phosphorylation sites are replaced by alanine, shows significant defect in targeting. Moreover, expression of the mutant p85 betaPIX 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 betaPIX complex via the ERK cascade and subsequent translocation of this complex.
Shin
Eun-Young
EY
Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju 361-763, Korea.
Shin
Kyung-Sun
KS
Lee
Chan-Soo
CS
Woo
Kyung-Nam
KN
Quan
Song-Hua
SH
Soung
Nak-Kyun
NK
Kim
Young Gyu
YG
Cha
Choong Ik
CI
Kim
Seung-Ryul
SR
Park
Dongeun
D
Bokoch
Gary M
GM
Kim
Eung-Gook
EG
D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D004789:Enzyme Activation; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D016716:PC12 Cells; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D021381:Protein Transport; D051381:Rats; D064067:Rho Guanine Nucleotide Exchange Factors; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013752:Tetracycline / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D020033:Protein Isoforms; D064067:Rho Guanine Nucleotide Exchange Factors; D016222:Fibroblast Growth Factor 2; D013912:Threonine; D012694:Serine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D013752:Tetracycline