Abstract
p21-activated kinase (PAK) targeting to the plasma membrane is essential for PC12 cell neurite outgrowth. Phospholipase C-gamma1 (PLC-gamma1) can mediate the PAK translocation in response to growth factors, since PLC-gamma1 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-gamma1 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-gamma1 SH3 was able to bind to PAK1 but not GST alone. However, examination of PLC-gamma1 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-gamma1 is functionally relevant for PAK targeting.
Highlights
Basic fibroblast growth factor, a member of FGF family, is a potent differentiation factor in the pheochromocytoma cell model PC12. bFGF exerts its effects through the activation of its high-affinity FGF receptor (FGFR), a receptor tyrosine kinase
We examined whether Phospholipase C-γ1 (PLC-γ1) can be a mediator for the p21-activated kinase (PAK) recruitment to the plasma membrane, since PLC-γ1 contains a SH3 domain that interacts with PAK
To determine whether bFGF-induced translocation of PAK involves the tyrosine phosphorylation and the activation of PLC-γ1, we generated stable PC12 cell lines that overexpress in a tetracycline-inducible manner either the wild-type FGFR-1 or the Y766F FGFR-1 mutant, in which Tyr 766 has been replaced by phenylalanine
Summary
Basic fibroblast growth factor (bFGF), a member of FGF family, is a potent differentiation factor in the pheochromocytoma cell model PC12. bFGF exerts its effects through the activation of its high-affinity FGF receptor (FGFR), a receptor tyrosine kinase. One of the target molecules of FGFR is phospholipase C gamma1 (PLC-γ1), which, upon bFGF stimulation, binds to the receptor and becomes tyrosine phosphorylated and activated. It has been shown that PLC-γ1-mediated phosphatidylinositol (PI) hydrolysis is not essential for FGF-induced neuronal differentiation of PC12 cells (Spivak-Kroizman et al, 1994).
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