An essential role for the SHIP2-dependent negative feedback loop in neuritogenesis of nerve growth factor–stimulated PC12 cells

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Abstract
The local accumulation of phosphatidylinositol (3,4,5) trisphosphate (PIP3) and resulting activation of Rac1/Cdc42 play a critical role in nerve growth factor (NGF)–induced neurite outgrowth. To further explore the mechanism, we visualized PIP3, phosphatidylinositol (3,4) bisphosphate, and Rac1/Cdc42 activities by fluorescence resonance energy transfer (FRET) imaging in NGF-stimulated PC12 cells. Based on the obtained FRET images, and with the help of in silico kinetic reaction model, we predicted that PI-5-phosphatase negatively regulates PIP3 upon NGF stimulation. In agreement with this model, depletion of Src homology 2 domain–containing inositol polyphosphate 5-phosphatase 2 (SHIP2) markedly potentiated NGF-induced Rac1/Cdc42 activation and PIP3 accumulation and considerably increased the number and the length of neurites in phosphate and tensin homologue–depleted PC12 cells. Further refinement of the computational model predicted Rac1 regulation of PI3-kinase and SHIP2, which was also validated experimentally. We propose that the SHIP2-mediated negative feedback on PIP3 coordinately works with the PI3-kinase–mediated positive feedback to form an initial protrusive pattern and, later, to punctuate the PIP3 accumulation to maintain proper neurite outgrowth.