Phosphatidylinositol 4-phosphate 5-kinase alpha negatively regulates nerve growth factor-induced neurite outgrowth in PC12 cells

Neurite outgrowth, a cell differentiation process involving membrane morphological changes, is critical for neuronal network and development. The membrane lipid, phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), is a key regulator of many important cell surface events of membrane signaling, trafficking and dynamics. This lipid is produced mainly by the type I PI 4-phosphate 5-kinase (PIP5K) family members. In this study, we addressed whether PIP5Kalpha, an isoform of PIP5K, could have a role in neurite outgrowth induced by nerve growth factor (NGF). For this purpose, we knocked down PIP5Kalpha in PC12 rat pheochromocytoma cells by stable expression of PIP5Kalpha microRNA that significantly reduced PIP5Kalpha expression and PIP2 level. Interestingly, NGF-induced neurite outgrowth was more prominent in PIP5Kalpha-knockdown (KD) cells than in control cells. Conversely, add-back of PIP5Kalpha into PIP5Kalpha KD cells abrogated the effect of NGF on neurite outgrowth. NGF treatment activated PI 3-kinase (PI3K)/Akt pathway, which seemed to be associated with reactive oxygen species generation. Similar to the changes in neurite outgrowth, the PI3K/Akt activation by NGF was potentiated by PIP5Kalpha KD, but was attenuated by the reintroduction of PIP5Kalpha. Moreover, exogenously applied PIP2 to PIP5Kalpha KD cells also suppressed Akt activation by NGF. Together, our results suggest that PIP5Kalpha acts as a negative regulator of NGF-induced neurite outgrowth by inhibiting PI3K/Akt signaling pathway in PC12 cells.

Pleckstrin homology domain of phospholipase C-gamma1 directly binds to 68-kDa neurofilament light chain

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.