Phospholipase activity of phospholipase C-gamma1 is required for nerve growth factor-regulated MAP kinase signaling cascade in PC12 cells.
Phospholipase C-gamma1 (PLC-gamma1) hydrolyzes phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). PLC-gamma1 is implicated in a variety of cellular signalings and processes including mitogenesis and calcium entry. However, numerous studies demonstrate that the lipase activity is not required for PLC-gamma1 to mediate these events. Here, we report that the phospholipase activity of PLC-gamma1 plays an essential role in nerve growth factor (NGF)-triggered Raf/MEK/MAPK pathway activation in PC12 cells. Employing PC12 cells stably transfected with an inducible form of wild-type PLC-gamma1 or lipase inactive PLC-gamma1 with histidine 335 mutated into glutamine in the catalytic domain, we show that NGF provokes robust activation of MAP kinase in wild-type but not in lipase inactive cells. Both Ras/C-Raf/MEK1 and Rap1/B-Raf/MEK1 pathways are intact in the wild-type cells. By contrast, these signaling cascades are diminished in the mutant cells. Pretreatment with cell permeable DAG analog 1-oleyl-2-acetylglycerol rescues the MAP kinase pathway activation in the mutant cells. These observations indicate that the lipase activity of PLC-gamma1 mediates NGF-regulated MAPK signaling upstream of Ras/Rap1 activation probably through second messenger DAG-activated Ras and Rap-GEFs.
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Rong
R
Rong
Jee-Yin
JY
Ahn
Peng
P
Chen
Pann-Ghill
PG
Suh
Keqiang
K
Ye
D000818:Animals; D020134:Catalytic Domain; D004789:Enzyme Activation; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D020932:Nerve Growth Factor / Q000378:metabolism*; D016716:PC12 Cells; D051966:Phospholipase C gamma; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D006639:Histidine; D020932:Nerve Growth Factor; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D020827:rap1 GTP-Binding Proteins