Sustained activation of extracellular signal-regulated kinase by nerve growth factor regulates c-fos protein stabilization and transactivation in PC12 cells.
The duration of intracellular signaling is thought to be a critical component in effecting specific biological responses. This paradigm is demonstrated by growth factor activation of the extracellular signal-regulated kinase (ERK) signaling cascade in the rat pheochromocytoma cell line (PC12 cells). In this model, sustained ERK activation induced by nerve growth factor (NGF) results in differentiation, whereas transient ERK activation induced by epidermal growth factor (EGF) results in proliferation in these cells. Recently, the immediate early gene product c-fos has been proposed to be a sensor for ERK signaling duration in fibroblasts. In this study, we ask whether this is true for NGF and EGF stimulation of PC12 cells. We show that NGF, but not EGF, can regulate both c-fos stability and activation in an ERK-dependent manner in PC12 cells. This is achieved through ERK-dependent phosphorylation of c-fos. Interestingly, distinct sites regulate enhanced stability and transactivation of c-fos. Phosphorylation of Thr325 and Thr331 are required for maximal NGF-dependent transactivation of c-fos. In addition, a consensus ERK binding site (DEF domain) is also required for c-fos transactivation. However, stability is controlled by ERK-dependent phosphorylation of Ser374, while phosphorylation of Ser362 can induce conformational changes in protein structure. We also provide evidence that sustained ERK activation is required for proper post-translational regulation of c-fos following NGF treatment of PC12 cells. Because these ERK-dependent phosphorylations are required for proper c-fos function, and occur sequentially, we propose that c-fos is a sensor for ERK signaling duration in the neuronal-like cell line PC12.
Pellegrino
Michael J
MJ
Neuroscience Graduate Program, Oregon Health Sciences University, Portland, Oregon, USA.
Stork
Philip J S
PJ
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002070:Butadienes / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects*; D008156:Luciferases / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009570:Nitriles / Q000494:pharmacology; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection / Q000379:methods
D002070:Butadienes; D004791:Enzyme Inhibitors; D009570:Nitriles; D016760:Proto-Oncogene Proteins c-fos; D018808:Transcription Factor AP-1; C113580:U 0126; D013912:Threonine; D012694:Serine; D020932:Nerve Growth Factor; D008156:Luciferases; D048049:Extracellular Signal-Regulated MAP Kinases; D019278:Matrix Metalloproteinase 3