Nerve growth factor, but not epidermal growth factor, increases Fra-2 expression and alters Fra-2/JunD binding to AP-1 and CREB binding elements in pheochromocytoma (PC12) cells

In pheochromocytoma (PC12) cells nerve growth factor (NGF) and epidermal gr owth factor (EGF) activate similar receptor tyrosine kinase signaling pathw ays but evoke strikingly different biological outcomes: NGF induces differe ntiation and EGF acts as a mitogen. A novel approach was developed for iden tifying transcription factor activities associated with NGF-activated, but not EGF-activated, signaling, using random oligonucleotide clones from a DN A recognition library to isolate specific DNA binding proteins from PC12 nu clear extracts. A protein complex from NGF-treated, but not EGF-treated, ce lls was identified that exhibits increased mobility and DNA binding activit y in gel mobility shift assays. The binding complex was identified in super shift assays as Fra-2/JunD. The clones used as probes contain either AP-1 o r cAMP response element binding (CREB) recognition elements. Time course ex periments revealed further differences in NGF and EGF signaling in PC12 cel ls. NGF elicits a more delayed and sustained ERK phosphorylation than EGF, consistent with previous reports. Both growth factors transiently induce c- fos, but NGF evokes a greater response than EGF. NGF specifically increases Fra-1 and Fra-2 levels at 4 and 24 hr. The latter is represented in Wester n blots by bands in the 40-46 kDa range. NGF, but not EGF, enhances the upp er bands, corresponding to phosphorylated Fra-2. These findings suggest tha t prolonged alterations in Fra-2 and subsequent increases in Fra-2/JunD bin ding to AP-1 and CREB response elements common among many gene promoters co uld serve to trigger broadly an NGF-specific program of gene expression.