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 growth factor (EGF) activate similar receptor tyrosine kinase signaling pathways but evoke strikingly different biological outcomes: NGF induces differentiation and EGF acts as a mitogen. A novel approach was developed for identifying transcription factor activities associated with NGF-activated, but not EGF-activated, signaling, using random oligonucleotide clones from a DNA recognition library to isolate specific DNA binding proteins from PC12 nuclear extracts. A protein complex from NGF-treated, but not EGF-treated, cells was identified that exhibits increased mobility and DNA binding activity in gel mobility shift assays. The binding complex was identified in supershift assays as Fra-2/JunD. The clones used as probes contain either AP-1 or cAMP response element binding (CREB) recognition elements. Time course experiments revealed further differences in NGF and EGF signaling in PC12 cells. 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 Western blots by bands in the 40-46 kDa range. NGF, but not EGF, enhances the upper bands, corresponding to phosphorylated Fra-2. These findings suggest that prolonged alterations in Fra-2 and subsequent increases in Fra-2/JunD binding to AP-1 and CREB response elements common among many gene promoters could serve to trigger broadly an NGF-specific program of gene expression.
Boss
V
V
Departments of Pharmacology and Pathology, Emory University School of Medicine, Atlanta, Georgia 30329, USA.
Roback
J D
JD
Young
A N
AN
Roback
L J
LJ
Weisenhorn
D M
DM
Medina-Flores
R
R
Wainer
B H
BH
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D051921:Fos-Related Antigen-2; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020932:Nerve Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D016716:PC12 Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000187:drug effects / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis*
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D051921:Fos-Related Antigen-2; C497874:Fosl2 protein, rat; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D004815:Epidermal Growth Factor; D020932:Nerve Growth Factor; D020928:Mitogen-Activated Protein Kinases