Estrogen activates raf-1 kinase and induces expression of Egr-1 in MCF-7 breast cancer cells.
We have investigated whether the raf-1 kinase, a downstream mediator of both receptor tyrosine kinase and protein kinase C signalling, is activated by estrogen (E2) in an estrogen receptor positive human breast cancer cell line. Autophosphorylation of raf-1 kinase was studied after treatment of MCF-7 cells with E2. E2-deprived cells contained low levels of raf-1 kinase activity. Treatment of cells for 1 min with E2 resulted in raf-1 autophosphorylation which was maximal within 5 min. Western blot analysis showed that raf-1 undergoes an electrophoretic mobility shift following E2 treatment. Egr-1 is a zinc finger-containing transcription factor which is expressed in association with raf-1 activation. Untreated MCF-7 cells expressed low levels of Egr-1 while E2 treatment resulted in an induction of egr-1 mRNA expression. These kinetics followed closely behind the E2 induction of c-myc mRNA. Egr-1 protein was similarly low in E2-deprived MCF-7 cells and was transiently increased following E2 treatment. Several studies have suggested that kinase activity may play a role in estrogen receptor (ER) activation. While activated v-raf failed to augment ER activation of transcription in transient transfection assays, a dominant negative mutant of raf-1 inhibited E2-induced transcription by 50% primarily as a result of increased baseline levels of E2 independent transcription. The results show that E2 can induce raf-1 kinase activity in MCF-7 breast cancer cells associated with the expression of an early growth response gene and modulation of ER signalling.
Pratt
M A
MA
Department of Pharmacology, University of Ottawa, Ontario, Canada.
Satkunaratnam
A
A
Novosad
D M
DM
D015152:Blotting, Northern; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D017874:Immediate-Early Proteins; D009154:Mutation; D027384:Oncogene Proteins v-raf; D010766:Phosphorylation; D011233:Precipitin Tests; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D011960:Receptors, Estrogen; D015682:Retroviridae Proteins, Oncogenic; D014157:Transcription Factors; D004958:Estradiol; D027384:Oncogene Proteins v-raf; D019908:Proto-Oncogene Proteins c-raf