Downregulation of c-fos gene transcription in cells transformed by E1A and cHa-ras oncogenes: a role of sustained activation of MAP/ERK kinase cascade and of inactive chromatin structure at c-fos promoter.
REF cells transformed by oncogenes E1A and cHa-ras reveal high and constitutive DNA-binding activity of AP-1 factor lacking in c-Fos protein. Consistently, the transcription of c-fos gene has been found to be downregulated. To elucidate the mechanisms of c-fos downregulation in E1A+cHa-ras transformants, we studied the levels of activity of ERK, JNK/SAPK and p38 kinases and phosphorylation state of Elk-1 transcription factor involved in regulation of c-fos gene. Using two approaches, Western blot analysis with phospho-specific antibodies to MAP kinases and in vitro kinase assay with specific substrates, we show here that ectopic expression of E1A and ras oncogenes leads to a sustained activation of ERK and p38 kinases, whereas JNK/SAPK kinase activity is similar to that in non-transformed REF52 cells. Due to sustained activity of the MAP kinase cascades, Elk-1 transcription factor is being phosphorylated even in serum-starved E1A+cHa-ras cells; moreover, serum does not additionally increase phosphorylation of Elk-1, which is predominant TCF protein bound to SRE region of c-fos gene promoter in these cells. Although the amount of ternary complexes SRE/SRF/TCF estimated by EMSA was similar both in serum-starved and serum-stimulated transformed cells, serum addition still caused a modest activation of c-fos gene transcription at the level of 20% to normal REF cells. In attempt to determine how serum caused the stimulatory effect, we found that PD98059, an inhibitor of MEK/ERK kinase cascade, completely suppressed serum-induced c-fos transcription both in REF and E1A+cHa-ras cells, implicating the ERK as primary kinase for c-fos transcription in these cells. In contrast, SB203580, an inhibitor of p38 kinase, augmented noticeably serum-stimulated transcription of c-fos gene in REF cells, implying the involvement of p38 kinase in negative regulation of c-fos. Furthermore, sodium butyrate, an inhibitor of histone deacetylase activity, was capable of activating c-fos transcription both in serum-stimulated and even in serum-starved E1A+cHa-ras cells. Conversely, serum-starved REF cells fail to respond to sodium butyrate treatment by c-fos activation confirming necessity of prior Elk-1 phosphorylation. Taken together, these data suggest that downregulation of c-fos in E1A+cHa-ras cells seems to occur due to a maintenance of a refractory state that arises in normal REF cells after serum-stimulation. The refractory state of c-fos in E1A+cHa-ras cells is likely a consequence of Ras-induced sustained activation of MAPK (ERK) cascade and persistent phosphorylation of TCF (Elk-1) bound to SRE. Combination of these events eventually does contribute to formation of an inactive chromatin structure at c-fos promoter mediated through recruitment of histone deacetylase activity.
Kukushkin
Alexander N
AN
Institute of Cytology, Russian Academy of Sciences, 194064 St-Petersburg, Russia.
Abramova
Maria V
MV
Svetlikova
Svetlana B
SB
Darieva
Zalfia A
ZA
Pospelova
Tatiana V
TV
Pospelov
Valery A
VA
D017500:Adenovirus E1A Proteins / Q000235:genetics*; D000818:Animals; D002087:Butyrates / Q000494:pharmacology; D002460:Cell Line; D002461:Cell Line, Transformed; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016762:Genes, fos; D011905:Genes, ras; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D058610:Isobutyrates; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D027602:Serum Response Element; D014157:Transcription Factors; D014158:Transcription, Genetic; D050807:ets-Domain Protein Elk-1; D048051:p38 Mitogen-Activated Protein Kinases
D017500:Adenovirus E1A Proteins; D002087:Butyrates; D002843:Chromatin; D004268:DNA-Binding Proteins; C494112:ELK1 protein, human; C494114:Elk1 protein, rat; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D058610:Isobutyrates; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; C020380:isobutyric acid; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases