Transient activation of RAF-1, MEK, and ERK2 coincides kinetically with ternary complex factor phosphorylation and immediate-early gene promoter activity in vivo.
We have investigated the early in vivo signaling events triggered by serum that lead to activation of the c-fos proto-oncogene in HeLa cells. Both RAF-1 and MEK kinase activities are fully induced within 3 min of serum treatment and quickly decrease thereafter, slightly preceding the activation and inactivation of p42MAPK/ERK2. ERK2 activity correlates tightly with a transient phosphatase-sensitive modification of ternary complex factor (TCF), manifested by the slower electrophoretic mobility of TCF-containing protein-DNA complexes. These induced complexes in turn correlate with the activity of the c-fos, egr-1, and junB promoters. Phorbol ester treatment induces the same events but with slower and prolonged kinetics. Inhibition of serine/threonine phosphatase activities by okadaic acid treatment reverses the repression of the c-fos promoter either after induction or without induction. This corresponds to the presence of the induced complexes and of ERK2 activity, as well as to the activation of a number of other kinases. Inhibition of tyrosine phosphatase activities by sodium vanadate treatment delays but does not block ERK2 inactivation, TCF dephosphorylation, and c-fos repression. The tight linkage in vivo between the activity of MAP kinase, TCF phosphorylation, and immediate-early gene promoter activity is consistent with the notion that a stable ternary complex over the serum response element is a direct target for the MAP kinase signaling cascade. Furthermore, serine/threonine phosphatases are implicated in regulating the kinase cascade, as well as the state of TCF modification and c-fos promoter activity, in vivo.
Hipskind
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RA
Institute for Molecular Biology, Hannover Medical School, Germany.
Baccarini
M
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Nordheim
A
A
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D017781:Genes, Immediate-Early; D006367:HeLa Cells; D006801:Humans; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors; D014158:Transcription, Genetic; D050807:ets-Domain Protein Elk-1
D004268:DNA-Binding Proteins; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D015682:Retroviridae Proteins, Oncogenic; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; C072545:p62TCF protein, human; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases