Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate requires Raf and is necessary for transformation.
The protein kinase cascade Raf-MAPKK/MEK-MAPK/ERK connects protein tyrosine kinase receptors in the membrane with control of transcription factor activity in the nucleus. We have examined whether Raf is obligatory for activation of this cascade and whether this signaling pathway is relevant to transformation. By use of transient assays with epitope-tagged ERK-1 cDNA and a dominant inhibitory mutant of Raf-1 we found that serum and 12-O-tetradecanoylphorbol-13-acetate as well as representatives of three classes of oncogenes (protein tyrosine kinases abl/src, Ras, and protein serine/threonine kinases mos/cot) were all Raf-dependent for stimulation of MAPK. All of the MAPK stimulating oncogenes were also activators of Raf kinase as judged by shift induction. It thus appears that there is little or no redundancy in pathways used by growth regulators for activation of MAPK/ERK. Furthermore, the ability to stimulate MAPK/ERK appears to be critical for transformation by oncogenic Raf-1 and ERK-1 and -2 synergized with v-raf in a focus induction assay on NIH3T3 cells and kinase dead mutants of ERK-2 were inhibitory. Raf/ERK synergism was also observed in transcriptional transactivation of the oncogene-response element in the polyoma enhancer. We conclude that this Raf signaling pathway, which connects to many upstream activators and downstream effectors, is essential for transformation by most oncogenes.
Troppmair
J
J
Viral Pathology Section, Laboratory of Viral Carcinogenesis, Frederick, Maryland 21702-1201.
Bruder
J T
JT
Munoz
H
H
Lloyd
P A
PA
Kyriakis
J
J
Banerjee
P
P
Avruch
J
J
Rapp
U R
UR
D016475:3T3 Cells; D000818:Animals; D001769:Blood; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D003470:Culture Media; D006801:Humans; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D009857:Oncogenes; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D003470:Culture Media; D011518:Proto-Oncogene Proteins; D015500:Chloramphenicol O-Acetyltransferase; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D013755:Tetradecanoylphorbol Acetate