Reconstitution of signal transduction from the membrane to the nucleus in a baculovirus expression system: activation of Raf-1 leads to hypermodification of c-jun and c-fos via multiple pathways.
We have attempted to dissect signaling pathways involved in transmitting activating signals from the cell surface to the nucleus by reconstituting them in the baculovirus/Sf9 cell system. We have used this system to coexpress different combinations of the critical signaling proteins pp60v-src, p21v-ras, Raf-1 and ERK-1 and assayed the effects of resulting signaling cascades on the modifications of coexpressed transcription factors c-jun or c-fos. We observe that activation of ERK-1 via Raf-1 and p21ras dependent signals can result in the hyperphosphorylation of c-jun. In contrast, c-fos appears to be the target of two Raf-1 activated modifying signals: one independent of ERK-1 and the other dependent on ERK-1 stimulation. Thus, coexpression of c-fos with pp60v-src, p21v-ras or constitutively active forms of Raf-1 results in a dramatic reduction of its electrophoretic mobility in the absence of coexpressed ERK-1. Activation of this ERK-1-independent pathway together with the ERK-1 dependent pathway that modifies c-jun results in additional modification of c-fos. Our observation of a Raf-1 activated, ERK-independent signaling pathway is consistent with previous reports that constitutively active Raf-1 can, in some cell types, result in transformation or differentiation without activation of ERKs. Our data indicate the presence of multiple Raf-1 activated pathways that lead to modification of transcription factors.
Agarwal
S
S
Dana Farber Cancer Institute, Harvard Medical School, Boston Massachusetts 02115, USA.
Corbley
M J
MJ
Roberts
T M
TM
D016475:3T3 Cells; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010449:Peptide Mapping; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011994:Recombinant Proteins; D015398:Signal Transduction; D018411:Spodoptera
D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D016392:Proto-Oncogene Proteins pp60(c-src); D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D016283:Proto-Oncogene Proteins p21(ras)