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
Agarwal, S.; Corbley, M.J.; Roberts, T.M.
Oncogene 11(3): 427-438
1995
ISSN/ISBN: 0950-9232 PMID: 7543194 Document Number: 439957
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 pp60-v-src, p21-v-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 p21-ras 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 pp60-v-src, p21-v-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 ERK1-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.