Abstract
The activity of the transcription factor NF-kappaB can be modulated by members of the Rho family of small GTPases (Perona, R., Montaner, S., Saniger, L., Sánchez-Pérez, I., Bravo, R., and Lacal, J. C. (1997) Genes Dev. 11, 463-475). Ectopic expression of RhoA, Rac1, and Cdc42Hs proteins induces the translocation of NF-kappaB dimers to the nucleus, triggering the transactivation of the NF-kappaB-dependent promoter from the human immunodeficiency virus. Here, we demonstrate that activation of NF-kappaB by RhoA does not exclusively promote its nuclear translocation and binding to the specific kappaB sequences. NF-kappaB is also involved in the regulation of the transcriptional activity of the c-fos serum response factor (SRF), since the activation of a SRE-dependent promoter by RhoA can be efficiently interfered by the double mutant IkappaBalphaS32A/S36A, an inhibitor of the NF-kappaB activity. We also present evidence that RelA and p50 NF-kappaB subunits cooperate with the transcription factor C/EBPbeta in the transactivation of the 4 x SRE-CAT reporter. Furthermore, RhoA increases the levels of C/EBPbeta protein, facilitating the functional cooperation between NF-kappaB, C/EBPbeta, and SRF proteins. These results strengthen the pivotal importance of the Rho family of small GTPases in signal transduction pathways which modulate gene expression and reveal that NF-kappaB and C/EBPbeta transcription factors are accessory proteins for the RhoA-linked regulation of the activity of the SRF.
Highlights
We aimed to investigate whether activation of nuclear factor-B (NF-B) by Rho proteins was functionally related to the activation of the serum response element (SRE)-dependent transcriptional activity
We were able to demonstrate a more than 10-fold activation by serum when the NIH 3T3 cell system was used instead of the COS-7 cell sytem using the same reporter vectors. These results demonstrate that these three members of the Rho family of GTPases mediate in the signal transduction pathways implicated in the activation of the transcription factor NF-B and in the up-regulation of intracellular cascades which promote gene expression through the SRE-binding site in the simian fibroblast-like COS-7 cells
Since transactivation of the 4 ϫ SRE-chloramphenicol acetyltransferase (CAT) induced by RhoA seemed to be dependent on the NF-B activity (Fig. 2A), we investigated whether the expression of the IB␣S32A/S36A mutant had any effect in the activation of the SRE by lysophosphatidic acid (LPA), a physiological activator of the RhoA signaling cascade
Summary
The prototypic c-fos SRE binds a ternary complex composed of a homodimer of p67SRF (serum response factor) and a third subunit, p62TCF (ternary complex factor), which belongs to the Ets family of accessory proteins These TCF factors have the ability to bind a purine-rich motif 5Ј to the SRF-binding site, known as the Ets recognition domain, only when SRF is bound to DNA, and include Elk-1, SAP-1, and SAP-2/ERP/NET proteins. Ent sequences which can inhibit its own transactivation capability [25], it is possible that such interaction could relieve the transactivation domain from this inhibitory effect This putative recognition factor should be a critical target for Rho family-mediated signal transduction pathways
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