The Rho family of small GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to the cell nucleus, including the JNK/SAPK signaling pathway, the c-/os serum response factor, and the p70 S6 kinase. Here we report a novel signaling pathway activated by the Rho proteins that may be responsible for their biological activities, including cytoskeleton organization, transformation, apoptosis, and metastasis. The human RhoA, CDC42, and Rac-1 proteins efficiently induce the transcriptional activity of nuclear factor KB (NF-KB) by a mechanism that involves phosphorylation of iKBa and translocation of p50/p50 and p50/p65 dimers to the nucleus, but independent of the Ras GTPase and the Raf-1 kinase. We also show that activation of NF-KB by TNFa depends on CDC42 and RhoA, but not Rac-1 proteins, because this activity is drastically inhibited by their respective dominant-negative mutants. In contrast, activation of NF-KB by UV light was not affected by Rho, CDC42, or Rac-1 dominant-negative mutants. Thus, members of the Rho family of GTPases are involved specifically in the regulation of NF-KB-dependent transcription.
Recent progress in deciphering the molecular basis of carcinogenesis is of utmost importance to the development of new anticancer strategies. To this end, it is essential to understand the regulation of both normal cell proliferation and its alterations in cancer cells. We have previously demonstrated that in ras-transformed cells there is an increased level of phosphorylcholine (PCho) resulting from a constitutive activation on choiline kinase (ChoK). The importance of ChoK for the regulation of cell proliferation has also been proposed since an inhibitor for this enzyme, hemicholinium-3 (HC-3), drastically reduces entry into the S phase after stimulation with growth factors. Here we report the synthesis of several new compounds which are highly speci®c inhibitors for ChoK, with up to 1000-fold or 600-fold increased inhibitory activity, compared to HC-3 under ex vivo or in vitro conditions respectively. These novel compounds also drastically reduce entry into the S phase after stimulation with speci®c growth factors. A more profound inhibition of cell proliferation was observed in ras-, src-and mos-transformed cells in the presence of ChoK inhibitors, compared to their parental, untransformed NIH3T3 cells. By contrast, this e ect was not observed in fos-transformed cells. While ras, src and mos transformation is associated with elevated levels of ChoK activity, fos-induced transformation does not a ect ChoK activity. The inhibitory e ect on proliferation of the new compounds correlates with their ability to inhibit the production of phosphorylcholine in whole cells, a proposed novel second messenger for cell proliferation. These results strongly support a critical role of choline kinase in the regulation of cell growth and makes this enzyme a novel target for the design of new antiproliferative and anticancer drugs.
The activity of the transcription factor NF-B can be modulated by members of the Rho family of small GTPases (Perona, R., Montaner, S., Saniger, L., Sá nchez-Pé rez, 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-B dimers to the nucleus, triggering the transactivation of the NF-B-dependent promoter from the human immunodeficiency virus. Here, we demonstrate that activation of NF-B by RhoA does not exclusively promote its nuclear translocation and binding to the specific B sequences. NF-B 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 IB␣S32A/S36A, an inhibitor of the NF-B activity. We also present evidence that RelA and p50 NF-B subunits cooperate with the transcription factor C/EBP in the transactivation of the 4 ؋ SRE-CAT reporter. Furthermore, RhoA increases the levels of C/EBP protein, facilitating the functional cooperation between NF-B, C/EBP, 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-B and C/EBP transcription factors are accessory proteins for the RhoA-linked regulation of the activity of the SRF.Gene expression is regulated by the interplay of different transcription factors which bind to specific DNA recognition motifs and cooperate with the basal machinery to initiate transcription. During the last few years, an emerging body of evidence is revealing the importance of crossed interactions between members of distinct families of transcription factors to form higher complexes, enabling the accurate regulation of this process. The serum response element (SRE) 1 is a specific DNA sequence which is found in the promoter of several immediateearly genes (2). The prototypic c-fos SRE binds a ternary complex composed of a homodimer of p67 SRF (serum response factor) and a third subunit, p62 TCF (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. The formation of this SRE bindingternary complex requires the conserved B-box motif of the Ets subunits and sequences located in the core domain of the SRF protein (core SRF ), which is a region that is also responsible of its DNA binding and dimerization capabilities (2-8).The SRE has shown to be necessary and sufficient for the rapid induction of the c-fos proto-oncogen in response to different external stimuli such as serum, growth factors, and phorbol esters (2, 9). Furthermore, this DNA motif is a point of convergence of different signal transduction cascades activated by an extensive range of agonists. The regulat...
The juvenile-to-adult transition is a complex and poorly understood process in plant development required to reach reproductive competence. For woody plants, knowl
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