Cell signaling affects gene expression by regulating the activity of transcription factors. Here, we report that mitogen-activated protein kinase (MAPK) phosphorylation of Ets-1 and Ets-2, at a conserved site N terminal to their Pointed (PNT) domains, resulted in enhanced transactivation by preferential recruitment of the coactivators CREB binding protein (CBP) and p300. We discovered this phosphorylation-augmented interaction in an unbiased affinity chromatography screen of HeLa nuclear extracts by using either mock-treated or ERK2-phosphorylated ETS proteins as ligands. Binding between purified proteins demonstrated a direct interaction. Both the phosphoacceptor site, which lies in an unstructured region, and the PNT domain were required for the interaction. Minimal regions that were competent for induced CBP/p300 binding in vitro also supported MAPK-enhanced transcription in vivo. CBP coexpression potentiated MEK1-stimulated Ets-2 transactivation of promoters with Ras-responsive elements. Furthermore, CBP and Ets-2 interacted in a phosphorylation-enhanced manner in vivo. This study describes a distinctive interface for a transcription factor-coactivator complex and demonstrates a functional role for inducible CBP/p300 binding. In addition, our findings decipher the mechanistic link between Ras/MAPK signaling and two specific transcription factors that are relevant to both normal development and tumorigenesis.The connection of cell signaling to changes of gene expression represents a central step in many types of biological regulation. The Ras/mitogen-activated protein kinase (MAPK) pathway exemplifies the relevance of signaling to both normal development and disease. In this pathway, Ras, a GTPase, transmits extracellular signaling from receptor tyrosine kinases to two serine/threonine kinases (Raf and MEK) and, finally, to the activation of MAPKs. Upon nuclear import, MAPKs phosphorylate many different transcription factors, modulating DNA binding affinity, nuclear localization, stability, and interactions with coregulators (20, 58), thereby regulating gene expression. Genetic studies of Drosophila (54) and Caenorhabditis elegans (48) demonstrate that Ras/MAPK signaling plays a role in normal development. Furthermore, ϳ15 to 20% of all human tumors have an activating mutation in one of three ras genes (N-, K-, or H-ras), and oncogenic mutations in the B-raf gene have been identified for a wide variety of cancers, with 66% of all melanomas being affected (6,8). Although many of the components of Ras/MAPK signaling have been characterized, the full array of transcription factors affected is not known. Furthermore, the detailed mechanisms by which phosphorylation modulates transcription factors remain unclear in many cases.Multiple members of the ETS family of transcription factors are phosphorylated upon activation of the Ras/MAPK signaling pathway. For example, Elk-1 phosphorylation results in recruitment of the mediator complex via its Sur-2 (MED23) subunit (49), enhanced interaction with the coactivator p30...
We present evidence that Escherichia coli RNA polymerase  subunit may be a transcriptional activator contact site. Stimulation of the activity of the p R promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage . We found that DnaA activates the p R promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the p R promoter was scrambled by in vitro mutagenesis, the p R promoter was no longer activated by DnaA both in vivo and in vitro. Therefore, we conclude that DnaA may contact the  subunit of RNA polymerase during activation of the p R promoter. A new classification of prokaryotic transcriptional activators is proposed.Activation of transcription is a common way to regulate gene expression in both prokaryotes and eukaryotes (for reviews, see refs. 1 and 2). In bacterial cells, transcription activation at a given promoter is achieved usually by a direct contact between a transcriptional activator and RNA polymerase. The initiation of replication of plasmids derived from bacteriophage , known as plasmids (a map of the replication region present in standard plasmids is presented in Fig. 1), requires transcription at or near the origin of replication ori (for a review, see ref. 9). Transcription starting from the p R promoter and provides mRNA for production of the replication proteins O and P. Moreover, this transcription serves in the so called transcriptional activation of ori. It seems that the main regulatory role in the initiation of plasmid replication is played by the transcriptional activation of ori rather than by binding of the initiator protein O to ori (10-15). Therefore, regulation of the activity of the p R promoter is crucial for the control of plasmid DNA replication.It has been reported that E. coli DnaA protein is important for replication of bacteriophage (16) and plasmids (17,18). Subsequent studies demonstrated that transcription starting at p R and proceeding through ori is depressed in certain dnaA mutants, which led to the conclusion that this promoter is positively regulated by DnaA (19). The above mentioned conclusion (18) were strengthened by observations that wildtype plasmids cannot replicate in certain temperaturesensitive dnaA mutants even at temperatures permissive for bacterial growth (30 or 37°C) and that this defect may be suppressed by a mutation of the type in P gene. It was proposed that transcriptional activation of ori is coupled with the chaperone-mediated rearrangement of the preprimosomal complex (liberation of DnaB from P inhibition) and insertion of the preprimosome between transiently separated DNA strands (16,18). Because the product of the P gene harboring a mutation interacts significantly weaker with the host DnaB helicase than does the wild-type P protein (20), it was proposed that impaired transcript...
Ras/MAPK signaling is often aberrantly activated in human cancers. The downstream effectors are transcription factors, including those encoded by the ETS gene family. Using cell-based assays and biophysical measurements, we have determined the mechanism by which Ras/MAPK signaling affects the function of Ets1 via phosphorylation of Thr38 and Ser41. These ERK2 phosphoacceptors lie within the unstructured N-terminal region of Ets1, immediately adjacent to the PNT domain. NMR spectroscopic analyses demonstrated that the PNT domain is a four-helix bundle (H2-H5), resembling the SAM domain, appended with two additional helices (H0-H1). Phosphorylation shifted a conformational equilibrium, displacing the dynamic helix H0 from the core bundle. The affinity of Ets1 for the TAZ1 (or CH1) domain of the coactivator CBP was enhanced 34-fold by phosphorylation, and this binding was sensitive to ionic strength. NMR-monitored titration experiments mapped the interaction surfaces of the TAZ1 domain and Ets1, the latter encompassing both the phosphoacceptors and PNT domain. Charge complementarity of these surfaces indicate that electrostatic forces act in concert with a conformational equilibrium to mediate phosphorylation effects. We conclude that the dynamic helical elements of Ets1, appended to a conserved structural core, constitute a phospho-switch that directs Ras/MAPK signaling to downstream changes in gene expression. This detailed structural and mechanistic information will guide strategies for targeting ETS proteins in human disease.MAP kinase | protein structure/dynamics | transcriptional regulation | protein-protein interaction | Ets2
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