The PEA3 Ets Transcription Factor Comprises Multiple Domains That Regulate Transactivation and DNA Binding

Abstract

PEA3, a member of the Ets family of transcription factors, is a nuclear phosphoprotein capable of activating transcription. Mouse PEA3 comprises 480 amino acids and bears an ϳ85-amino acid ETS domain near its carboxyl terminus. Whereas analyses of bacterially expressed PEA3 revealed that the ETS domain is required for sequence-specific DNA binding, little is known of the functional domains in the protein required for its activity in mammalian cells. To this end, we defined the location of the PEA3 functional domains in COS cells. PEA3 bears a strong activation domain near its amino terminus, which is flanked by two regions that independently negatively regulate its activity. PEA3 expressed in COS cells was incapable of binding to DNA in vitro. However, DNA binding activity could be unmasked by incubation with a PEA3-specific antibody. Analyses of the DNA binding activity of PEA3 deletion mutants revealed that two regions flanking the ETS domain independently inhibited DNA binding; deletion of both regions was required to detect DNA binding in the absence of a PEA3-specific antibody. Under these conditions, the ETS domain was sufficient for sequence-specific DNA binding. These findings suggest that the activity of PEA3 is exquisitely controlled at multiple functional levels.The Ets family of transcription factors are defined by an evolutionarily conserved ϳ85-amino ETS domain (1). These proteins are found exclusively in multicellular organisms and are thought to play cardinal roles in development and oncogenesis (2). Multiple ets genes have been identified in individual organisms; over 20 mammalian genes have been discovered thus far (3). Ets proteins are sequence-specific DNA-binding proteins that regulate transcription (reviewed in Ref. 2). Generally, these proteins activate transcription, but several members of the family are known to repress this process. DNA binding is achieved by interaction between the ETS domain and an ϳ10-base pair sequence element termed the Ets binding site comprising a highly conserved central core sequence, 5Ј-GGA(A/T)-3Ј. Individual Ets proteins demonstrate specificity for sequences flanking this core, but it is not uncommon for different Ets proteins to bind to the same Ets binding site. Structural analyses of the ETS domain reveal a winged-helixturn-helix structure akin to that of the Escherichia coli catabolite activator protein and the HNF3/forkhead and heat shock transcription factors (4 -7).Mouse pea3 (8) (the human gene is named ETV4 and has also been termed E1A-F) (9, 10) is the founding member of the pea3 subfamily of ets genes. This subfamily also includes er81 (ETV1) (11-13) and erm (ETV5) (14, 15). Each of these genes is located on a different chromosome (16), but all three genes share a common architecture comprising 14 equivalently sized exons that encode similar sequences of the respective proteins (16 -18). The overall amino acid sequence similarity of the PEA3 1 subfamily is ϳ50% (17). The three longest stretches of greatest sequence similarity include the 85-amino ...

Identification of Novel Domains within Sox-2 and Sox-11 Involved in Autoinhibition of DNA Binding and Partnership Specificity

“…It is also interesting to note that in the case of both Sox-11F and Sox-11F⌬TAD, the addition of the M2 antibody led to an increase in the intensity of the DNA-protein complex (compare lanes 3 and 4 or lanes 6 and 7), indicating that antibody binding may in some way neutralize the autoinhibitory region. We believe this further supports the hypothesis that an autoinhibitory region exists within Sox-11F because antibody binding has been found to release autoinhibition in both PEA3 and p53 (34,35). Taken together our findings suggest that a novel autoregulatory domain is present in Sox-11, which we suspect plays an important role in its function as a transcription factor in vivo.…”

“…Evidence that these autoinhibitory domains also regulate in vivo recruitment of PEA3 was observed when the ability of full-length PEA3 to activate a reporter gene was compared with a deletion construct in which one autoinhibitory domain was removed. In these studies, removal of the autoinhibitory domain increased activation of the reporter gene ϳ2-fold, which indicates that the optimal capability of full-length PEA3 to activate gene expression was reduced (34).…”

HER2/Neu- and TAK1-mediated Up-regulation of the Transforming Growth Factor β Inhibitor Smad7 via the ETS Protein ER81

“…This suggests that DNA binding of ER81 is intramolecularly inhibited by N-terminal amino acids that contain its phosphorylation sites, similar to what has been observed for the related ETS protein PEA3 (65,66). HER2/Neu induces the phosphorylation of ER81 within its N terminus (42) and may thereby alleviate this intramolecular repression.…”

Pea3 expression is regulated by FGF signaling in developing retina

“…Thus, the expression of Pea3 is not sufficient to cause ganglion cell development, and other factors must also regulate this process. DNA binding by Pea3 subfamily members is regulated by cisacting autoinhibitory domains on either side of the DNA binding domain (Bojovic and Hassell, 2001). Phosphorylation of Erm by PKA adjacent to the DNA-binding domain regulates its ability to bind DNA, such that phosphorylated Erm binds DNA much less effectively than its unphosphorylated form (Baert et al, 2002).…”