Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site

Abstract

The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies, protein interactions and posttranslational modifications, respectively. By using NMR, we have determined the structure of a 110-residue fragment of murine Ets-1 that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architecture is distinct from that of any known DNA-or proteinbinding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly f lexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphorylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site.Transcription factor families are defined by highly conserved DNA-binding domains that display similar DNA recognition properties. The means by which individual family members control different genes therefore must be determined by regulatory mechanisms that enhance the specificity of DNA binding. In the ets gene family, which includes at least 18 members in the human genome, partnerships with additional transcription factors, as well as posttranslational modifications, help dictate specificity for distinct targets (1). These regulatory mechanisms converge on a highly conserved Ϸ80-aa region termed the Pointed (PNT) domain (2).The PNT domain occurs in approximately one-third of the ets proteins, including Ets-1, Ets-2, GABP␣, and TEL from vertebrates, and PNT-P2 and Yan from Drosophila (Fig. 1). This domain is proposed to mediate protein-protein interactions and to be regulated by ras-dependent signaling because of the presence of an adjacent mitogen-activated protein (MAP) kinase phosphorylation site (1). In particular, the PNT domain is implicated in the self-association of chimeric oncoproteins, identified in human leukemias, that result from chromosomal translocations of the gene encoding the ets protein TEL with segments of genes encoding several tyrosine kinases or the acute myeloid leukemia (AML)-1B transcription factor (3-9).To date sequence conservation has defined the PNT domain, yet it has not been established that this region is a structural module that acts in a biological context. To create a framework for understanding the role of the PNT domain in the regulation of a variety of ets proteins and in the oncogenic potential of TEL fusion proteins, we have characterized structurally a fragment of Ets-1 that includes this domain and the adjacent MAP kinase phosphorylation site. MATERIALS AND METHODSProtein Samples. DNA sequences encoding Ets-1 , Ets-1 (29 -138) , and Ets-1 were PCR-amplified from the full-length murine ets-1 cDNA and...

Regulation of the Ets-1 transcription factor by sumoylation and ubiquitinylation

“…Ets-1 transcriptional activity is known to be regulated by phosphorylation via RAS and Ca 2 þ -dependent protein kinase pathways (Rabault and Ghysdael, 1994;Rabault et al, 1996;Wasylyk et al, 1997;Slupsky et al, 1998;Cowley and Graves, 2000;Paumelle et al, 2002). Here, we demonstrated that Ets-1 is also modified by other post-translational modifications, that is sumoylation and ubiquitinylation.…”

“…The N-terminal sequence of Ets-1 is phosphorylated at threonine 38, within a MAPK phosphorylation motif activated following RAS activation. This phosphorylation enhances Ets-1 transcriptional activity (Rabault et al, 1996;Wasylyk et al, 1997;Slupsky et al, 1998;Paumelle et al, 2002). In particular, we demonstrated that such activation of Ets-1 is triggered by hepatocyte growth factor/scatter factor (HGF/SF) through a RAS-ERK pathway, leading to transcriptional activation of Ets-1 and activation of promoters containing RAS-Responsive Elements constituted by combined EBS/AP1-binding sites (Fafeur et al, 1997;Paumelle et al, 2002).…”

“…Nonetheless, the specific disappearance of a sumoylated form of Ets-1 for K15 and K227 mutants at a different molecular weight was not expected (K15R and K227R leading to disappearance of the 75 and 90 kDa band, respectively). According to these molecular weights, it is possible that (Slupsky et al, 1998) and DNAbinding domain (DB, black) (Donaldson et al, 1996) are indicated. The transactivation domain (gray) and the central regulatory domain corresponding to exon VII are also indicated.…”

Characterization of the chronic myelomonocytic leukemia associated TEL-PDGFβR fusion protein

“…Our ®ndings thus suggest that the pointed/B domain homophilic interaction is a candidate target for drug development. In this context, the recently reported structures of the pointed/B domain of Ets-1, and of the structurally related SAM domains of the Eph4A and EphB2 receptor tyrosine kinases, are of great interest (Slupsky et al, 1998, Stapleton et al, 1999Thanos et al, 1999).…”

Dual function of the epithelial specific ets transcription factor, ELF3, in modulating differentiation

“…ets-1, ets-2,¯i-1, TEL, GABPa, and ELF3, share a homologous region at the N-terminus, referred to as the pointed domain. This domain appears to be important for the transcriptional regulatory mechanisms mediated by these ets factors and might be involved in protein ± protein interactions (Slupsky et al, 1998;Oettgen et al, 1999).…”