Multiple aromatic side chains within a disordered structure are critical for transcription and transforming activity of EWS family oncoproteins

Abstract

Chromosomal translocations involving the N-terminal approximately 250 residues of the Ewings sarcoma (EWS) oncogene produce a group of EWS fusion proteins (EFPs) that cause several distinct human cancers. EFPs are potent transcriptional activators and interact with other proteins required for mRNA biogenesis, indicating that EFPs induce tumorigenesis by perturbing gene expression. Although EFPs were discovered more than a decade ago, molecular analysis has been greatly hindered by the repetitive EWS activation domain (EAD) structure, containing multiple degenerate hexapeptide repeats (consensus SYGQQS) with a conserved tyrosine residue. By exploiting total gene synthesis, we have been able to systematically mutagenize the EAD and determine the effect on transcriptional activation by EWS/ATF1 and cellular transformation by EWS/Fli1. In both assays, we find the following requirements for EAD function. First, multiple tyrosine residues are essential. Second, phenylalanine can effectively substitute for tyrosine, showing that an aromatic ring can confer EAD function in the absence of tyrosine phosphorylation. Third, there is little requirement for specific peptide sequences and, thus, overall sequence composition (and not the degenerate hexapeptide repeat) confers EAD activity. Consistent with the above findings, we also report that the EAD is intrinsically disordered. However, a sensitive computational predictor of natural protein disorder (PONDR VL3) identifies potential molecular recognition features that are tyrosine-dependent and that correlate well with EAD function. In summary we have uncovered several molecular features of the EAD that will impact future studies of the broader EFP family and molecular recognition by complex intrinsically disordered proteins.

Fig. 1.

Structure of EFPs and the EAD. (A) EFPs. The normal EWS protein contains an N-terminal region (EAD) and a C-terminal RNA-binding domain (RBD). A simplified structure of the two oncogenic EFPs analyzed in the current study (EWS/Fli1 and EWS/ATF1) is shown. EWS/Fli1 and EWS/ATF1 are associated with Ewings sarcoma (EWS) (3, 4) and clear cell sarcoma (CCS) (46), respectively. The extended EFP family (2, 3) includes EWS/WT1 (desmoplastic small round cell tumor), EWS/CHOP (myxoid liposarcoma), EWS/CHN (myxoid chondrosarcoma), and EWS/ZSG (small round cell tumor). All EFPs contain the EAD (residues 1–264) and are lacking the EWS RBD. The distinct EWS fusion partners noted above are all transcription factors that in each case contribute at least a DNA-binding domain to the EFP. Intact ATF1 is a PKA-inducible activator (47), whereas, in contrast, EWS/ATF1 is a potent constitutive activator of ATF-dependent promoters (10) dependent on the EAD. (B) Primary structure of the EAD. There are multiple degenerate hexapeptide repeats (DHRs, purple boxes) with consensus sequence SYGQQS (4) and seven additional Tyr residues (dark gray boxes). The location of SH2-binding sites (YxxP, yellow boxes) and SH3-binding sites (PxxP, green boxes) are indicated. Spaces between DHRs are generally only a few residues except for S1 and S2, which are 12 and 25 residues, respectively. (C) DHRs. The sequence variation for EAD DHRs is indicated. Numbers are % occurrence and the absolutely conserved tyrosine (position 2) and well conserved glutamine (position 4) are highlighted in red.

Fig. 2.

Effect of EAD mutations on transactivation by EWS/ATF1. All proteins contain the ATF1 portion of EWS/ATF1, but this is not shown. (Left) WT is an EWS/ATF1 fusion (Δ287C) containing the intact EAD (residues 1–287) and the region of ATF1(residues 66–271) present in oncogenic EWS/ATF1. DHRs (light gray boxes) and other non-DHR tyrosines (dark gray) are shown. Tyr to Ala changes (DA, OA, CA, N3OA, and NA) are indicated by green boxes. Gln to Ala changes (QA, light blue boxes) include all conserved DHR Gln residues in EAD1-176. Ser/Thr to Ala changes (STA, orange boxes) include one change in each DHR within EAD1-176 (see Materials and Methods). N3 (expressed from pΔ167C; ref. 11) contains the N-terminal 166 residues of the EAD and Δ78m is lacking the N-terminal 78 residues of the EAD. DAR contains the WT EAD region (present in N3) and the corresponding region (N3DA, green box) from DA. (Center) Quantitation of transactivation relative to 100% for WT with the SEM as error bar. In cases without error bars, the experiment was performed only twice. DA protein is expressed at much higher levels than WT, and the number indicated (1.6% of WT) overstates DA activity. (Right) Autoradiograms of representative CAT assays are shown (c, chloramphenicol; ac, acetylated chloramphenicol) together with the corresponding Western blot showing activator levels.

Fig. 3.

More detailed mutagenesis of the EAD. Transactivation assays, data presentation, and mutant depiction are as described for Fig. 2. Tyr to Ile (DI, red boxes) and Tyr to Phe (DF and TF, blue boxes) are indicated. Protein 57Z contains EAD1-57 (including six DHRs) fused to ATF1 and the zta bZIP domain (12). 57ZA (A), 57ZI (I), 57ZF (F), 57ZW (W), and 57ZH (H) have all six DHR tyrosines changed to Ala, Ile, Phe, Trp, and His, respectively. Transactivation assays for 57Z derivatives were performed by using a zta-CAT reporter (12). MSP protein has all spacer residues (white) between DHRs converted to two residues (see Results for details). For SCR, the relative positions of all 17 DHRs present in EAD1-176 are exchanged in a random manner. For REV, the peptide sequence between adjacent tyrosines present in EAD1-176 is inverted. ∗, REV protein level is lower than WT and normalization (under linear assay conditions) shows that REV exhibits >84% of WT activity.

Fig. 4.

Predictions of the presence of potential MoRFs in the EAD by using PONDR VL3. Higher PONDR scores reflect propensity for disorder (low probability for forming MoRFs), and lower scores reflect the propensity for order (higher probability for forming MoRFs). For (Y/A), (Y/I), (Y/H), and (Y/F), all EAD tyrosines are changed to Ala, Ile, His, or Phe, respectively. Color code is as follows: black, WT EAD; green, Y/A; red, Y/I; yellow, Y/H; blue, Y/F. PONDR plots derived for EAD mutants REV (dark green), SCR (purple), STA (dashed gray), and QA (dashed light blue) also are shown. Except for STA, REV, and SCR, the PONDR curve color code approximates that of the mutant proteins (Figs. 2 and 3).

Fig. 5.

Effect of EAD mutations on transformation by EWS/Fli1. Transformation of NIH 3T3 cells was tested by using retroviral introduction of EWS/Fli1 (ESF) and selected EAD mutants, followed by soft agar colony assay. (Upper) Representative colony formation under low-serum conditions is shown. (Lower) Expression analysis was performed by Western blot with anti-Fli1 antibody. All EWS/Fli1 mutants contain EAD1-264, harboring the same mutations described for EWS/ATF1. Thus, DAF corresponds to the EWS/ATF1 mutant DA (Fig. 2) and likewise for TFF, DFF, DIF, STAF, QAF, MSPF, and SCRF.