Nelson Mary L, Kang Hyun-Seo, Lee Gregory M, Blaszczak Adam G, Lau Desmond K W, McIntosh Lawrence P, Graves Barbara J
Department of Oncological Sciences, University of Utah School of Medicine, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, 84112, USA.
Proc Natl Acad Sci U S A. 2010 Jun 1;107(22):10026-31. doi: 10.1073/pnas.0915137107. Epub 2010 May 13.
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.
Ras/MAPK信号通路在人类癌症中常常被异常激活。其下游效应器是转录因子，包括由ETS基因家族编码的那些转录因子。通过基于细胞的分析和生物物理测量，我们确定了Ras/MAPK信号通路通过对Thr38和Ser41的磷酸化来影响Ets1功能的机制。这些ERK2磷酸化位点位于Ets1的无结构N端区域内，紧邻PNT结构域。核磁共振光谱分析表明，PNT结构域是一个四螺旋束（H2 - H5），类似于SAM结构域，并附加有另外两个螺旋（H0 - H1）。磷酸化改变了构象平衡，使动态螺旋H0从核心束中移位。Ets1与共激活因子CBP的TAZ1（或CH1）结构域的亲和力通过磷酸化提高了34倍，并且这种结合对离子强度敏感。核磁共振监测的滴定实验绘制了TAZ1结构域和Ets1的相互作用表面，后者包括磷酸化位点和PNT结构域。这些表面的电荷互补性表明静电力与构象平衡协同作用以介导磷酸化效应。我们得出结论，Ets1的动态螺旋元件附加到一个保守的结构核心上，构成了一个磷酸化开关，将Ras/MAPK信号通路导向基因表达的下游变化。这一详细的结构和机制信息将指导针对人类疾病中ETS蛋白的靶向策略。
