Institut für Genetik (240), University of Hohenheim, Garbenstr. 30, 70599, Stuttgart, Germany.
Core Facility Hohenheim, Mass Spectrometry Unit University of Hohenheim, 70599, Stuttgart, Germany.
Sci Rep. 2017 Sep 19;7(1):11820. doi: 10.1038/s41598-017-11952-0.
Notch signalling activity governs cellular differentiation in higher metazoa, where Notch signals are transduced by the transcription factor CSL, called Suppressor of Hairless [Su(H)] in Drosophila. Su(H) operates as molecular switch on Notch target genes: within activator complexes, including intracellular Notch, or within repressor complexes, including the antagonist Hairless. Mass spectrometry identified phosphorylation on Serine 269 in Su(H), potentially serving as a point of cross-regulation by other signalling pathways. To address the biological significance, we generated phospho-deficient [Su(H)] and phospho-mimetic [Su(H)] variants: the latter displayed reduced transcriptional activity despite unaltered protein interactions with co-activators and -repressors. Based on the Su(H) structure, Ser269 phosphorylation may interfere with DNA-binding, which we confirmed by electro-mobility shift assay and isothermal titration calorimetry. Overexpression of Su(H) during fly development demonstrated reduced transcriptional regulatory activity, similar to the previously reported DNA-binding defective mutant Su(H). As both are able to bind Hairless and Notch proteins, Su(H) and Su(H) provoked dominant negative effects upon overexpression. Our data imply that Ser269 phosphorylation impacts Notch signalling activity by inhibiting DNA-binding of Su(H), potentially affecting both activation and repression. Ser269 is highly conserved in vertebrate CSL homologues, opening the possibility of a general and novel mechanism of modulating Notch signalling activity.
Notch 信号通路活动在高等后生动物的细胞分化中起调控作用，在果蝇中，Notch 信号由转录因子 CSL（称为 Suppressor of Hairless [Su(H)]）转导。Su(H)作为 Notch 靶基因的分子开关发挥作用：在激活复合物中，包括细胞内 Notch；或在抑制复合物中，包括拮抗剂 Hairless。质谱分析鉴定了 Su(H)上丝氨酸 269 的磷酸化，可能作为其他信号通路交叉调节的一个点。为了阐明其生物学意义，我们生成了磷酸化缺陷型 [Su(H)]和磷酸化模拟型 [Su(H)]变体：尽管与共激活因子和共抑制因子的蛋白相互作用没有改变，但后者的转录活性降低。基于 Su(H)结构，推测 Ser269 磷酸化可能会干扰 DNA 结合，我们通过电泳迁移率变动分析和等温滴定量热法证实了这一点。在果蝇发育过程中过表达 Su(H)显示出降低的转录调控活性，与先前报道的 DNA 结合缺陷突变体 Su(H)相似。由于两者都能够结合 Hairless 和 Notch 蛋白，因此 Su(H)和 Su(H)过表达时会引发显性负效应。我们的数据表明，Ser269 磷酸化通过抑制 Su(H)的 DNA 结合来影响 Notch 信号通路活性，可能会影响激活和抑制。Ser269 在脊椎动物 CSL 同源物中高度保守，为调节 Notch 信号通路活性提供了一种普遍而新颖的机制的可能性。
