Context-specific regulation of Notch signalling activity by phosphorylation of Suppressor of Hairless in Drosophila

果蝇无毛抑制子磷酸化对 Notch 信号活性的上下文特异性调节

• 批准号: 400152242
• 负责人: Dr. Anja Christina Nagel
• 金额: --
• 依托单位: Fachgebiet Allgemeine Genetik (190g)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/400152242?language=en
• 关键词: Context; specific; regulation; Notch; signalling

Notch signalling activity governs cellular differentiation in higher metazoa, and has been involved e.g. in leukaemia genesis. We use Drosophila as a model system thanks to its genetic accessibility. Here, Notch signals are transduced by the transcription factor Su(H). Mass spectrometry identified in vivo phosphorylation on Serine 269 in Su(H), potentially serving as a point of cross-regulation by other signalling pathways. Phospho-deficient [Su(H)S269A] and phospho-mimetic [Su(H)S269D] variants were made. Su(H)S269D affected DNA binding and showed reduced transcriptional activity upon overexpression in vivo. In contrast Su(H)S269A is slightly overactive. Apparently, S269 phosphorylation impedes Su(H) activity. For further analysis, we introduced these mutations into the native Su(H) locus by genome engineering. Su(H)S269D resemble null mutants due to the DNA binding defect, whereas Su(H)S269A animals resemble wild type. Hematopoiesis is disturbed, however, since crystal cells (one of three blood cell types) accumulate in larvae. Notch regulates embryonic and imaginal hematopoiesis. Su(H) phosphorylation in this context may inhibit Notch activity and hence influence the proportion of blood cell types. Mammalian hematopoiesis is also under the control of Notch. Conservation of the S269 phospho-site favours a similar regulatory mechanism in mammals, which we want to address. Using cell markers we may distinguish, whether excess crystal cells are present in embryos or appear only in larvae by trans-differentiation (due to an external signal?). Central to the project is the identification and analysis of the responsible kinase/s. Potential candidates will be assayed for crystal cell number in respective kinase mutants or RNAi-lines. Those with excess numbers will be re/combined with the Su(H) mutants: as Su(H) is expected to act downstream, Su(H) mutant crystal cell numbers should not change in the double mutant. Next we ask, whether the selected kinases are able to phosphorylate Su(H) in vitro, and whether pseudo-activated or dominant-negative versions will change Notch activity in S2 cells or in vivo. To follow pS269-Su(H) directly, we want to establish the Phos-Tag method as well as generate phospho-specific antibodies. The former may resolve phospho-Su(H) within tissue, the latter ideally within cells. To address the conservation of this regulatory mechanism in mammals, we have now generated flies with murine RBPJ in place of Su(H), and next want to analyse hematopoiesis in respective phospho-specific mutants (RBPJS221A, RBPJS221D). In collaboration with F. Oswald (Uni Ulm) these mutants may be also functionally tested in T-cells. Moreover, homologues of the identified kinases shall be analysed by overexpression and with inhibitors. Finally, leukemia data bases may be screened for existing mutations in related kinases.

Notch信号传导活性控制高等后生动物中的细胞分化，并且已经参与例如白血病发生。我们使用果蝇作为模型系统，这要归功于它的遗传可及性。在此，Notch信号由转录因子Su（H）转导。质谱法鉴定了Su（H）中丝氨酸269的体内磷酸化，可能作为其他信号传导途径的交叉调节点。制备磷酸缺陷型[Su（H）S269A]和磷酸模拟型[Su（H）S269D]变体。Su（H）S269D影响DNA结合，并在体内过表达时显示出降低的转录活性。相比之下，Su（H）S269A稍微过度活跃。显然，S269磷酸化阻碍了Su（H）活性。为了进一步分析，我们通过基因组工程将这些突变引入天然Su（H）基因座。由于DNA结合缺陷，Su（H）S269D类似于无效突变体，而Su（H）S269A动物类似于野生型。然而，由于晶体细胞（三种血细胞类型之一）在幼虫中积累，造血受到干扰。Notch调节胚胎和成虫的造血。在这种情况下，Su（H）磷酸化可以抑制Notch活性，从而影响血细胞类型的比例。哺乳动物的造血也受Notch的控制。S269磷酸化位点的保守有利于哺乳动物中类似的调节机制，这是我们想要解决的问题。使用细胞标记，我们可以区分，是否过量的晶体细胞存在于胚胎中或仅出现在幼虫通过转分化（由于外部信号？）。该项目的核心是鉴定和分析负责的激酶。将测定潜在候选物在相应激酶突变体或RNAi系中的晶体细胞数。具有过量数量的那些将与Su（H）突变体重新组合：由于预期Su（H）作用于下游，所以Su（H）突变体晶体细胞数量在双突变体中不应改变。接下来，我们要问的是，所选激酶是否能够在体外磷酸化Su（H），以及假激活或显性阴性版本是否会改变S2细胞或体内的Notch活性。为了直接跟踪pS269-Su（H），我们想要建立Phos-Tag方法以及产生磷酸化特异性抗体。前者可以在组织内分解磷酸-Su（H），后者理想地在细胞内。为了解决哺乳动物中这种调节机制的保守性，我们现在已经用鼠RBPJ代替Su（H）产生了果蝇，接下来想要分析相应磷酸特异性突变体（RBPJS 221 A，RBPJS 221 D）中的造血作用。与F合作。Oswald（Uni乌尔姆）也可以在T细胞中对这些突变体进行功能测试。此外，应通过过表达和抑制剂分析已鉴定激酶的同源物。最后，可以对白血病数据库中相关激酶的现有突变进行筛选。