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

Notch信号活动控制着高等后生动物的细胞分化，并参与了白血病的发生。我们使用果蝇作为模型系统，这要归功于它的基因可及性。在这里，Notch信号由转录因子SU(H)转导。质谱仪在体内发现了SU(H)丝氨酸269的磷酸化，这可能是其他信号通路的交叉调节点。合成了缺磷[SU(H)S269A]和拟磷[SU(H)S269D]突变体。SU(H)S269D影响DNA结合，并在体内过表达时表现出转录活性降低。相比之下，SU(H)S269A略显过度。显然，S269的磷酸化抑制了SU(H)的活性。为了进一步分析，我们通过基因组工程将这些突变引入了天然的SU(H)基因座。由于DNA结合缺陷，SU(H)S269D类似于零突变体，而SU(H)S269A类似于野生型。然而，由于晶体细胞(三种血细胞中的一种)在幼虫体内积累，造血受到了干扰。Notch调节胚胎和想象中的造血。在这种情况下，SU(H)的磷酸化可能会抑制Notch活性，从而影响血细胞类型的比例。哺乳动物的造血也在Notch的控制下。S269磷酸位点的保护有利于哺乳动物中类似的调控机制，这是我们想要解决的问题。使用细胞标记物，我们可以区分胚胎中是否存在过多的晶体细胞，还是仅在幼虫中通过转分化(由于外部信号？)而出现。该项目的核心是确定和分析负责的激酶/S。潜在的候选者将在各自的激酶突变体或RNA干扰系中进行晶体细胞数量的分析。那些数量过多的将与SU(H)突变体重新组合：由于SU(H)预计将向下游作用，SU(H)突变体的晶体细胞数量在双重突变体中应该不会改变。接下来，我们要问的是，在体外，这些被选择的激酶是否能够磷酸化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)，我们希望建立PHOS-Tag方法并产生磷酸化特异性抗体。前者可以在组织内分解磷酸苏(H)，后者理想情况下在细胞内。为了解决哺乳动物中这种调节机制的保守问题，我们现在已经用小鼠RBPJ代替SU(H)产生了苍蝇，接下来我们想要分析各自磷酸特异性突变体(RBPJS221A，RBPJS221D)中的造血作用。在与F.Oswald(Uni Ulm)的合作中，这些突变体也可能在T细胞中进行功能测试。此外，应通过过度表达和使用抑制剂来分析已识别的激酶的同系物。最后，可以对白血病数据库进行筛选，以寻找相关激酶的现有突变。

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.