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

• Applicant: Privatdozentin Dr. Anja Christina Nagel
• Subject Area: Developmental Biology ; General Genetics and Functional Genome Biology ; Cell Biology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 400152242

• Final Report Year: 2023

Final Report Abstract

Cellular differentiation and homeostasis in metazoan is under the control of the finely coordinated interplay of a few signaling cascades, including the Notch signaling pathway. The exact temporal and spatial regulation of Notch signaling activity is of utmost importance, as perturbations may cause severe diseases including cancer. Signal transduction relies on the transcription factor CSL/Su(H), regulating the expression of Notch target genes, and thus relaying Notch signaling activity within the cell. In Drosophila melanogaster Su(H) is phosphorylated at Serine 269 (S269), which impedes its DNA binding, and hence Notch activity. We aimed to study this novel regulatory mechanism of the Notch pathway in more detail, as it could entail a context specific, temporal inactivation of Notch activity in response to internal or external signals via protein kinases. We concentrated on hematopoiesis, since we originally identified the Su(H) phosphorylation in cells with blood cell character. There, the Notch signal promotes the formation of so-called crystal cells, one of three blood cell types in Drosophila. We generated specific Su(H) mutants, in which the wild type Su(H) gene was replaced via genome engineering with a phospho-deficient (Su(H)S269D) and a phosphomimetic (Su(H)S269A) variant, respectively. As expected, the Su(H)S269D mutant showed a total loss of Notch activity, since DNA-binding is a premise for Su(H) function. In contrast, the Su(H)S269A allele displayed too many crystal cells in all phases of blood cell development. No influence of the general Notch-antagonist Hairless on crystal cell formation was observed, confirming the specificity of the kinase-mediated repression of Notch activity. Apparently, S269 phosphorylation represents a general mechanism of regulating Notch activity in the context of the entire hematopoiesis. Next, we replaced endogenous Su(H) with the orthologous murine RBPJ-gene carrying the respective mutations: these displayed likewise blood cell defects in support of a conservation of phospho-mediated Notch-regulation. Blood cells in Drosophila primarily serve the immune response. Infestation with parasitoid wasps results in a powerful multiplication of lamellocytes, i.e. blood cells that can encapsulate the wasp’s egg. These are absent from healthy animals, however, are formed at the expense of crystal cells in response to wasp attack. To this end, the Notch signaling activity needs to be downregulated, which could be mediated by Su(H) phosphorylation, hence representing a stress response to the infection by inhibiting the Notch signal in favor of lamellocyte formation. In support of this working hypothesis, the immune response of the Su(H)S269A allele is indeed impaired. Moreover, Su(H) protein is phosphorylated at S269 in response to wasp infestation. In silico, in vitro and in vivo screens finally led to the identification of protein kinase Pkc53E, involved in the stress-induced phosphorylation of Su(H). Experiments in human cell culture as well as the high structural and functional conservation of CSL proteins and the PKC family support the idea that mammalian CSL is subjected to a negative regulation by context specific phosphorylation as well.

Publications

• An RBPJ-Drosophila Model Reveals Dependence of RBPJ Protein Stability on the Formation of Transcription–Regulator Complexes. Cells, 8(10), 1252.
  Gahr, Bernd M.; Brändle, Franziska; Zimmermann, Mirjam & Nagel, Anja C.
  
• Limited Availability of General Co-Repressors Uncovered in an Overexpression Context during Wing Venation in Drosophila melanogaster. Genes, 11(10), 1141.
  Nagel, Anja C.; Maier, Dieter; Scharpf, Janika; Ketelhut, Manuela & Preiss, Anette
  
• Nucleo-cytoplasmic shuttling of murine RBPJ by Hairless protein matches that of Su(H) protein in the model system Drosophila melanogaster. Hereditas, 158(1).
  Wolf, Dorina B.; Maier, Dieter & Nagel, Anja C.
  
• Phospho-Site Mutations in Transcription Factor Suppressor of Hairless Impact Notch Signaling Activity During Hematopoiesis in Drosophila. Frontiers in Cell and Developmental Biology, 9.
  Frankenreiter, Lisa; Gahr, Bernd M.; Schmid, Hannes; Zimmermann, Mirjam; Deichsel, Sebastian; Hoffmeister, Philipp; Turkiewicz, Aleksandra; Borggrefe, Tilman; Oswald, Franz & Nagel, Anja C.
  
• The Membrane-Bound Notch Regulator Mnr Supports Notch Cleavage and Signaling Activity in Drosophila melanogaster. Biomolecules, 11(11), 1672.
  Nagel, Anja C.; Müller, Dominik; Zimmermann, Mirjam & Preiss, Anette
  
• The Binding of CSL Proteins to Either Co-Activators or Co-Repressors Protects from Proteasomal Degradation Induced by MAPK-Dependent Phosphorylation. International Journal of Molecular Sciences, 23(20), 12336.
  Fechner, Johannes; Ketelhut, Manuela; Maier, Dieter; Preiss, Anette & Nagel, Anja C.
  
• Genetic and Molecular Interactions between HΔCT, a Novel Allele of the Notch Antagonist Hairless, and the Histone Chaperone Asf1 in Drosophila melanogaster. Genes, 14(1), 205.
  Maier, Dieter; Bauer, Milena; Boger, Mike; Sanchez, Jimenez Anna; Yuan, Zhenyu; Fechner, Johannes; Scharpf, Janika; Kovall, Rhett A.; Preiss, Anette & Nagel, Anja C.
  
• Inhibition of the Notch signal transducer CSL by Pkc53E-mediated phosphorylation to fend off parasitic immune challenge inDrosophila. Cold Spring Harbor Laboratory.
  Deichsel, Sebastian; Frankenreiter, Lisa; Fechner, Johannes; Gahr, Bernd M.; Zimmermann, Mirjam; Mastel, Helena; Preis, Irina; Preiss, Anette & Nagel, Anja C.