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

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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.