Phosphorylation of Suppressor of Hairless impedes its DNA-binding activity

Abstract

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)S269A] and phospho-mimetic [Su(H)S269D] 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)S269D during fly development demonstrated reduced transcriptional regulatory activity, similar to the previously reported DNA-binding defective mutant Su(H)R266H. As both are able to bind Hairless and Notch proteins, Su(H)S269D and Su(H)R266H 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.

Phospho-Site Mutations in Transcription Factor Suppressor of Hairless Impact Notch Signaling Activity During Hematopoiesis in Drosophila

“…Su(H) S269D mutants were developmentally delayed and died before pupation, comparable to what was observed for the null allele Su(H) attP , and in agreement with a functional loss due to impaired DNA-binding (Figure 1B) (Nagel et al, 2017;Praxenthaler et al, 2017). By contrast, the phospho-deficient Su(H) S269A mutants were nearly indistinguishable from the Su(H) gwt wild type control with respect to developmental timing and hatching rate (Figure 1B).…”

“…Generation of Phospho-Site Specific Su(H) Mutants in the Endogenous Locus Previously, we showed that Su(H) is phosphorylated at Serine 269 in Drosophila S2 cells, and that the introduction of a negative charge at this position interfered with the DNAbinding capability and transcriptional activity of Su(H). In addition, tissue-specific overexpression studies suggested that both, activation and repression of Notch signaling activity might be regulated by the phosphorylation of Su(H) at this site (Nagel et al, 2017). Based on the dominant negative character of the overexpression phenotypes, we were unable to decide on the biological relevance of the phosphorylation in vivo.…”

“…Furthermore, Gro and Su(H) are two other well defined substrates, whose activity is mitigated by MAPK-dependent phosphorylation in various N-dependent processes (Hasson et al, 2005;Auer et al, 2015). Recently, we identified a further phosphorylation to occur on Serine 269 (S269) in Su(H) isolated from Drosophila Schneider S2 cells, may impact Notch signaling by perturbing DNAbinding of Su(H) (Nagel et al, 2017). As this might influence both, N-dependent activation and repression, a careful spatiotemporal investigation of N-dependent processes regarding this modification is needed.…”

Decoding the PTM-switchboard of Notch

“…The authors also hypothesize this phosphorylation to be plausible in humans. Similarly another site in the beta-trefoil domain (BTD), on S269 (corresponding to S195 in humans) was also discovered in Drosophila melanogaster and is shown to inhibit the binding of BTD to DNA [ 174 ].…”

Hes1 Notch Response Elements and their Roles in RBPJ-K Binding

“…Additionally, in Drosophila, Su(H) has been discovered to have a supplementary phosphorylation site on the Serine 269 residue. This residue is highly conserved and may influence interactions with the NICD or DNA (Nagel et al, 2017). Further examination in relation to RBPJ-k binding on the NRE sites within the SPS on Hes1 must be conducted;…”