Constitutively active protein kinase D acts as negative regulator of the Slingshot-phosphatase in Drosophila.

The mammalian protein kinase D family is involved in manifold cellular processes including cell migration and motility. Recently it was shown that human PKD1 and PKD2 phosphorylate and thereby inhibit Slingshot 1 Like (SSH1L), a phosphatase which is central to the regulation of actin cytoskeletal dynamics. We noted before that the overexpression of a constitutively active form of Drosophila PKD (PKD-SE) affects the fly retina and the resultant phenotypes suggest underlying defects in the actin cytoskeleton. Drosophila Slingshot, however, does not possess the phosphorylation site known to be targeted in SSH1L by human PKD1. Here we show that Drosophila PKD, despite this lack of conservation, nevertheless negatively regulates Slingshot. Overexpression of the active PKD-SE protein causes cellular defects that are similar to those of slingshot mutants. These include aberrant bristle morphology and positioning of photoreceptor nuclei. Interestingly, the observed nuclear mispositioning is due to a disturbance of the cytoskeleton rather than the epithelial organization. In accordance, overexpression of PKD-SE results in an accumulation of filamentous actin. This enrichment is modified by changes in slingshot gene doses, in line with an antagonistic relationship between PKD and slingshot. We conclude that similar to mammals, Drosophila PKD is a negative regulator of Ssh, with the premise of a different target phosphorylation site in Ssh.

Phosphorylation of Suppressor of Hairless impedes its DNA-binding activity.

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.

Local overexpression of Su(H)-MAPK variants affects Notch target gene expression and adult phenotypes in Drosophila.

In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. Here we address the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H) (MAPK-) (ko) and a phospho-mimetic Su(H) (MAPK-ac) isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ, vg (BE) -lacZ). In general, Su(H) (MAPK-) (ko) induced a stronger response than wild-type Su(H), whereas the response to Su(H) (MAPK-ac) was very weak. Notch target genes cut, wingless and vg (BE) -lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DER (act) ) or the MAPK (rl (SEM) ) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones.

MAPK-dependent phosphorylation modulates the activity of Suppressor of Hairless in Drosophila.

Cell differentiation strictly depends on the epidermal growth factor receptor (EGFR)- and Notch-signalling pathways, which are closely intertwined. Here we address the molecular cross talk at the level of Suppressor of Hairless [Su(H)]. The Drosophila transcription factor Su(H) mediates Notch signalling at the DNA level: in the presence of signalling input Su(H) assembles an activator complex on Notch target genes and a repressor complex in its absence. Su(H) contains a highly conserved mitogen activated protein kinase (MAPK) target sequence. Here we provide evidence that Su(H) is phosphorylated in response to MAPK activity. Mutation of the Su(H) MAPK-site modulated the Notch signalling output: whereas a phospho-deficient Su(H)(MAPK-ko) isoform provoked a stronger Notch signalling activity, a phospho-mimetic Su(H)(MAPK-ac) mutant resulted in its attenuation. In vivo assays in Drosophila cell culture as well as in flies support the idea that Su(H) phosphorylation affects the dynamics of repressor or activator complex formation or the transition from the one into the other complex. In summary, the phosphorylation of Su(H) attenuates Notch signalling in vivo in several developmental settings. Consequently, a decrease of EGFR signal causes an increase of Notch signalling intensity. Hence, the antagonistic relationship between EGFR- and Notch-signalling pathways may involve a direct modification of Su(H) by MAPK in several developmental contexts of fly development. The high sequence conservation of the MAPK target site in the mammalian Su(H) homologues supports the idea that EGFR signalling impacts on Notch activity in a similar way in humans as well.