Analysis of timekeeper implicates antagonism between CK2 and PP2A during Drosophila neurogenesis

It is increasingly becoming apparent that dynamic control of phosphorylation and dephosphorylation underlies the proper spatial and temporal activities of many signaling circuits including Notch (N). Emerging evidence suggests that protein kinase CK2 mediates the effects of N during Drosophila eye and bristle development via phosphorylation of E(spl) repressors. Previous studies in our laboratory shown that reducing levels/activity of CK2 elicits rough eyes due to excess R8 founders and ectopic and split bristles due to excess SOP\u27s, both indicative of impaired lateral inhibition and N signaling. While these studies implicate a role for CK2 in the regulation of E(spl), the identity and/or involvement of a cognate phosphatase remain unknown. A dominant mutation of CK2alpha, Timekeeper (Tik), harbors two amino acid substitutions, M161K and E165D. The former substitution is thought to predominantly contribute to the dominant behavior of Tik, since it lies in the ATP binding pocket. However, the E165D mutation might also contribute to the behavior of Tik, given that it resides in a region, which harbors the HEN/HRKL motif, mediates recruitment of the phosphatase PP2A. Therefore, the possibility arises that the analysis of Tik might uncover the relationship between CK2 and PP2A. To test this hypothesis, we first generated two variants of CK2 that harbor either of the Tik substitutions, which were then used in vivo in Drosophila. We find that misexpression of either the MK or the ED variant elicits ectopic bristles whose frequency and penetrance closely approximate that seen with Tik. We have also tested for the ability of these variants to suppress the neural defects of Nspl flies and found that MK and ED suppress the retinal defects of N spl. Then, we have tested the role of PP2A during eye and bristle development in wild type and/or Nspl flies. We find that increasing PP2A dosage (UAS-mts) elicits ectopic and missing bristles in N+ flies, both indicative of a loss of N signaling. In addition, we find that increased dosage of PP2A suppresses the rough and reduced eye phenotypes of Nspl flies, a result that is also associated with decreased CK2 levels/activity. Together, these results suggest that PP2A activity might attenuate, whereas CK2 augments, inhibitory N signaling. Given the previously described genetic and direct biochemical interactions between CK2 and E(spl)M8/M5/M7, PP2A might serve in an antagonistic manner to CK2