The aforementioned studies, in turn, raise the question on control of E-M8 by dephosphorylation. A candidate enzyme is the phosphatase PP2A, whose role emerged in assays for impaired signaling in wild type and mutant Notch backgrounds. Specifically, increased dosage of microtubule star, the unique catalytic LY294002 subunit of Drosophila PP2A, elicits twinned R8s/ SOPs, defects that closely mimic loss of Notch or CK2. The possibility thus arises that PP2A antagonizes Notch signaling. However, the participating PP2A regulatory subunit remained to be identified, and it was unknown if this phosphatase impacted E-M8 activity in vivo. The modulation of the IOB defects of M8 appears more straightforward given that the P-domain is unperturbed. In contrast, the modulation of the eye/R8 defects of the CK2 phospho-mimetic M8-S159D may seem paradoxical, as this Asp variant should not have been responsive to phosphatase activity if PP2A were to target the CK2 site. As shown in Fig. 7B, the P-domain is populated by a number of Ser residues that are highly conserved in M8, M7, M5, three of which are also conserved in the human, mouse and Anolis HES6. The importance of the CK2 site is now well understood for fly M8 and mouse HES6, but the roles of the other highly conserved Ser residues are beginning to be resolved. In the case of HES6, the PGSP motif is targeted by MAPK, but its developmental role remains unknown. Likewise, the additional Ser residues in M8 appear to be subject to phosphorylation. In support, our ongoing studies reveal that replacement of the MAPK site of M8-S159D with PLAP neutralizes repression of Ato and the R8 fate, raising the prospect that M8, like HES6, requires multi-site phosphorylation. In light of these findings, PP2A may target the MAPK site or modifications at the other Ser residues, thereby controlling repressor activity. The possibility arises that coordinated functions of the participatory kinases and PP2A control M8 phosphorylation levels and/or activity. Future biochemical studies are required to test this model for regulation, determine if Wdb permits PP2A to dephosphorylate M8, and identify which residue is a target of this phosphatase. Remarkably, the CK2 Site Is Conserved and Similarly Located in Chicken/Frog HES6-1, as Well as in HER13.1/2 from Fish. Even Though They Lack a Recognizable MAPK Site, the P-Domain in HES6-1/HER13.1/ 2 Harbors Several Conserved Ser/Thr Residues, Many of Which Meet the Consensus for CK2. Because CK2 Is an Acidophilic Kinase That Utilizes Pser/Pthr as Surrogates for Asp/Glu, the Possibility Remains That HES6-1/HER13.1/2 Are More Extensively Phosphorylated by This Enzyme. This Possibility May Explain a Report That Mutation of the Primary CK2 Site in Chicken HES6-1 to Ala Does Not Affect Repression of HES5. Direct Biochemical Studies Are Needed to Determine If CK2 Targets a Single/Multiple Sites in HES6-1 and HER13.1/2. The E/HES/HER proteins display length and sequence heterogeneity of the CtD. Computational analysis of fly E proteins reveals that the P-domain and its flanking sequences are intrinsically.