E(spl)-Mγ harbors multiple consensus sites for CK2.
We first aligned E(spl)-Mγ homologues from 12 species that represent ~ 50x106 years (MYR) of Drosophila evolution . All E(spl)-Mγ homologues harbor three conserved consensus sites for CK2 (Fig. 1A), fully meeting the strict recognition requirements for this kinase . Unique among these are Thr45 and Ser48, which reside within the loop of the HLH domain (Fig. 1A). Modification of Ser48 is predicted to elicit hierarchical phosphorylation of Thr45, because Asp/Glu or pSer/pThr promote recognition by CK2 with equal potency . Interestingly, of the seven E(spl) bHLH-O proteins in Drosophila, E(spl)-Mγ is the sole member harboring potential site(s) for CK2 within the HLH domain. The third CK2 site (Ser195) is juxtaposed to the WRPW motif (Fig. 1A), a feature also unique to E(spl)-Mγ. Notably, numerous Asp/Glu, a hallmark of 'high likelihood' targets of CK2, flank Ser195. Although E(spl)-Mγ homologues from several Drosophila species harbor the insertion of one/two amino acids immediately N-terminal to Ser195, in no case is the DS195EDEE motif perturbed (Fig. 1A, numbering reflects the D. melanogaster sequence).
We next determined if the insertion/deletion of two amino acids correlates to specific branches of the Drosophila family. Given the known evolutionary relationships between 12 species representing six major groups of fruit flies , it appears likely that E(spl)-Mγ from Hawaiian, Virilis, Repleta and Obscura groups (harboring an insertion of two residues) are the ancestral form (Fig. 1B). A one residue deletion occurred ~ 37x106 MYR ago with the emergence of the Willistoni group, and both residues were lost ~ 13x106 MYR ago with the emergence of the Melanogaster group. The strong conservation of the C-terminal CK2 site despite the insertion/deletion, suggests that the one/two residue insertion (in the ancestral species) is unlikely to alter protein structure and/or function.
Phosphorylation of E(spl)-Mγ by CK2.
We next determined if E(spl)-Mγ is phosphorylated by CK2. This kinase exits as an α2β2 holoenzyme in all eukaryotes , and phosphorylates Ser/Thr/Tyr in a messenger-independent manner. Although phosphorylation is catalyzed by CK2α, the regulatory CK2β subunit promotes or inhibits recognition in a target-specific manner [19, 25]. We thus used native α2β2 holoenzyme (CK2-HoloE) purified from Drosophila embryos and monomeric (recombinant) CK2α purified from yeast rescued by a cDNA encoding Drosophila CK2α . Both enzyme forms are pure based on Coomassie staining (Fig. 2, Gel). In the case of CK2-HoloE, the weak staining of CK2β relative to CK2α (Fig. 2) does not reflect sub-stoichiometric levels, as this enzyme preparation has a sedimentation coefficient of 6.4S (data not shown), consistent with the α2β2 conformation, and reflects weak staining of CK2β from flies and mammals [26, 27]. Both forms display kinetic properties and ATP/GTP-dependency identical to those reported by others and us [20, 28].
The two isoforms of CK2 were used to phosphorylate E(spl)-Mγ. In these assays, 20 ng of CK2α or CK2-HoloE were used and thus proteins with the mobility of CK2-α/β are not seen in SDS-PAGE gels stained with Coomassie (Fig. 2). E(spl)-Mγ was tested as a GST fusion, while GST-alone and GST-E(spl)-M8 served as negative and positive controls, respectively. As shown in Fig. 2, E(spl)-Mγ was robustly phosphorylated by both CK2α and CK2-HoloE. Phosphorylation is specific to E(spl)-Mγ, as GST was not phosphorylated. The band intensities suggest that CK2-HoloE more efficiently phosphorylates E(spl)-Mγ than does CK2α. This difference likely reflects (~ 4-fold) greater activity of the holoenzyme , rather than a requirement for CK2β. Phosphorylation of E(spl)-M8 (positive control) was observed with both isoforms, although CK2β appeared to modestly diminish phosphorylation. Thus, E(spl)-Mγ is targeted by native Drosophila CK2 independent of CK2β.
CK2-specific targeting of E(spl)-Mγ revealed through effectors.
It remained formally possible that a minor enzyme contaminant (other than CK2) was responsible for phosphorylation of Mγ. To exclude this, Mγ phosphorylation by CK2-HoloE was tested in the presence of competitor (GTP), inhibitor (Heparin), or polybasic activators, as previously reported . We exploited the unusual property that CK2 can use ATP/GTP as phosphate donors . Assays contained 15 µM [γ32P]-ATP (1xKm) and supplemented with 60 µM cold-GTP. In this assay, competition by GTP should diminish phosphorylation, if the enzyme is CK2. Indeed, GTP strongly decreased 32P-incorporation into GST-Mγ (Fig. 3A). Likewise, Heparin (1 µg/mL), a CK2 inhibitor , strongly diminished phosphorylation of GST-E(spl)-Mγ. We next tested polybasic compounds that stimulate CK2-HoloE through interaction with CK2β . All three (protamine, spermine and poly-lysine) modestly increased phosphorylation of GST-Mγ. As GST-alone is not phosphorylated by CK2-HoloE in the absence of these effectors (Fig. 3), or in their presence (data not shown and see ), the phosphorylation observed in Figs. 2 and 3 reveal a specific interaction of CK2 with Mγ. These studies confirm E(spl)-Mγ as a bona fide substrate of CK2.
Identify the CK2 site(s) in E(spl)-Mγ.
We next sought to identify CK2 target sites. Because Mγ harbors three predicted CK2 sites, it was divided into two parts; Mγ-NTD contains the bHLH-O domains, whereas Mγ-CtD contains residues of the CtD including WRPW (Fig. 4A). These constructs separate the CK2 sites within the HLH domain (T45LES48EGE) from that (S195EDE) proximal to WRPW.
Wild type and Ala mutants of Mγ were tested for phosphorylation by CK2-HoloE. Analysis with monomeric CK2α was deemed unnecessary given CK2β-independent targeting of Mγ (Fig. 2). In brief, 20 ng of CK2-HoloE were used to phosphorylate 0.5-1.0 µg of Mγ variants under conditions (10 min) where enzyme activity is linear. As shown in Fig. 4B, Mγ-CtD was efficiently phosphorylated by CK2, and this was abrogated by replacing Ser195 with Ala (Fig. 4B). Under similar conditions CK2 did not phosphorylate GST-alone. We next tested Mγ-NTD and its Ala mutants. While Mγ-NTD-S48A was very weakly phosphorylated, neither Mγ-NTD-T45A nor Mγ-NTD-T45 + S48A were phosphorylated (Fig. 4A). We thus tested full-length Mγ with the S195A mutant, and find that, unlike wild-type E(spl)-Mγ (Fig. 2), the single-site mutant FL-S195A was not phosphorylated (Fig. 4B). Therefore, the faint band with Mγ-NTD-S48A (boxed area in Fig. 4B) more likely represent an in vitro artifact. These results demonstrate that CK2 modifies Mγ only at Ser195 in its CtD (Fig. 4A). Localized folding may render Ser48/Thr45 (in the HLH domain) refractory/inaccessible to CK2.
The importance of PTM of E(spl)/HES proteins.
The phosphorylation of E(spl)-Mγ reveals that a preponderance (four out of seven) of E(spl)C repressors are targeted by CK2. The physiological consequences of phosphorylation are best understood for E(spl)-M8, whose regulation by phosphorylation has been studied during eye and bristle development [13, 15]. In either context, the CK2 and MAPK sites auto-inhibit the O-domain via protein-protein interactions. Phosphorylation overrides auto-inhibition to uncover the O-domain, thereby permitting E(spl)-M8 repressor activity. Accordingly, removal of the CtD or just the P-domain elicits constitutive (PTM-independent) repression by E(spl)-M8 . More recent studies (, and Majot and Bidwai, unpublished) are revealing that M8 is likely to be regulated through PTM-dependent degradation. The P-domain of M8 harbors two additional Ser residues (Ser154 and Ser155), which meet the consensus for CK1, a kinase that phosphorylates Ser with pSer/Asp/Glu at the n+ 4-to-n+ 9 positions [30, 31]. CK2 targeting of M8 (at Ser159) may thereby favor CK1-dependent modification of Ser154 and Ser155. This, in turn, generates the pSpSGYHpSDCD 'phospho-degron' (Fig. 4C), which meets the strict consensus for degradation via Slimb/βTrCP . The M8-Slimb/βTrCP interaction has been reported , but its phospho-dependency remains unresolved. A similar sequence is conserved in M5 and M7 (Fig. 4C). The spacing of the kinase motifs in the P-domain is likely to be germane to understanding the function and regulation of these bHLH repressors. HES6, the mammalian homologue of E(spl)-M8 also conserves similarly positioned CK2, MAPK, and CK1 sites in its P-domain (not shown). We interpret our findings on Mγ in light of those with M8.
While the E(spl)/HES proteins are conserved across the bHLH-O region, sequence divergence is largely restricted to the CtD, which is of variable length and composition. Might CtD divergence underlie functional diversity amongst this family of repressors? Several lines of evidence support this possibility. 1) The CtDs of E(spl)-M8/M5/M7/Mγ are notable in that they are rich in Ser/Thr residues; of these the CK2 site is present in all four members, whereas that for MAPK has been lost from only M7 (Fig. 4C). This pattern of PTM-site maintenance/loss is invariant in homologues across 12 Drosophila species (grey box in Fig. 4C), a strong evolutionary argument that this correlation is not merely incidental. 2) It is increasingly being realized that PTM often targets ‘intrinsically disordered’ (ID) regions, which are rich in Ser/Thr/Asp/Glu residues [33, 34]. Accordingly, the CtDs of M8/M5/M7/Mγ (and HES6) are predicted to be disordered , but not for those lacking PTM sites such as E(spl)-Mδ . While the CK2 and/or MAPK sites of M8/M5/M7 are contained within the ID-region, Mγ is unique in that the CK2 and MAPK sites flank the ID-region . The differential placement of PTM sites relative to regions of ID raises the prospect that phosphorylation effects on Mγ are unlikely to mimic those with M5, M7, M8 and HES6. Indeed, in vivo studies in the eye/bristle reveal that Asp mutations at the CK2 and MAPK sites on M8 potently enhance repressor activity, whereas they have no discernible effects on M5 (, Kim and Bidwai, unpublished). 3) Unlike the P-domain of M8, which directly mediates auto-inhibition, the CK2 and MAPK sites of Mγ are separated by 31 residues, making it likely that CtD-Orange interactions would be different from those in M8. Evidence in favor of this will require knowledge of residues of the O-domain that directly contact the CtD and participate in auto-inhibition. 4) As mentioned above, M8/M5/M7 conserve a CK2 + CK1-dependent phospho-degron, pSpSGYHpS (Fig. 4C). It is difficult to reconcile a similar mechanism for Mγ-turnover, because its SSYAGS sequence does not meet the consensus for Slimb-binding (Fig. 4C), and the absence of Asp/Glu residues C-terminal to this sequence and displacement of the CK2 site preclude the formation of a 'phospho-degron' through hierarchical phosphorylation. Degradation may therefore be of lesser importance for Mγ. We do, however, note numerous conserved Ser/Thr in the linker separating the CK1 and CK2 sites (see Fig. 4C), raising the prospect that distinct kinases may impact Mγ. 5) The juxtaposition of the CK2 site to WRPW is unique to Mγ (Fig. 4C), and it is unknown if CK2 influences Groucho binding. If this were the case, CK2 would modulate formation of an Mγ-Groucho complex, thereby imposing regulation distinct from that for M8, whose interaction with Groucho is phosphorylation-independent . 6) The ability of E(spl) proteins to repress other bHLH factors such as Atonal hinges upon many factors, one of which is the formation of homo/heterodimers . The possibility that phosphorylation of Mγ may bias partner preference for dimerization remains unexplored. Systematic biochemical and genetic analyses are required to identify which E(spl) proteins hetero-dimerize with Mγ, and if phosphorylation influences their binding with bHLH factors other than Atonal.
In summary, our studies reveal that a preponderance of E(spl) proteins are targets of CK2 and conserve a site for MAPK, implicating these kinases in regulating Notch signaling pathway output. These sites of phosphorylation co-localize with regions predicted to be intrinsically disordered, but are positioned differently in each isoform, raising the prospect that these bHLH repressors do not serve redundant (anti-neural) functions. Although the roles of multi-site phosphorylation of M7, M5, Mγ and HES6 remain unresolved, our studies on Mγ suggest that PTM is more central to these proteins than has been recognized. Future efforts are needed to clarify the roles of phosphorylation in dimerization of E(spl) proteins, their ability to bind and repress proneural bHLH factors, and determine if these modifications underlie tissue-specific roles of Notch signaling during development of the nervous system and elsewhere.