Protein kinase CK2 phosphorylates a conserved motif in the Notch effector E(spl)-Mγ

Area of Science:

• Molecular Biology
• Developmental Biology
• Genetics

Background:

• Notch signaling pathway effects are mediated by Enhancer of Split (E(spl)/HES) basic Helix-Loop-Helix-Orange (bHLH-O) repressors across metazoans.
• While E(spl)/HES repressors are conserved, sequence diversity is mainly in the C-terminal domain (CtD), impacting co-repressor Groucho binding.
• Kinases CK2 and MAPK are known to target CtD in specific E(spl)/HES repressors, but regulation across the family is unclear.

Purpose of the Study:

• To investigate the broader impact of protein phosphorylation on the E(spl)/HES bHLH-O repressor family.
• To identify new targets of CK2 and MAPK kinases within this repressor family.

Main Methods:

• Bioinformatics analysis to identify conserved phosphorylation sites.
• Evolutionary analysis to track conserved motifs over time.
• Biochemical assays using purified native CK2 from Drosophila embryos to test phosphorylation.

Main Results:

• E(spl)-Mγ identified as a novel target of Drosophila CK2.
• Phosphorylation of E(spl)-Mγ by CK2 is specific and independent of the CK2-β subunit.
• The CK2 phosphorylation site is adjacent to the WRPW motif, a feature conserved in Mγ homologues over 50 million years.

Conclusions:

• A significant portion of Drosophila E(spl) homologues (four out of seven) are targets for CK2 phosphorylation.
• The distinct positioning of CK2 and MAPK phosphorylation sites suggests a role in functional diversification of bHLH-O proteins.
• This study expands the understanding of post-translational regulation in the Notch signaling pathway.