Drosophila melanogaster Casein Kinase II Interacts with and Phosphorylates the Basic Helix-Loop-Helix Proteins m5, m7, and m8 Derived from the Enhancer of split Complex

• Published in: The Journal of biological chemistry Vol. 276; no. 3; pp. 2159 - 2167
• Main Authors: Trott, Regina L., Kalive, Madhavi, Paroush, Zeev, Bidwai, Ashok P.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 19-01-2001; American Society for Biochemistry and Molecular Biology
• Subjects: casein kinase II; Drosophila melanogaster; enhancer of split complex (E(spl)C) gene
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M005996200

Drosophila melanogastercasein kinase II (DmCKII) is composed of catalytic (α) and regulatory (β) subunits associated as an α2β2heterotetramer. Using the two-hybrid system, we have screened aD. melanogaster embryo cDNA library for proteins that interact with DmCKIIα. One of the cDNAs isolated in this screen encodes m7, a basic helix-loop-helix (bHLH)-type transcription factor encoded by the Enhancer of split complex (E(spl)C), which regulates neurogenesis. m7 interacts with DmCKIIα but not with DmCKIIβ, suggesting that this interaction is specific for the catalytic subunit of DmCKII. In addition to m7, we demonstrate that DmCKIIα also interacts with two otherE(spl)C-derived bHLH proteins, m5 and m8, but not with other members, such as m3 and mC. Consistent with the specificity observed for the interaction of DmCKIIα with these bHLH proteins, sequence alignment suggests that only m5, m7, and m8 contain a consensus site for phosphorylation by CKII within a subdomain unique to these three proteins. Accordingly, these three proteins are phosphorylated by DmCKIIα, as well as by the α2β2 holoenzyme purified fromDrosophila embryos. In line with the prediction of a single consensus site for CKII, replacement of Ser159 of m8 with either Ala or Asp abolishes phosphorylation, identifying this residue as the site of phosphorylation. We also demonstrate that m8 forms a direct physical complex with purified DmCKII, corroborating the observed two-hybrid interaction between these proteins. Finally, substitution of Ser159 of m8 with Ala attenuates interaction with DmCKIIα, whereas substitution with Asp abolishes the interaction. These studies constitute the first demonstration that DmCKII interacts with and phosphorylates m5, m7, and m8 and suggest a biochemical and/or structural basis for the functional equivalency of these bHLH proteins that is observed in the context of neurogenesis.