Drosophila melanogaster casein kinase II interacts with and phosphorylatesthe basic helix-loop-helix proteins m5, m7, and m8 derived from the Enhancer of split complex

Drosophila melanogaster casein kinase II (DmCKII) is composed of catalytic (alpha) and regulatory (beta) subunits associated as an alpha (2)beta (2) h eterotetramer. Using the two-hybrid system, we have screened a D. melanogas ter embryo cDNA library for proteins that interact with DmCKII alpha. One o f the cDNAs isolated in this screen encodes m7, a basic helix-loop-helix (b HLH)-type transcription factor encoded by the Enhancer of split complex (E( spl)C), which regulates neurogenesis. m7 interacts with DmCKII alpha but no t with DmCKII beta, suggesting that this interaction is specific for the ca talytic subunit of DmCKII. In addition to m7, we demonstrate that DmCKII al pha also interacts with two other E(spl)C-derived bHLH proteins, m5 and m8, but not with other members, such as m3 and mC. Consistent with the specifi city observed for the interaction of DmCKII alpha with these bHLH proteins, sequence alignment suggests that only m5, m7, and m8 contain a consensus s ite for phosphorylation by CKII within a subdomain unique to these three pr oteins. Accordingly, these three proteins are phosphorylated by DmCKII alph a, as well as by the alpha (2)beta (2), holoenzyme purified from Drosophila embryos. In line with the prediction of a single consensus site for CKII, replacement of Ser(159) of m8 with either Ala or Asp abolishes phosphorylat ion, identifying this residue as the site of phosphorylation. We also demon strate that m8 forms a direct physical complex with purified DmCKII, corrob orating the observed two-hybrid interaction between these proteins. Finally , substitution of Ser(159) of m8 with Ala attenuates interaction with DmCKI I alpha, whereas substitution with Asp abolishes the interaction. These stu dies constitute the first demonstration that DmCKII interacts with and phos phorylates 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.