DNA binding by cut homeodomain proteins is down-modulated by casein kinase II.

The Drosophila and mammalian Cut homeodomain proteins contain, in addition to the homeodomain, three other DNA binding regions called Cut repeats. Cut-related proteins thus belong to a distinct class of homeodomain proteins with multiple DNA binding domains. Using nuclear extracts from mammalian cells, Cut-specific DNA binding was increased following phosphatase treatment, suggesting that endogenous Cut proteins are phosphorylated in vivo. Sequence analysis of Cut repeats revealed the presence of sequences that match the consensus phosphorylation site for casein kinase II (CKII). Therefore, we investigated whether CKII can modulate the activity of mammalian Cut proteins. In vitro, a purified preparation of CKII efficiently phosphorylated Cut repeats causing an inhibition of DNA binding. In vivo, overexpression of the CKII alpha and beta caused a decrease in DNA binding by Cut. The CKII phosphorylation sites within the murine Cut (mCut) protein were identified by in vitro mutagenesis as residues Ser400, Ser789, and Ser972 within Cut repeat 1, 2, and 3, respectively. Cut homeodomain proteins were previously shown to function as transcriptional repressors. Overexpression of CKII reduced transcriptional repression by mCut, whereas a mutant mCut protein containing alanine substitutions at these sites was not affected. Altogether our results indicate that the transcriptional activity of Cut proteins is modulated by CKII.