DNA-BINDING BY CUT HOMEODOMAIN PROTEINS IS DOWN-MODULATED BY CASEIN KINASE-II

The Drosophila and mammalian Cut homeodomain proteins contain, in addi tion to the homeodomain, three other DNA binding regions called Cut re peats. Cut-related proteins thus belong to a distinct class of homeodo main proteins with multiple DNA binding domains. Using nuclear extract s from mammalian cells, Cut-specific DNA binding was increased followi ng 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 fo r 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 phosphoryla tion sites within the murine Cut (mCut) protein were identified by in vitro mutagenesis as residues Ser(400) Ser(789), and Ser(972) within C ut repeat 1, 2, and 3, respectively. Cut homeodomain proteins were pre viously shown to function as transcriptional repressors. Overexpressio n 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 act ivity of Cut proteins is modulated by CKII.