DNA-BINDING BY CUT HOMEODOMAIN PROTEINS IS DOWN-MODULATED BY PROTEIN-KINASE-C

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 protein kinase C (PKC). Therefore, we investigated whether PKC can m odulate the activity of mammalian Cut proteins. In vitro, a purified p reparation of PKC efficiently phosphorylated Cut repeats, which inhibi ted DNA binding. In vivo, a brief treatment of cells with calphostin C , a specific inhibitor of PKC, led to an increase in Cut-specific DNA binding, whereas phorbol 12-myristate 13-acetate, a specific activator of PKC, caused a decrease in DNA binding. The PKC phosphorylation sit es within the murine Cut (mCut) protein were identified by in vitro mu tagenesis as residues Thr(415), Thr(804), and Ser(987) within Cut repe ats 1-3, respectively. Cut homeodomain proteins were previously shown to function as transcriptional repressors. Activation of PKC by phorbo l 12-myristate 13-acetate reduced transcriptional repression by mCut, whereas a mutant mCut protein containing alanine substitutions at thes e sites was not affected. Altogether, our results indicate that the tr anscriptional activity of Cut proteins is modulated by PKC.