DNA binding by cut homeodomain proteins is down-modulated by protein kinase C.
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 protein kinase C (PKC). Therefore, we investigated whether PKC can modulate the activity of mammalian Cut proteins. In vitro, a purified preparation of PKC efficiently phosphorylated Cut repeats, which inhibited 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 sites within the murine Cut (mCut) protein were identified by in vitro mutagenesis as residues Thr415, Thr804, and Ser987 within Cut repeats 1-3, respectively. Cut homeodomain proteins were previously shown to function as transcriptional repressors. Activation of PKC by phorbol 12-myristate 13-acetate 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 PKC.
Coqueret
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O
Molecular Oncology Group, McGill University, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec H3A 1A1, Canada.
Bérubé
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G
Nepveu
A
A
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D029721:Drosophila Proteins; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009281:Naphthalenes / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors
D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D018398:Homeodomain Proteins; D009281:Naphthalenes; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C066381:ct protein, Drosophila; D004247:DNA; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate