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.
Coqueret
O
O
Molecular Oncology Group, McGill University, Royal Victoria Hospital, Montreal, Quebec H3A 1A1, Canada.
Martin
N
N
Bérubé
G
G
Rabbat
M
M
Litchfield
D W
DW
Nepveu
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D047390:Casein Kinase II; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D004330:Drosophila; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012097:Repressor Proteins; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases