Abstract
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.
Highlights
The cDNAs for several mammalian homologues of the Drosophila Cut homeodomain protein have recently been isolated (1– 4)
Expressed Cut Repeats Are Phosphorylated in Vitro by casein kinase II (CKII)—The evolutionarily conserved Cut repeats were previously shown to function as specific DNA binding domains either alone or in conjunction with the Cut homeodomain
As a first step to investigate whether CKII can modulate Cut repeat activity, we verified if CKII can phosphorylate glutathione S-transferase (GST) fusion proteins containing either Cut repeat 1 (GST/CR1) or Cut repeat 3 and homeodomain (GST/Cut repeat 3/homeodomain (CR3HD))
Summary
The cDNAs for several mammalian homologues of the Drosophila Cut homeodomain protein have recently been isolated (1– 4). A Cut-related protein was independently identified either as a CCAAT displacement protein or as a protein that binds to an Sp1-like site within the c-Myc promoter and represses its expression (1, 2). When Cut expression was artificially elevated in embryos, all precursor cells gave rise to external sensory organs (6). The human Cut protein (hCut) was found to bind to upstream regulatory sequences of the gp91-phox gene and the expression of this gene was shown to coincide with down-regulation of hCut binding activity upon differentiation of granulocytic cells (16 –18). From the analysis of their deduced amino acid sequences, mammalian Cut proteins are almost identical over their entire
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
