DNA Binding by Cut Homeodomain Proteins Is Down-modulated by Protein Kinase C

doi:10.1074/jbc.271.40.24862

DNA Binding by Cut Homeodomain Proteins Is Down-modulated by Protein Kinase C*

From the Molecular Oncology Group, Departments of ^‡ Medicine,
^¶ Oncology, and
^∥ Biochemistry, McGill University, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec H3A 1A1, Canada

^″ Recipient of a scholarship from the Fonds de la Recherche en Santé du Québec. To whom correspondence should be addressed. Tel.: 514-842-1231 (ext. 5832); Fax: 514-843-1478; E-mail: alain{at}lan1.molonc.mcgill.ca

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 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.

Footnotes

↵^§ Recipient of a fellowship from The Royal Victoria Hospital Department of Medicine and Research Institute.
↵* This research was supported by Grant MT-11590 from the Medical Research Council of Canada and Grant 3497 from the National Cancer Institute of Canada (to A. N.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

hCut

human Cut

mCut

mouse Cut

CR

Cut repeat

HD

homeodomain

PKC

protein kinase C

GST

glutathione S-transferase

DMEM

Dulbecco's modified Eagle's medium

FBS

fetal bovine serum

PMA

phorbol 12-myristate 13-acetate

EMSA

electrophoretic mobility shift analysis

CAT

chloramphenicol acetyltransferase

tk

thymidine kinase.
↵² O. Coqueret and A. Nepveu, manuscript in preparation.
↵³ O. Coqueret and A. Nepveu, manuscript in preparation.
↵⁴ N. Martin and A. Nepveu, manuscript in preparation.
↵⁵ O. Coqueret and A. Nepveu, unpublished data.
- Received April 26, 1996.
- Revision received July 5, 1996.