Increased Affinity of c-Myb for CREB-binding Protein (CBP) after CBP-induced Acetylation

Yuji Sano,Shunsuke Ishii

doi:10.1074/jbc.m006896200

Abstract

The c-myb proto-oncogene product (c-Myb) is a sequence-specific DNA-binding protein that functions as a transcriptional activator. The transcriptional coactivator CREB-binding protein (CBP) binds via its KIX domain to the activation domain of c-Myb and mediates c-Myb-dependent transcriptional activation. CBP possesses intrinsic histone acetyltransferase activity, and can acetylate not only histones but also certain transcriptional factors such as GATA1 and p53. Here we demonstrate that the C/H2 domain of CBP, which is critical for the acetyltransferase activity, also directly interacts with the negative regulatory domain (NRD) of c-Myb. Consistent with this observation, CBP acetylated c-Myb in vitro at Lys(438) and Lys(441) within the NRD. In addition, CBP acetylated c-Myb in vivo not only at the sites found in this study but also at the p300-induced acetylation sites reported recently. Replacement of lysine by arginine at all of these sites dramatically decreased the trans-activating capacity of c-Myb. The results of transcriptional activation assays with c-Myb acetylation site mutants suggested that acetylation of c-Myb at each of these five sites synergistically enhances c-Myb activity. Mutations of these acetylation sites reduced the strength of the interaction between c-Myb and CBP. Thus, acetylation of c-Myb by CBP increases the trans-activating capacity of c-Myb by enhancing its association with CBP. These results demonstrate a novel molecular mechanism of regulation of c-Myb activity.

Highlights

The c-myb proto-oncogene product (c-Myb) is a sequence-specific DNA-binding protein that functions as a transcriptional activator
Direct Interaction between the C/H2 Region of cAMP response element-binding protein (CREB)-binding protein (CBP) and the negative regulatory domain (NRD) of c-Myb—We previously reported that CBP directly binds to the transcriptional activation domain of c-Myb via the N-terminal KIX domain [17]
In a more precise analysis of the interaction between c-Myb and CBP, we obtained evidence suggesting that a region other than the KIX domain directly interacts with c-Myb

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—The plasmids pGEX-KIX, pGEX-Bromo, pGEX-C/H2, and pGEX-C/H3 to express GST fusions containing various portions of CBP were described previously [45]. About 1–2 ␮g of the purified GST fusion protein as substrate and 1 ␮l of [14C]acetyl-CoA (51 mCi/mmol, Amersham Pharmacia Biotech) were added to the protein G-Sepharose beads containing CBP, and the reaction volume was adjusted to 30 ␮l with acetylation buffer. To examine the trans-activation capacity of the Gal4-Myb fusion, the Gal site containing luciferase reporter, in which the thymidine kinase promoter is linked to three tandem repeats of the Gal4-binding site, was transfected together with plasmid expressing various forms of Gal4-Myb fusions or the control vector and the internal control plasmid pact-␤-gal. Cell lysates were prepared using TNE buffer (10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10 mM sodium butyrate) containing a protease inhibitor mixture and the lysates were centrifuged. The immunocomplexes were separated on 8% SDS gels and analyzed by Western blotting using the anti-c-Myb monoclonal antibody 5-1 and a chemiluminescent detection regent (New England Biolabs)

RESULTS

DISCUSSION

Yuji Sano and Shunsuke Ishii