Abstract
CtBP family members, CtBP1 and CtBP2, are unique transcriptional regulators that adapt a metabolic enzyme fold, and their activities are regulated by NAD(H)-binding. CtBP1 is both cytoplasmic and nuclear, and its subcellular localization is regulated by sumoylation, phosphorylation, and binding to a PDZ protein. In contrast, we showed that CtBP2 is exclusively nuclear. CtBP1 and CtBP2 are highly similar, but differ at the N-terminal 20 amino acid region. Substitution of the N-terminal domain of CtBP1 with the corresponding CtBP2 domain confers a dominant nuclear localization pattern to CtBP1. The N-terminal domain of CtBP2 contains three Lys residues. Our results show that these Lys residues are acetylated by the nuclear acetylase p300. Although all three Lys residues of CtBP2 (Lys-6, Lys-8, and Lys-10) appear to be acetylated, acetylation of Lys-10 is critical for nuclear localization. CtBP2 with a single amino acid substitution at Lys-10 (K10R) is predominantly localized in the cytoplasm. The cytoplasmic localization of the K10R mutant is correlated with enhanced nuclear export that is inhibited by leptomycin B. Furthermore, lack of acetylation at Lys-10 renders CtBP2 to be more efficient in repression of the E-cadherin promoter. Our studies have revealed the important roles of acetylation in regulating subcellular localization and transcriptional activity of CtBP2.
Highlights
Unlike other transcription factors, CtBPs share a high degree of sequence homology with a group of metabolic enzymes of the 2-hydroxy acid dehydrogenase family [4]
We show that CtBP2 has a dominant nuclear localization pattern, which is dictated by the N-terminal 20 amino acid domain
We examined the localization patterns of transiently expressed CtBPs (Fig. 1E), which were found to be highly similar to patterns of endogenous CtBPs and the patterns of CtBPs expressed in stably transfected cell lines (Fig. 1D)
Summary
CtBPs share a high degree of sequence homology with a group of metabolic enzymes of the 2-hydroxy acid dehydrogenase family [4]. The C-terminal region of CtBP1 has a PDZ-binding motif [1], which mediates interaction with a PDZ domain containing protein, neuronal nitric-oxide synthase, changing the localization pattern of CtBP1 from nuclear to cytoplasmic [36]. The specificities of the antibodies used in these studies were confirmed by their differential reactivity toward CtBP1 or CtBP2 by Western blot analysis of N-terminally tagged versions (Flag-HA) of CtBP1 or CtBP2 immunoprecipitated from stably transfected cell lines (Fig. 1A).
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