No abstract

No abstract

CCAAT/enhancer-binding proteins (C/EBPs) are basic region/leucine zipper transcription factors that function as regulators of cell growth and differentiation in numerous cell types. We previously localized transcriptional activation and inhibitory regions in one family member, C/EBP⑀. Here we describe the further characterization of a C/EBP⑀ inhibitory domain termed regulatory domain I. We show that functionally related domains are present in C/EBP␣, C/EBP␤, and C/EBP␦. These domains contain an evolutionarily conserved five-amino acid motif (the regulatory domain motif (RDM)) that conforms to the consensus sequence (I/V/ L)KXEP. Mutagenesis studies revealed that the residues at positions 1, 2, and 4 of the RDM are critical for inhibitory domain function. Data base searches identified RDM-like sequences in a number of nuclear proteins. We found that small regions from c-Jun, JunB, and JunD containing this sequence also function as transcriptional inhibitory domains. Importantly, the RDM is similar to the recognition sequence for attachment of the ubiquitin-like protein, small ubiquitin-like modifier-1 (SUMO-1), and the conserved lysine residue of each C/EBP RDM served as an attachment site for SUMO-1. SUMO-1 attachment decreased the inhibitory effect of the C/EBP⑀ regulatory domain, suggesting that sumoylation may play an important role in modulating C/EBP⑀ activity as well as that of the other C/EBP family members.

The transcription factor CCAAT/enhancer-binding protein delta (C/EBP␦, CEBPD) is a tumor suppressor that is downregulated during breast cancer progression but may also promote metastasis. Here, we have investigated the mechanism(s) regulating C/EBP␦ expression and its role in human breast cancer cells. We describe a novel pathway by which the tyrosine kinase Src downregulates C/EBP␦ through the SIAH2 E3 ubiquitin ligase. Src phosphorylates SIAH2 in vitro and leads to tyrosine phosphorylation and activation of SIAH2 in breast tumor cell lines. SIAH2 interacts with C/EBP␦, but not C/EBP␤, and promotes its polyubiquitination and proteasomal degradation. Src/SIAH2-mediated inhibition of C/EBP␦ expression supports elevated cyclin D1 levels, phosphorylation of retinoblastoma protein (Rb), motility, invasive properties, and survival of transformed cells. Pharmacological inhibition of Src family kinases by SKI-606 (bosutinib) induces C/EBP␦ expression in an SIAH2-dependent manner, which is necessary for "therapeutic" responses to SKI-606 in vitro. Ectopic expression of degradation-resistant mutants of C/EBP␦, which do not interact with SIAH2 and/or cannot be polyubiquitinated, prevents full transformation of MCF-10A cells by activated Src (Src truncated at amino acid 531 [Src-531]) in vitro. These data reveal that C/EBP␦ expression can be regulated at the protein level by oncogenic Src kinase signals through SIAH2, thus contributing to breast epithelial cell transformation.