ABSTRACT
Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt)) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt) embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt) ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt) ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.
Subject(s)
Embryonic Development , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Cycle/genetics , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Embryo, Mammalian/metabolism , Embryonic Development/genetics , Embryonic Stem Cells/metabolism , Female , Male , Mice , Mutation , Phenotype , Protein Stability , Protein Structure, Tertiary , Transcriptome , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/geneticsABSTRACT
Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERalpha and ERbeta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERalpha and TGF-beta/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERalpha inhibits TGF-beta signaling by decreasing Smad protein levels. ERalpha-mediated reductions in Smad levels did not require the DNA binding ability of ERalpha, implying that ERalpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ERalpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERalpha.
Subject(s)
Breast Neoplasms/metabolism , Estrogens/pharmacology , Signal Transduction/drug effects , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Transforming Growth Factor beta/metabolism , Biomarkers, Tumor/metabolism , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Movement , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Female , Gene Expression Profiling , Humans , Immunoprecipitation , Neoplasm Invasiveness , Oligonucleotide Array Sequence Analysis , Plasminogen Activator Inhibitor 1/genetics , Plasminogen Activator Inhibitor 1/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Transforming Growth Factor beta/genetics , Tumor Cells, Cultured , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
CHIP is a U-box-type ubiquitin ligase that induces ubiquitylation and degradation of its substrates, which include several oncogenic proteins. The relationship between CHIP and tumour progression, however, has not been elucidated. Here, we show that CHIP suppresses tumour progression in human breast cancer by inhibiting oncogenic pathways. CHIP levels were negatively correlated with the malignancy of human breast tumour tissues. In a nude mouse xenograft model, tumour growth and metastasis were significantly inhibited by CHIP expression. In contrast, knockdown of CHIP (shCHIP) in breast cancer cells resulted in rapid tumour growth and metastastic phenotypes in mice. In cell-based experiments, anchorage-independent growth and invasiveness of shCHIP cells was significantly elevated due to increased expression of Bcl2, Akt1, Smad and Twist. Proteomic analysis identified the transcriptional co-activator SRC-3 (refs 13, 14, 15, 16, 17, 18, 19) as a direct target for ubiquitylation and degradation by CHIP. Knocking down SRC-3 in shCHIP cells reduced the expression of Smad and Twist, and suppressed tumour metastasis in vivo. Conversely, SRC-3 co-expression prevented CHIP-induced suppression of metastasis formation. These observations demonstrate that CHIP inhibits anchorage-independent cell growth and metastatic potential by degrading oncogenic proteins including SRC-3.
