The conserved WRPW motif of Hes6 mediates proteasomal degradation☆
Section snippets
Materials and methods
Plasmids. Construction of expression vector plasmids was performed using standard molecular cloning techniques. The full length sequence of Hes6 was amplified from a cDNA plasmid and inserted upstream of IRES-GFP cassette in a shuttle vector. The DNA fragment containing the wild type Hes6 sequence and IRES-GFP (Hes6WT-IRES-GFP) was then cloned into pcDNA3.1. To generate the WRPW motif deletion mutant of Hes6 with IRES-GFP (Hes6MT-IRES-GFP), a downstream PCR primer bearing a single nucleotide
Results and discussion
In the course of generating a WRPW motif deletion mutant as a potential dominant negative derivative of Hes6, we noticed that the protein level was much higher for the deletion mutant than for the full length protein. To examine the phenomenon more precisely, we generated a point mutant of Hes6 that contains a stop codon in place of W221 (TGG → TGA). The full length gene (Hes6WT) and the WRPW deletion mutant (Hes6MT) were inserted upstream of IRES-GFP cassette which would function as an internal
Acknowledgments
We thank S.Y. Lee for his critical comments on the manuscript. We thank D.J. Anderson for permitting the use of anti-Hes6 antiserum. This research was supported by the Ewha Womans University Research Grant of 2003, by the Korea Science and Engineering Foundation through the Center for Cell Signaling Research at Ewha Womans University, and by a grant (M103KV010010 04K2201 01020; to J. Kim) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science
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The E3 ubiquitin ligase SCF<sup>FBXL14</sup> complex stimulates neuronal differentiation by targeting the Notch signaling factor HES1 for proteolysis
2017, Journal of Biological ChemistryCitation Excerpt :Therefore, the fine-tuning of oscillatory behavior of HES1 makes it a rhythmic composer to regulate pluripotent cell (ES, EC, neural stem cell) maintenance, proliferation, and differentiation. It has been reported that the WRPW motif mediates the proteasome degradation of HES6 (50). The WRPW motif has been shown to be sufficient for acceleration of proteolysis because it fused to two heterologous proteins, the GFP of Aequoria victoria, and Gal4 DNA-binding domain of Saccharomyces cerevisiae (50).
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2013, Experimental Cell ResearchCitation Excerpt :Reduction in HES6 mRNA levels was noticeable as early as 6 h following transfection and lasted for at least 72 h (data not shown). As HES6 has a short half-life it is likely the protein levels were also significantly diminished at 24 h after siRNA transfection [22]. In an attempt to confirm knockdown of HES6 protein levels, multiple commercial and in-house antibodies against HES6 were tested using RH30, which expresses a high level of HES6, and HX170C, an ARMSn cell line in which HES6 expression is absent due to a chromosomal deletion.
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2010, Current Topics in Developmental BiologyCitation Excerpt :Thus, Hes proteins act as transcriptional repressors by binding to the target sequences. The orange domain also regulates the selection of bHLH heterodimer partners (Dawson et al., 1995; Taelman et al., 2004), while the WRPW sequence acts as a polyubiquitination signal, controlling the half-life of Hes protein by promotion of proteasome-mediated degradation (Kang et al., 2005). It is well established that the Notch–RBPj–Hes pathway regulates many biological events by repressing target gene expression (Honjo, 1996; Kageyama et al., 2007).
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Abbreviations: Hes, Hairy and Enhancer of split; TLE, transducin-like element; IRES, internal ribosome entry site; EGFP, enhanced green fluorescent protein.