The conserved WRPW motif of Hes6 mediates proteasomal degradation

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Abstract

Hes6 belongs to a subfamily of basic helix–loop–helix transcription factors that includes Drosophila Hairy and Enhancer of split genes. Like other members of the family, Hes6 features the WRPW motif which is consisted just of four amino acids at its C-terminus. Here, we show that WRPW motif deletion mutant protein is substantially stabilized in comparison to the full length protein and that the enhanced stability is due to its resistance to proteasomal degradation. The WRPW motif also appears to be sufficient for acceleration of proteolysis as its fusion to two heterologous proteins, the green fluorescent protein (GFP) of Aequoria victoria and Gal4 DNA binding domain of Saccharomyces cerevisiae, significantly destabilized the proteins. These findings demonstrate a novel function of this conserved motif as a degradation signal and raise the possibility of utilizing it for controlling the level of ectopically expressed gene products.

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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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Abbreviations: Hes, Hairy and Enhancer of split; TLE, transducin-like element; IRES, internal ribosome entry site; EGFP, enhanced green fluorescent protein.

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