Knockout HAX 1 mice show increased apoptosis of neurons and postnatal le thality. Hax 1 is a multifunctional protein that Bicalutamide plays roles in calcium homeostasis, cell migration and apoptotic regulation. It was reported that Hax 1 protects cells against various stimuli and has been shown to interact with a number of cellular and viral proteins to suppress their pro death proper ties. In addition, Hax 1 has been found to be up regulated in breast cancer, lung cancer and melan oma, suggesting that it also has a role in oncogenesis. A PEST sequence is a peptide sequence which is rich in proline, glutamic acid, serine, and threo nine. It is known that the PEST sequence functions as a proteolytic signal to target proteins for degradation resulting in short intracellular half lives.
For example, the PEST sequence of NF kappa B is respon sible for its cleavage by calpain. It was reported that c myc, a protein with a PEST sequence, has a half life shorter than one hour. Notch 1, another short lived protein, is ubiquitinated by an E3 ligase sel 10 and degraded by the proteasome dependent on its PEST se quence. Hax 1 was predicted to contain a PEST sequence, however, it is still unknown whether this PEST sequence effects its turnover rate. In this study, we investigated the stability of Hax 1 in differ ent cells and explored the role of the PEST sequence in its degradation and biological function. Results Rapid degradation of Hax 1 In addition to its BH domains and a trans membrane domain, Hax 1 has a PEST sequence. The PEST re gion in Hax 1 is highly conserved in mammalian animals.
We tested the degradation profile of Hax 1 using a cycloheximide chase experiment in both human lung cancer cell line H1299 and mouse neuro blastoma cell line N2a. Hax 1 was found to have a much shorter half life than other two pro survival Bcl 2 family proteins, Bcl 2 and Bcl xL, suggesting that the Hax 1 protein is unstable and is rapidly degraded. PEST sequence dependent degradation of Hax 1 We next tested whether the PEST sequence in Hax 1 is responsible for its rapid degradation. A deletion mutant of Hax 1 was constructed in which the PEST sequence was deleted. The CHX chase experiments showed that the PEST Hax 1 level remained largely unchanged up to 3 hours, whereas WT Hax 1 level rap idly decreased to 50 % within 3 hours, suggesting that the PEST sequence in Hax 1 is neces sary for its rapid degradation.
Degradation of Hax 1 by the ubiquitin proteasome pathway Proteasome and autophagy systems are two main path ways for protein degradation. Here we tested which pathway is involved in the fast turnover of Hax 1. Cells were treated with MG132, a proteasome inhibitor, or Bafilomycin A1, an autophagy inhibitor. The level of EGFP Hax 1 increased in cells treated GSK-3 with MG132 for 3 hours, whereas in cells treated with Bafilo mycin A1 the protein level remained unchanged up to 18 hours.
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