Hax-1 is rapidly degraded by the proteasome dependent on its PEST sequence

Li et al. BMC Cell Biology 2012, 13:20 http://www.biomedcentral.com/1471-2121/13/20 RESEARCH ARTICLE Open Access Hax-1 is rapidly degraded by the proteasome dependent on its PEST sequence Bin Li1,2, Qingsong Hu1,2, Ranjie Xu1,2, Haigang Ren1, Erkang Fei2, Dong Chen1 and Guanghui Wang1* Abstract Background: HS-1-associated protein X-1 (Hax-1), is a multifunctional protein that has sequence homology to Bcl-2 family members. HAX-1 knockout animals reveal that it plays an essential protective role in the central nervous system against various stresses. Homozygous mutations in the HAX-1 gene are associated with autosomal recessive forms of severe congenital neutropenia along with neurological symptoms. The protein level of Hax-1 has been shown to be regulated by cellular protease cleavage or by transcriptional suppression upon stimulation. Results: Here, we report a novel post-translational mechanism for regulation of Hax-1 levels in mammalian cells. We identified that PEST sequence, a sequence rich in proline, glutamic acid, serine and threonine, is responsible for its poly-ubiquitination and rapid degradation. Hax-1 is conjugated by K48-linked ubiquitin chains and undergoes a fast turnover by the proteasome system. A deletion mutant of Hax-1 that lacks the PEST sequence is more resistant to the proteasomal degradation and exerts more protective effects against apoptotic stimuli than wild type Hax-1. Conclusion: Our data indicate that Hax-1 is a short-lived protein and that its PEST sequence dependent fast degradation by the proteasome may contribute to the rapid cellular responses upon different stimulations. Keywords: Hax-1, Proteasome, Ubiquitin, PEST sequence, Bcl-2 family protein Background HS-1-associated protein X-1, Hax-1, is a 35 kDa protein with two Bcl-2 homology (BH) domains that was identified in a yeast two hybrid screen where it was found to interact with HS-1, a Src kinase substrate [1]. Hax-1 is ubiquitously expressed in most tissues and is reported to be localized in mitochondria as well as the endoplasmic reticulum (ER) and nuclear membrane [1-3]. Mutations identified in the human HAX-1 gene have been shown to cause neutropenia and neurodevelopmental abnormalities [4-6]. Knockout HAX-1 mice show increased apoptosis of neurons and postnatal lethality. [7]. Hax-1 is a multifunctional protein that plays roles in calcium homeostasis [8], cell migration [9] and apoptotic regulation [10,11]. It was reported that Hax-1 protects cells against various stimuli and has been shown to interact with a number of cellular * Correspondence: 1 Laboratory of Molecular Neuropathology, Department of Pharmacology, Soochow University College of Pharmaceutical Sciences, Suzhou, Jiangsu 201203, People's Republic of China Full list of author information is available at the end of the article and viral proteins to suppress their pro-death properties [12-15]. In addition, Hax-1 has been found to be up-regulated in breast cancer, lung cancer and melanoma [16], suggesting that it also has a role in oncogenesis. A PEST sequence is a peptide sequence which is rich in proline (P), glutamic acid (E), serine (S), and threonine (T). It is known that the PEST sequence functions as a proteolytic signal to target proteins for degradation resulting in short intracellular half lives [17]. For example, the PEST sequence of NF-kappa B is responsible for its cleavage by calpain [18]. It was reported that c-myc, a protein with a PEST sequence, has a half-life shorter than one hour [17]. Notch 1, another short-lived protein, is ubiquitinated by an E3 ligase sel-10 and degraded by the proteasome dependent on its PEST sequence [19,20]. Hax-1 was predicted to contain a PEST sequence (aa 104–117) [1], however, it is still unknown whether this PEST sequence effects its turnover rate. In this study, we investigated the stability of Hax-1 in different cells and explored the role of the PEST sequence in its degradation and biological function. © 2012 Li et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Li et al. BMC Cell Biology 2012, 13:20 http://www.biomedcentral.com/1471-2121/13/20 Figure 1 (See legend on next page.) Page 2 of 10 Li et al. BMC Cell Biology 2012, 13:20 http://www.biomedcentral.com/1471-2121/13/20 Page 3 of 10 (See figure on previous page.) Figure 1 Rapid degradation of Hax-1 is dependent on its PEST sequence. A. Schematic representation of a PEST sequence in Hax-1 protein. The PEST sequence was identified using Pestfind service on “emboss.bioinformatics.nl/cgi-bin/emboss/pestfind”. The PEST sequence in Hax-1 is conserved among different mammals. B. Chase-time experiment of Hax-1 and other Bcl-2 proteins. H1299 cells treated with CHX (100 ug/ml) for different time points were harvested for immunoblot analysis using indicated antibodies. C. Data from three independent experiments in B were quantified. D. Similar experiments as B were carried out using mouse N2a cells. E. An EGFP-tagged WT Hax-1 or ΔPEST Hax-1 was transiently transfected into H1299 cells. Forty-eight hours later, CHX chase experiments were carried out. F. Quantitative analysis of data from E with three independent experiments. Results Rapid degradation of Hax-1 In addition to its BH domains and a trans-membrane domain, Hax-1 has a PEST sequence [1]. The PEST region in Hax-1 is highly conserved in mammalian animals (Figure 1A). We tested the degradation profile of Hax-1 using a cycloheximide (CHX) chase experiment in both human lung cancer cell line H1299 and mouse neuroblastoma 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 (Figure 1B-D), 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 (aa 103–118) was deleted. The CHX chase experiments showed that the ΔPEST Hax-1 level remained largely unchanged up to 3 hours, whereas WT Hax-1 level rapidly decreased to < 50 % within 3 hours (Figure 1E and F), suggesting that the PEST sequence in Hax-1 is necessary for its rapid degradation. Degradation of Hax-1 by the ubiquitin-proteasome pathway Proteasome and autophagy systems are two main pathways 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 with MG132 for 3 hours (Figure 2A), whereas in cells treated with Bafilomycin A1 the protein level remained unchan (...truncated)