Stress-Inducible Caspase Substrate TRB3 Promotes Nuclear Translocation of Procaspase-3 Kouhei Shimizu1, Shoukichi Takahama 2 , Yaeta Endo1,2,3,4, Tatsuya Sawasaki1,2,3,4* 1 The Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Japan, 2 The Venture Business Laboratory, Ehime University, Matsuyama, Japan, 3 Proteo-Medicine Research Center, Ehime University, Toon, Japan, 4 RIKEN Systems and Structural Biology Center, Yokohama, Japan Abstract Pseudokinase TRB3 is a stress-inducible nuclear protein, which has recently been shown to be involved in ER stress-induced apoptosis. However, it remains unclear how TRB3 contributes to the process. We recently demonstrated that TRB3 was cleaved by caspase-3 (CASP3) in vitro and also in apoptosis-induced cells. Thus, we investigate the role of TRB3 cleavage in the apoptotic process to address the above question. Overexpression studies revealed that the cleavage of TRB3 promoted CASP3/7 activation and apoptosis. In contrast, the anti-apoptotic effects were found under TRB3 non-cleavable conditions, such as ER stress, and also when the CASP3/7 activation was enhanced by knockdown of endogenous TRB3 expression. Interestingly, nuclear translocation of procaspase-3 (proCASP3) was observed in cells either overexpressing TRB3 or under tunicamycin-induced ER stress. Although forced cytoplasmic expression of proCASP3 enhanced apoptosis significantly, its nuclear expression did not produce any pro-apoptotic effect, suggesting that nuclear distribution of proCASP3 is not critical for the execution of apoptosis. Thus, TRB3 might prevent cytoplasmic activation of CASP3 by promoting proCASP3 entry into the nucleus, and thereby inhibit apoptosis. Taken together, our results suggest that TRB3, through its own cleavage, functions as a molecular switch between the cell survival and apoptotic pathways under stressful conditions. Citation: Shimizu K, Takahama S, Endo Y, Sawasaki T (2012) Stress-Inducible Caspase Substrate TRB3 Promotes Nuclear Translocation of Procaspase-3. PLoS ONE 7(8): e42721. doi:10.1371/journal.pone.0042721 Editor: Dhyan Chandra, Roswell Park Cancer Institute, United States of America Received March 28, 2012; Accepted July 10, 2012; Published August 9, 2012 Copyright: 2012 Shimizu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was partly supported by a Grant-in-Aid for Scientific Research (B) (Dr. Sawasaki) and a Grant-in-Aid for JSPS Fellows (Dr. Shimizu) from Japan Society for the Promotion of Science, and a Grant-in-Aid for Scientific Research on Innovative Areas (Dr. Sawasaki) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sawasaki@ehime-u.ac.jp Introduction TRB3 (also known as TRIB3, NIPK, SINK, or SKIP), one of the mammalian orthologues of Drosophila Tribbles, was identified as a pseudokinase, because it contains a Ser/Thr protein kinase- like domain that lacked the ATP-binding domain and core catalytic residues, therefore, dose not have any kinase activity [1]. Despite a lack of characteristic functional domain, TRB3 has been shown to be involved in multiple cellular processes such as glucose and lipid metabolism, muscle and adipocyte differentiation, and stress response by interacting with various functional proteins (e.g. kinase: AKT, MAPK; transcription factor: ATF4, CHOP, PPARc; E3 ubiquitin ligase: COP1) [2–9]. Endoplasmic reticulum (ER) stress has recently been recognized as another key pathway for triggering apoptosis [10,11]. The adaptive phase of ER stress promotes cell survival by reducing the accumulation of unfolded proteins through global transcriptional control, well known as the unfolded protein response (UPR) [12]. However, apoptosis is considered selected when the apoptotic pathway gains ascendancy over the adaptive pathway by over- whelming the ER stress. During ER stress, TRB3 is upregulated by an ER stress-inducible transcription factor, ATF4/CHOP [6]. Excess expression of TRB3 downregulates its own expression by negative feedback via the repression of ATF4/CHOP transcrip- tional activity [13]. Several studies suggest that CHOP and its transcriptional target, BH3-only proteins such as Bim and PUMA, promote ER stress-induced apoptosis [14,15]. TRB3 has been shown to be involved in ER stress-induced apoptosis via these regulatory processes [6,16]. TRB3 expression is also induced in a PI3K-dependent manner by nutrient deficiency, such as the lack of glucose or amino acids [17]. Results of a transient over- expression study suggest that TRB3 plays an apoptosis inhibitory role under glucose depletion condition. Thus, the expression of TRB3 could be both up- and down-regulated by various cellular stresses [18]. Taken together, these studies indicate that TRB3 functions as an important component of the stress response mechanism, namely, regulates stress-induced apoptosis. However, it remains to be elucidated how TRB3 contributes to the stress responses. Caspase-3 (CASP3), one of the most downstream components of the caspase cascade, is known to cleave many critical proteins such as lamin, PARP, ICAD/DFF45 and PAK2, and in turn induces irreversible apoptosis that involves substrate proteolysis and positive feedback of caspase cascade [19,20]. Recently, we have demonstrated that TRB3 is a substrate for CASP3 [21]. To investigate the role of TRB3 cleavage in the apoptotic process, we carried out cell-based analysis using the wild type and a non- cleavable mutant of TRB3. In this study, we have shown a TRB3 cleavage-dependent pro-apoptotic response, and have also pre- sented evidence for a novel anti-apoptotic mechanism involving TRB3-mediated nuclear translocation of procaspase-3 (pro- CASP3). This dual function of TRB3 may serve as a key switch between the cell survival and apoptosis pathways depending on the cellular context. PLoS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42721