Transportin 2 Regulates Apoptosis through the RNA-binding Protein HuR

Abstract

In response to severe stress, apoptotic cell death is engaged. Apoptosis is a well orchestrated process that involves the activation and implication of many factors. In this study, we identified a role for the nuclear trafficking factor TRN2 (transportin 2) in cell death. TRN2 is normally responsible for the nuclear import of the RNA-binding protein HuR. During apoptosis, however, HuR accumulates in the cytoplasm. This is due to the caspase-mediated cleavage of the cytoplasmic fraction of HuR. One of the cleavage fragments generated by this processing of HuR interacts with TRN2 and thus blocks the re-import of HuR into the nucleus. This concentrates HuR in the cytoplasm, advancing apoptosis. Therefore, increasing or decreasing the levels of TRN2 has an inverse consequential effect on cell death, demonstrating for the first time the role of a nucleocytoplasmic transport factor in apoptosis.When confronted with stress, eukaryotic cells undergo different responses depending on the severity and duration of the stress (1). Although adaptation and survival are at one end of these responses, cell death can also be the outcome. Apoptotic cell death is a complex pathway involving the activation and cleavage of several proteins, some of the most important of which are cysteine-aspartic proteases (caspases). This family of proteases then cleaves specific substrates, allowing the progression of cell death (2-5). In addition to causing the organized destruction of a cell, caspase-mediated cleavage can also serve to activate target proteins that have important roles in advancing apoptosis themselves. Recently, the RNA-binding protein HuR was identified as one such factor (6). Under normal conditions, HuR is an important regulator of gene expression, controlling the localization, stabilization, and translation of many different mRNAs (7,8). The targets of HuR are involved in many cellular processes, including cell growth, differentiation, and survival and apoptotic cell death. Likewise, the importance of HuR in regulating these processes has been shown, given that its absence or overexpression in different systems can have potent effects on cell physiology (6, 9 -12).The role of HuR as a promoter of cell death was identified when it was found that caspase-3 and caspase-7 cleave HuR in response to lethal stress (6), such as with the use of the apoptotic stimulator drug staurosporine (STS). 4 The cleavage of HuR, occurring at Asp-226 of this protein, yields two cleavage products: HuR-CP1 (26 kDa) and HuR-CP2 (8 kDa). Mutation of this cleavage site prevents apoptosis, whereas providing these two cleavage products can stimulate cell death under nonlethal conditions (13). It was shown that HuR-CP2 helps promote apoptosis by binding to putative HLA-associated protein-I (PHAPI) (6), an activator of apoptosis and a known ligand of full-length HuR. However, the implication of other protein partners has not yet been explored.Intriguingly, prior to its cleavage, HuR, which typically shuttles between the nucleus and the cytoplasm, must ha...

Apoptotic-induced cleavage shifts HuR from being a promoter of survival to an activator of caspase-mediated apoptosis

“…25 HuR-CP1 causes a similar effect in response to lethal stress, by binding to the import factor transportin-2 and augmenting cytoplasmic localization of HuR. 41 Here we show that together the two CPs are capable of stabilizing caspase-9 mRNA, even to a greater extent than wtHuR, and our previous data also indicate that the two CPs of HuR are most potent for inducing apoptosis when present simultaneously. 24 Therefore, these data illustrate that the coordination between HuR and its CPs is an important aspect of the effects they have.…”

“…HuR shuttles between the nucleus and the cytoplasm, and while only a fraction of it (B10-20%) localizes to the cytoplasm, 36,37 it is in this compartment where HuR exercises its main biological effects. [15][16][17]30,[38][39][40] Our recent papers 18,24,25,41 strengthen this idea and show that the HuR-CPs, generated from B50% of cytoplasmic HuR, are both sufficient and necessary for HuR-mediated effects. 18 However, as not all cytoplasmic HuR is cleaved, wt HuR and both of its CPs are all present simultaneously, and thus may be affected by one another when exercising their effects.…”

HuR’s post-transcriptional regulation of death receptor 5 in pancreatic cancer cells

“…Other ongoing work includes attempting to further understand the function of CP1 in cells and uncover the exact signal by which CP1 may provide feedback to HuR and DR5 mRNA. 32 Our data along with others show that HuR can assert its influence and be influenced at many points in this powerful apoptotic pathway. 13 Confirmatory studies in other tumor systems will determine if all cancer cells select for HuR's ability to modulate or put the brakes on DR5-directed signaling in an effort to avoid apoptosis.…”

“…3,13 The precise role and/or relevance of HuR cleavage (CP1) in this pathway is currently under investigation, although others have linked this event to pp32's induction of apoptosis. 23,31,32 Clinically, we have previously shown that cytoplasmic HuR levels in PDA specimens can be a useful predictive marker for gemcitabine therapy. 19,20 Based on our current results, the cytoplasmic HuR levels could also be a predictive marker for DR5-targeted therapy, given the inverse relationship between cytoplasmic HuR abundance and DR5 intensity in patient tumors (p < 0.0001, Fig.…”

Modulation of Gene Expression by RNA Binding Proteins: mRNA Stability and Translation

“…Protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) p38 also modulate the association of HuR with target mRNA [38][39][40]. Although HuR is predominantly nuclear, it is capable of shuttling to the cytoplasm through its nucleoyctoplasmic shuttling sequence (HNS) and transport machinery including chromosome region maintenance 1(CRM1), transportins 1 and 2, and importin-1α [41][42][43][44]. In addition, under stress conditions such as arsenite or heat shock, HuR aggregates in the cytoplasm with RNAs, known as RNA granules or stress granules, where mRNAs are stored or translationally suppressed [37,45].…”