Nucleocytoplasmic shuttling of BID is involved in regulating its activities in the DNA-damage response

Abstract

The BH3-only BID protein acts as a sentinel to interconnect specific death signals to the core apoptotic pathway. Our previous data demonstrated that BID is important for both S-phase arrest and cell death following DNA damage, and that the cell cycle arrest function is regulated by its phosphorylation by the ATM kinase. We also showed that a portion of cellular BID localizes to the nucleus. Here, we demonstrate that etoposide and ionizing radiation induce the exit of BID from the nucleus and that leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, prevents the nuclear exit of BID. BID carries a nuclear export signal (NES) consensus motif; however, it does not seem to be functional. To examine the importance of BID nuclear export, we targeted BID to the nucleus by fusing it to a strong nuclear localization signal (NLS). NLS-BID is phosphorylated in a similar time course as wild-type BID, but does not exit the nucleus following etoposide treatment. Importantly, introducing NLS-BID into BID À/À cells failed to restore S-phase arrest and cell death in response to etoposide. These results implicate BID as a nuclear protein and raise the possibility that nucleocytoplasmic shuttling of BID is involved in regulating its activities in the DNAdamage response. Programmed cell death or apoptosis is critical for both the development and maintenance of tissues. Caspases, a family of cysteine proteases, are the major executioners of the apoptotic process, 1 whereas the BCL-2 family members are the major regulators of this process. 2 The cell death regulatory mechanisms of the BCL-2 proteins are largely unknown, although it is thought that their function depends mostly on their ability to modulate the release of proteins from the intermembrane space of the mitochondria. The BCL-2 family members are localized at multiple subcellular locations, that is, in the cytosol, nuclear outer membrane, endoplasmic reticulum membrane and mitochondrial outer membrane. In healthy cells, anti-apoptotic BCL-2 family members are mostly found associated with internal membranes, 3,4 the multidomain pro-apoptotic proteins are localized either at membranes (e.g. BAK 5 ) or in the cytoplasm (e.g. BAX 6,7 ), and the pro-apoptotic BH3-only proteins (see below) are predominantly found in the cytosol (e.g. BAD 8 ) or associated with the cytoskeleton (e.g. BIM 9 ). In response to an apoptotic signal, the pro-apoptotic proteins undergo a conformational change that enables them to target and integrate into membranes, especially the mitochondrial outer membrane, to trigger apoptosis. 10 An important subset of the pro-apoptotic BCL-2 family members are the BH3-only proteins, such as BID, which act as sensors of intracellular damage. 11 These proteins are held in check by diverse mechanisms, seemingly at cellular locations at which they can sense specific damage, and once activated, translocate to the mitochondria to communicate the damage signal. BID is a pivotal executioner of apoptosis in vivo, as it is essential for the TNFa/Fas death ...

Human Herpesvirus 8 Interferon Regulatory Factor-Mediated BH3-Only Protein Inhibition via Bid BH3-B Mimicry

“…4B). It is notable that some nuclear localization of Bid L was apparent, consistent with previous reports of nuclear localization and associated activities of Bid [47]–[49], but no nuclear staining was evident for tBid. In contrast to Bid, and consistent with previous findings [23], Bim distribution was altered in the presence of vIRF-1, with strong nuclear staining apparent exclusively with vIRF-1 co-expression (Fig.…”

The secret life of Bcl-2: Apoptosis-independent inhibition of DNA repair by Bcl-2 family members

“…Bid has two phosphorylation sites for ATM in mice (serines 61 and 78) and one in humans (serine 78), which are necessary for the cell cycle checkpoint. Bid is exported from the nucleus after its phosphorylation by ATM [59]. However, the established role of Bid in the DNA damage response has been challenged [60,61].…”

Bid can mediate a pro-apoptotic response to etoposide and ionizing radiation without cleavage in its unstructured loop and in the absence of p53

“…It has been proposed that Bid is activated in the nucleus to mediate DNA-damage-induced apoptosis, on the basis of several observations. First, full-length Bid was seen to translocate from the cytosol to the nucleus and from the nucleus to the cytosol in response to DNA-damaging anti-cancer drugs in HeLa cells (Oberkovitz et al , 2007; Anguissola et al , 2009). In addition, enforced nuclear retention of Bid through fusion of Bid with a nuclear localization signal inhibited etoposide-induced apoptosis (Oberkovitz et al , 2007).…”