Abstract
Anthrax is a life-threatening disease caused by infection with Bacillus anthracis, which expresses lethal factor and the receptor-binding protective antigen. These two proteins combine to form anthrax lethal toxin (LT), whose proximal targets are mitogen-activated kinase kinases (MKKs). However, the downstream mediators of LT toxicity remain elusive. Here we report that LT exposure rapidly reduces the levels of c-Jun, a key regulator of cell proliferation and survival. Blockade of proteasome-dependent protein degradation with the 26S proteasome inhibitor MG132 largely restored c-Jun protein levels, suggesting that LT promotes degradation of c-Jun protein. Using the MKK1/2 inhibitor U0126, we further show that MKK1/2-Erk1/2 pathway inactivation similarly reduces c-Jun protein, which was also restored by MG132 pre-exposure. Interestingly, c-Jun protein rebounded to normal levels 4 h following U0126 exposure but not after LT exposure. The restoration of c-Jun in U0126-exposed cells was associated with increased c-Jun mRNA levels and was blocked by inactivation of the JNK1/2 signaling pathway. These results indicate that LT reduces c-Jun both by promoting c-Jun protein degradation via inactivation of MKK1/2-Erk1/2 signaling and by blocking c-Jun gene transcription via inactivation of MKK4-JNK1/2 signaling. In line with the known functions of c-Jun, LT also inhibited cell proliferation. Ectopic expression of LT-resistant MKK2 and MKK4 variants partially restored Erk1/2 and JNK1/2 signaling in LT-exposed cells, enabling the cells to maintain relatively normal c-Jun protein levels and cell proliferation. Taken together, these findings indicate that LT reduces c-Jun protein levels via two distinct mechanisms, thereby inhibiting critical cell functions, including cellular proliferation.
Highlights
Anthrax is a life-threatening disease caused by infection with Bacillus anthracis, which expresses lethal factor and the receptor-binding protective antigen
Using the MKK1/2 inhibitor U0126, we further show that MKK1/2–Erk1/2 pathway inactivation reduces c-Jun protein, which was restored by MG132 pre-exposure
lethal factor (LF) cleavage of mitogen-activated kinase kinases (MKKs) at their docking sites (D sites) disrupts the activation of MAPKs, including Erk1/2, which are activated by MKK1 and MKK2; p38 MAPKs, which are activated by MKK3 and MKK6; and JNKs, which are activated by MKK4 and MKK7 [7, 12,13,14]
Summary
A previous study from our laboratory showed that anthrax LT inhibits the proliferation of T cells, which correlates with reduced AP-1 activity in LT-treated T cells [15]. Western blotting showed that inhibition of protein degradation largely corrected the LT-induced c-Jun reduction in both HepG2 and Hepa1c1c7 cells (Fig. 3A), suggesting that LT treatment promotes c-Jun protein degradation. To dissect the signaling pathways underlying the LT-dependent reduction in c-Jun protein levels, we treated cells with specific inhibitors that inhibit MKK1/2–Erk1/2 (U0126), JNK1/2 (SP600125), or p38 (SB203580). Inactivation of MKK1/2–Erk1/2 by U0126 induced a rapid reduction of c-Jun, which returned to normal levels by 3 h (Fig. 4C) This restoration did not occur in LT-treated cells. In line with the restoration of Erk1/2 and JNK1/2 function, cells ectopically dually expressing the LFresistant MKK2 (MKK2CR) and MKK7-4 showed higher levels of c-Jun protein after LT treatment compared with controls (Fig. 7B). These data support the conclusion that LT inhibits c-Jun by targeting both the MKK1/2–Erk1/2 and MKK4/7–JNK1/2 pathways, leading to arrest of cell proliferation
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