Anthrax lethal toxin rapidly reduces c-Jun levels by inhibiting c-Jun gene transcription and promoting c-Jun protein degradation

Abstract

Anthrax is a life-threatening disease caused by infection with , 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.

Erk1/2 inactivation promotes a rapid redistribution of COP1 and degradation of COP1 substrates

“…In contrast to conditions in which MKK4/MKK7-JNK-c-Jun signaling is fully intact, LT cleaves MKK4, presenting a challenge to predicting the functional role of the Erk1/2-TPR-COP1-c-Jun pathway during anthrax infection. To overcome this hurdle, we used Hepa1c1c7 cells ectopically expressing an LT-resistant MKK7-4 fusion protein, designed to preserve JNK1/2 signaling in LT-treated Hepa1c1c7 cells (12). Consistent with our previous report, ectopic expression of this MKK7-4 fusion protein in LTtreated cells maintained activation of the JNK1/2 signaling pathway following LT treatment, and cells with concomitant COP1 deletion preserved higher levels of c-Jun protein at baseline and following LT treatment (Fig.…”

“…Our previous study showed that Erk1/2 inactivation following treatment with anthrax LT or the MEK1/2 inhibitor, U0126, induces a rapid degradation of c-Jun protein (12). To identify the ubiquitin E3 ligase that mediates Erk1/2 inactivation-induced c-Jun degradation, we investigated a panel of E3 ligases, previously reported to target c-Jun for proteasomal degradation.…”

“…We previously reported that anthrax LT treatment causes a rapid degradation of c-Jun protein via inactivation of the MKK1/2-Erk1/2 signal pathway (12), but the underlying mechanisms were unknown. c-Jun, a basic leucine zip (bZIP) transcription factor, is known to be degraded by the proteasome following ubiquitination mediated by ubiquitin E3 ligases including FBW7 (16), ITCH (15), MEKK1 (19), SAG/ROC2 (18), and COP1 (17,20,29).…”

“…Studies from our laboratory have revealed that LT reduces levels of the c-Jun transcription factor protein by promoting its degradation via inactivation of MKK1/2-Erk1/2 signaling and blocking its gene transcription via inactivation of the MKK4-JNK1/2 signaling pathway (12). c-Jun is a key member of the AP-1 transcription factor family, which regulates a myriad of cellular activities, including cellular proliferation, differentiation, survival, death, and tumorigenesis (13,14).…”

Erk1/2 Inactivation-Induced c-Jun Degradation Is Regulated by Protein Phosphatases, UBE2d3, and the C-Terminus of c-Jun

“…c-Jun is degraded by the ubiquitination-dependent proteasome pathway [ 3 ]. Studies from our laboratory have revealed that inactivation of the MKK1/2–Erk1/2 signaling pathway by chemical MKK1/2 inhibitors or anthrax lethal toxin (LT) reduces levels of the c-Jun protein rapidly by promoting its degradation via a COP1-dependent pathway [ 4 , 5 ].…”