Bacillus anthracis Edema Toxin Activates Nuclear Glycogen Synthase Kinase 3β

Abstract

Bacillus anthracis edema toxin (ET) generates high levels of cyclic AMP and impacts a complex network of signaling pathways in targeted cells. In the current study, we sought to identify kinase signaling pathways modulated by ET to better understand how this toxin alters cell physiology. Using a panel of small-molecule inhibitors of mammalian kinases, we found that inhibitors of glycogen synthase kinase 3 beta (GSK-3␤) protected cells from ET-induced changes in the cell cycle. GSK-3␤ inhibitors prevented declines in cellular levels of cyclin D1 and c-Jun following treatment of macrophages with ET. Strikingly, cell fractionation experiments and confocal immunofluorescence microscopy revealed that ET activates a compartmentalized pool of GSK-3␤ residing in the nuclei, but not in the cytoplasm, of macrophages. To investigate the outcome of this event, we examined the cellular location and activation state of ␤-catenin, a critical substrate of GSK-3␤, and found that the protein was inactivated within the nucleus following intoxication with ET. To determine if ET could overcome the effects of stimuli that inactivate GSK-3␤, we examined the impact of the toxin on the Wnt signaling pathway. The results of these experiments revealed that by targeting GSK-3␤ residing in the nucleus, ET circumvents the upstream cytoplasmic inactivation of GSK-3␤, which occurs following exposure to Wnt-3A. These findings suggest ET arrests the cell cycle by a mechanism involving activation of GSK-3␤ residing in the nucleus, and by using this novel mechanism of intoxication, ET avoids cellular systems that would otherwise reverse the effects of the toxin.Bacillus anthracis edema factor (EF) is a calcium-and calmodulin-dependent adenylate cyclase that generates high levels of cyclic AMP (cAMP) after delivery into the cell by protective antigen (PA) (20). The three-dimensional structure of EF has been resolved, and its catalytic mechanism is well understood (10,15,16,32). Edema toxin (ET), the combination of PA and EF, suppresses immune responses during the development and progression of anthrax disease (3,26,36). ET has also been found to sensitize mice to anthrax lethal toxin (12), increase the expression of anthrax toxin receptors in monocytic cells (22), and reduce cell viability (11, 39). Thus, while EF generates a common second messenger, cAMP, the toxin does so in a way that disrupts normal cellular activities.ET generates supraphysiological levels of cAMP, which is thought to accumulate in the perinuclear region of the cell (7,20). In the original description of EF as an adenylate cyclase, Leppla found that treatment of CHO K1A cells with ET increased levels of cAMP by approximately 200-fold (20). In a more recent study by Dal Molin et al., EF was shown to be delivered into the cell via a late endosome pathway, and by remaining associated with this compartment, the toxin generates cAMP in the perinuclear region, with decreasing gradients of cAMP radiating to the periphery of the cell (7). These observations support a toxicity model in...

Clostridium difficile Toxin A Attenuates Wnt/β-Catenin Signaling in Intestinal Epithelial Cells

“…Additionally, Bacillus anthracis edema toxin inhibits the Wnt/␤-catenin pathway by protein kinase A induction. This reduces the levels of phosphorylated GSK3␤, allowing it to phosphorylate, and thereby inactivate, ␤-catenin and preventing it from binding to the Tcf/Lef transcription factor (44).…”

Adenomatous Polyposis Coli Protein Associates with C/EBP β and Increases Bacillus anthracis Edema Toxin-stimulated Gene Expression in Macrophages

“…In previously published work from our group, ET-exposed macrophages were found to down-regulate ␤-catenin-stimulated transcription because of the activation of nuclear GSK-3␤ and subsequent phosphorylation of ␤-catenin (12). Because ET modifies GSK-3 and ␤-catenin activity, we hypothesized that these proteins or associated proteins could interact with a well defined cAMP responsive pathway such as the CREB-C/EBP ␤ pathway.…”

Glycogen Synthase Kinase 3 Activation Is Important for Anthrax Edema Toxin-Induced Dendritic Cell Maturation and Anthrax Toxin Receptor 2 Expression in Macrophages

“…Although GSK-3 is commonly considered a constitutively active kinase, it is inactivated by phosphorylation at Ser-9 in the presence of serum (14) and is predominantly inactive in primary human macrophages (17). Interestingly, LPS treatment of macrophages results in further inactivation of GSK-3 (30), while treatment with ET results in activation of the nuclear pool of GSK-3 (22). Therefore, although MDDC activation and migration induced by LPS and ET appear similar in many respects (Fig.…”

“…Consistent with a role for CREB in these processes, ET-induced migration of macrophages requires CREB-dependent expression of syndecan-1 (20), and consensus cAMP response elements are present upstream of the ANTXR genes (51). Because ET activates GSK-3␤ and CREB in a PKA-dependent manner in macrophages (22), we hypothesized that ET-induced ANTXR expression (29) and modulation of MDDC maturation and migration are GSK-3␤ and CREB dependent. Here we test this hypothesis and investigate the role of GSK-3 and CREB in ET-mediated responses in monocyte-derived cells.…”

“…Two isoforms of GSK-3 exist, GSK-3␣ and GSK-3␤; these are highly homologous in the kinase domain, although little is known about their distinct contributions to cell physiology (19). We recently demonstrated that ET activates the nuclear pools of GSK-3␤ in a PKA-dependent manner (22). Targeting of the nuclear pools of GSK-3␤ by ET bypasses the upstream cytoplasmic inactivation of this kinase, suggesting an additional mechanism by which ET may alter host cell function.…”