ZAK: a MAP3Kinase that transduces Shiga toxin‐ and ricin‐induced proinflammatory cytokine expression

Summary Shiga toxins (Stxs) and ricin initiate damage to host cells by cleaving a single adenine residue on the α‐sarcin loop of the 28S ribosomal RNA. This molecular insult results in a cascade of intracellular events termed the ribotoxic stress response (RSR). Although Stxs and ricin have been shown to cause the RSR, the mitogen‐activated protein kinase kinase kinase (MAP3K) that transduces the signal from intoxicated ribosomes to activate SAPKinases has remained elusive. We show in vitro that DHP‐2 (7‐[3‐fluoro‐4‐aminophenyl‐(4‐(2‐pyridin‐2‐yl‐5,6‐dihydro‐4H‐pyrrolo[1,2‐b]pyrazol‐3‐yl))]‐quinoline), a zipper sterile‐α‐motif kinase (ZAK)‐specific inhibitor, blocks Stx2/ricin‐induced SAPKinase activation. Treatment of cells with DHP‐2 also blocks Stx2/ricin‐mediated upregulation of the proinflammatory cytokine interleukin‐8 and results in a modest but statistically significant improvement in cell viability following Stx2/ricin treatment. Finally we show that siRNA directed against the N‐terminus of ZAK diminishes Stx2/Ricin‐induced SAPKinase activation. Together, these data demonstrate that a ZAK isoform(s) is the MAP3Kinase that transduces the RSR. Therefore, ZAKα and/or β isoforms may act as potential therapeutic target(s) for treating Stx/ricin‐associated illnesses. Furthermore, a small molecule inhibitor like DHP‐2 may prove valuable in preventing the Stx/ricin‐induced proinflammatory and/or apoptotic effects that are thought to contribute to pathogenesis by Stx‐producing Escherichia coli and ricin.