A Toll-Like Receptor-Responsive Kinase, Protein Kinase R, Is Inactivated in Endotoxin Tolerance through Differential K63/K48 Ubiquitination

Abstract

ABSTRACT Overwhelming inflammation triggered by systemic infection in bacterial sepsis contributes to the pathology of this condition. Toll-like receptors (TLRs) are important in early septic inflammation. As a safeguard, the innate immune system has evolved to counter excessive inflammation through the induction of “tolerance.” In endotoxin tolerance, TLR signaling is inhibited and/or attenuated by multiple mechanisms that mitigate the ability of lipopolysaccharide (LPS) to activate critical kinases through TLR4. Here, we describe a novel mechanism. Protein kinase R (PKR), a kinase normally activated by a subset of TLRs, is rendered unresponsive to LPS in endotoxin-tolerized cells. In its naive state, PKR is subject to K63-linked ubiquitination (Ub), followed by K48-linked Ub, in response to LPS. In tolerance, the kinetics of this differential Ub is altered, resulting in a predominance of K48-linked chains, concomitant with a loss of PKR activation. These findings provide a novel mechanism by which a TLR-responsive kinase may be rendered inactive in tolerance. IMPORTANCE “Endotoxin tolerance” is a period of transient unresponsiveness to the lipopolysaccharide (LPS) outer membrane component of Gram-negative bacteria that is induced by prior exposure to LPS through Toll-like receptor 4 (TLR4). The loss of LPS-inducible cytokine production by macrophages from patients who have experienced Gram-negative sepsis is well documented, and the increased susceptibility of such patients to reinfection has been attributed to the development of endotoxin tolerance. Multiple mechanisms have been proffered to account for this attenuated response. Using the LPS-responsive kinase protein kinase R (PKR), we have identified differential K48 versus K63 ubiquitination as an additional molecular mechanism by which signal-transducing elements may be inactivated in a state of endotoxin tolerance. This work is highly significant because it links recent discoveries concerning the important role of ubiquitination of signaling molecules in regulating TLR signaling with the loss of LPS responsiveness in tolerance.