The scaffold MyD88 acts to couple protein kinase Cε to toll-like receptors

Abstract

Mice lacking protein kinase Cε(PKCε) are hypersensitive to both Gram-positive and Gram-negative bacterial infections; however, the mechanism of PKCε coupling to the Toll-like receptors (TLRs), responsible for pathogen detection, is poorly understood. Here we sought to investigate the mechanism of PKCε involvement in TLR signaling and found that PKCε is recruited to TLR4 and phosphorylated on two recently identified sites in response to lipopolysaccharide (LPS) stimulation. Phosphorylation at both of these sites (Ser-346 and Ser-368) resulted in PKCε binding to 14-3-3β. LPS-induced PKCε phosphorylation, 14-3-3β binding, and recruitment to TLR4 were all dependent on expression of the scaffold protein MyD88. In mouse embryo fibroblasts and activated macrophages from MyD88 knock-out mice, LPS-stimulated PKCε phosphorylation was reduced compared with wild type cells. Acute knockdown of MyD88 in LPS-responsive 293 cells also resulted in complete loss of Ser-346 phosphorylation and TLR4/PKCε association. By contrast, MyD88 overexpression in 293 cells resulted in constitutive phosphorylation of PKCε. A general role for MyD88 was evidenced by the finding that phosphorylation of PKCε was induced by the activation of all TLRs tested that signal through MyD88 (i.e. all except TLR3) both in RAW cells and in primary human macrophages. Functionally, it is established that phosphorylation of PKCε at these two sites is required for TLR4- and TLR2-induced NFκB reporter activation and IκB degradation in reconstituted PKCε-/- cells. This study therefore identifies the scaffold protein MyD88 as the link coupling TLRs to PKCε recruitment, phosphorylation, and downstream signaling. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.