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J. Biol. Chem., Vol. 283, Issue 27, 18591-18600, July 4, 2008

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The Scaffold MyD88 Acts to Couple Protein Kinase C to Toll-like Receptors^*

Amir Faisal, Adrian Saurin, Bernard Gregory, Brian Foxwell, and Peter J. Parker^¶1

From the Protein Phosphorylation Laboratory, London Research Institute, Cancer Research UK, London WC2A 3PX, Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, London SW7 2AZ, and ^¶King's College London Division of Cancer Studies, Guy's Hospital, London SE1 1UL, United Kingdom

Mice lacking protein kinase C (PKC) are hypersensitive to both Gram-positive and Gram-negative bacterial infections; however, the mechanism of PKC coupling to the Toll-like receptors (TLRs), responsible for pathogen detection, is poorly understood. Here we sought to investigate the mechanism of PKC involvement in TLR signaling and found that PKC 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 PKC binding to 14-3-3β. LPS-induced PKC 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 PKC 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/PKC association. By contrast, MyD88 overexpression in 293 cells resulted in constitutive phosphorylation of PKC. A general role for MyD88 was evidenced by the finding that phosphorylation of PKC 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 PKC at these two sites is required for TLR4- and TLR2-induced NFB reporter activation and IB degradation in reconstituted PKC^–/– cells. This study therefore identifies the scaffold protein MyD88 as the link coupling TLRs to PKC recruitment, phosphorylation, and downstream signaling.

Received for publication, December 19, 2007 , and in revised form, April 30, 2008.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 44 Lincoln's Inn Fields, WC2A 3PX London, UK. Tel.: 44-2072693513; Fax: 44-2072693094; E-mail: peter.parker{at}cancer.org.uk.

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