Abstract
Members of the Toll-like receptor (TLR) family are essential players in activating the host innate immune response against infectious microorganisms. All TLRs signal through Toll/interleukin 1 receptor domain-containing adapter proteins. MyD88 adapter-like (Mal) is one such adapter that specifically is involved in TLR2 and TLR4 signaling. When overexpressed we have found that Mal undergoes tyrosine phosphorylation. Three possible phospho-accepting tyrosines were identified at positions 86, 106, and 187, and two mutant forms of Mal in which tyrosines 86 and 187 were mutated to phenylalanine acted as dominant negative inhibitors of NF-kappaB activation by lipopolysaccharide (LPS). Activation of THP-1 monocytic cells with the TLR4 agonist LPS and the TLR2 agonist macrophage-activating lipopeptide-2 induced phosphorylation of Mal on tyrosine residues. We found that the Bruton's tyrosine kinase (Btk) inhibitor LFM-A13 could block the endogenous phosphorylation of Mal on tyrosine in cells treated with macrophage-activating lipopeptide-2 or LPS. Furthermore, Btk immunoprecipitated from THP-1 cells activated by LPS could phosphorylate Mal. Our study therefore provides the first demonstration of the key role of Mal phosphorylation on tyrosine during signaling by TLR2 and TLR4 and identifies a novel function for Btk as the kinase involved.
Highlights
The primary role of the Toll-like receptor (TLR)2 family is to recognize and signal the influx of invading pathogens, thereby activating the host innate immune response
We found that the Bruton’s tyrosine kinase (Btk) inhibitor LFM-A13 could block the endogenous phosphorylation of MyD88 adapter-like (Mal) on tyrosine in cells treated with macrophage-activating lipopeptide-2 or LPS
Prompt activation of the TLR signaling pathway has proven to be vital in the recognition of invading pathogens and in eliciting the host innate immune response
Summary
The primary role of the Toll-like receptor (TLR) family is to recognize and signal the influx of invading pathogens, thereby activating the host innate immune response. Once the TLRs are activated by their respective agonists, receptor dimerization via the TIR domain is thought to occur This leads to recruitment of cytosolic TIR domain-containing adapter proteins [1]. TRAM and TRIF recruitment leads to activation of the kinase receptor-interacting protein (RIP)-1, which engages with the IB kinase complex [17]. Both TRAM and TRIF can interact with TRAF-associated NF-B activator (TANK) binding kinase-1, which phosphorylates and activates interferon regulatory factor 3 [18, 19]. It has been shown that Btk interacts with the TIR domains of TLRs 4, 6, 8, and 9 and was found to associate with MyD88, Mal, and IL-1 receptor-associated kinase 1 [22]. Mal is the first substrate for Btk in TLR signaling to be identified, its phosphorylation by Btk being an important mechanism in signaling by TLR2 and TLR4
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