Abstract
Bruton's tyrosine kinase (Btk) has recently been shown to participate in the induction of nuclear factor kappaB (NFkappaB)-dependent gene expression by the lipopolysaccharide (LPS) receptor Toll-like receptor-4 (TLR4). In this study we have examined the mechanism whereby Btk participates in this response. Treatment of the murine monocytic cell line Raw264.7 with LFM-A13, a specific Btk inhibitor, blocked LPS-induced NFkappaB-dependent reporter gene expression but not IkappaB alpha degradation. Transient transfection of HEK293 cells with Btk had no effect on NFkappaB-dependent reporter gene expression but strongly promoted transactivation of a reporter gene by a p65-Gal4 fusion protein. IkappaB alpha degradation activated by LPS was intact in macrophages from X-linked immunodeficiency (Xid) mice, which contain inactive Btk. Transfection of cells with a dominant negative form of Btk (BtkK430R) inhibited LPS-driven p65 mediated transactivation. Additionally LFM-A13 impaired phosphorylation of serine 536 on p65 induced by LPS in HEK293-TLR4 cells, and in Xid macrophages this response was impaired. This study therefore reveals a novel function for Btk. It is required for the signaling pathway activated by TLR4, which culminates in phosphorylation of p65 on serine 536 promoting transactivation by NFkappaB.
Highlights
Bruton’s tyrosine kinase (Btk)1 is a member of the Tec family of non-receptor tyrosine kinases and is found in all cells of the hematopoietic lineage except plasma cells and T-cells [1]
Btk Is Involved in the Activation of nuclear factor B (NFB)-linked Reporter Gene Expression but Not IB␣ Degradation Activated by LPS—To gain insight into the function of Btk in NFB regulation by Toll-like receptor-4 (TLR4) we examined the effect of a kinase inactive form of Btk (Btk(K430R)) and the Btk-selective kinase inhibitor LFM-A13, on the induction of an NFB-linked reporter gene comprising five NFB sites linked to luciferase
There was no impairment of IB␣ degradation in X-linked immunodeficiency (Xid) peritoneal macrophages when compared with normal peritoneal macrophages, with complete degradation occurring from 15 min LPS stimulation (Fig. 1e, lane 3)
Summary
Bruton’s tyrosine kinase (Btk) is a member of the Tec family of non-receptor tyrosine kinases and is found in all cells of the hematopoietic lineage except plasma cells and T-cells [1]. Various mutations have been identified as being responsible for the XLA phenotype, whereas the mutation in Xid mice has been mapped to arginine 28 (R28C) in the PH domain [4] This prevents Btk associating with the membrane, inactivating it. MyD88 and Mal engage with IL-1 receptor-associated kinase (IRAK)-4 and IRAK-1 leading to activation of the IB kinase (IKK) complex via Traf-6 [9, 10]. In this pathway IB␣ becomes phosphorylated by the IKK complex and is subsequently targeted for ubiquitination and degradation [11, 12]. Our study elucidates the role of Btk in TLR4 signaling and implicates Btk for the first time in the transactivation pathway of NFB
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