Protein-tyrosine Kinase Activation Is Required for Lipopolysaccharide Induction of Interleukin 1β and NFκB Activation, but Not NFκB Nuclear Translocation

131

102

Bruton's tyrosine kinase (Btk) has recently been shown to participate in the induction of nuclear factor B (NFB)-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 NFB-dependent reporter gene expression but not IB␣ degradation. Transient transfection of HEK293 cells with Btk had no effect on NFB-dependent reporter gene expression but strongly promoted transactivation of a reporter gene by a p65-Gal4 fusion protein. IB␣ 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 NFB.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). In B-cells, activation of the B-cell receptor leads to Btk being rapidly recruited to the plasma membrane where it becomes phosphorylated and activated. Btk is required for normal B-cell development (2) and the gene encoding Btk was first identified as the mutated gene in X-linked agammaglobulinemia (XLA) in humans, an immune disease characterized by a lack of circulating B lymphocytes and an absence of Igs of all classes (3). Mutations in the Btk gene also result in the less severe Xlinked immunodeficiency (Xid) in mice. 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, thus inactivating it. The PH domain is important in the process of Btk activation, since it localizes Btk to the membrane through its interaction with phosphatidylinositol triphosphate (PIP 3 ), which is generated by phosphatidylinositol 3-kinase (PI3K).Due to the major phenotype of Btk deficiency being impaired B-cell development and function, the main focus of interest in Btk has centered around the B-cell. However, several studies have provided a more general role for Btk in immune regulation. Studies in Xid peritoneal macrophages have shown reduced responses to lipopolysaccharide (LPS) stimulation, with tumor necrosis factor ␣ (TNF␣) and IL-1␤ production decreased and macrophage effector functions impaired (5). We have shown that Btk is recruited to the LPS receptor Toll-like receptor-4 (TLR4) where it is activated and is required f...

Section: Discussionmentioning

confidence: 49%

Bruton's Tyrosine Kinase Is Involved in p65-mediated Transactivation and Phosphorylation of p65 on Serine 536 during NFκB Activation by Lipopolysaccharide

Doyle

Jefferies²,

O’Neill³

2005

131

102

“…Whether these phosphorylation events have the same functional outcome or whether they may lead to distinct functions is presently unclear. Recent data from our laboratory (30) and others (31) demonstrate that several inducers can control the transcriptional function of NF-B, independent of induced nuclear translocation.…”

Section: Discussionmentioning

confidence: 99%

Activation of Nuclear Factor-κB-dependent Transcription by Tumor Necrosis Factor-α Is Mediated through Phosphorylation of RelA/p65 on Serine 529

Wang

Baldwin

1998

350

271

Nuclear factor-B (NF-B) is an essential transcription factor in the control of expression of genes involved in immune and inflammatory responses. In unstimulated cells, NF-B complexes are sequestered in the cytoplasm through interactions with IB␣ and other IB proteins. Extracellular stimuli that activate NF-B, such as tumor necrosis factor ␣ (TNF␣), cause rapid phosphorylation of IB␣ at serines 32 and 36. The inducible phosphorylation of IB␣ is followed by its ubiquitination and degradation, allowing NF-B complexes to translocate into the nucleus and to activate gene expression. Previously, it has been shown that TNF␣ as well as other stimuli also lead to the phosphorylation of the RelA/p65 subunit of NF-B. In this report, we demonstrate that the TNF␣-induced phosphorylation of the RelA/p65 subunit occurs on serine 529, which is in the C-terminal (TA1) transactivation domain. Accordingly, the TNF␣-induced phosphorylation of Rel/p65 increases NF-B transcriptional activity but does not affect nuclear translocation or DNA binding affinity. NF-B1 /Rel transcription factors are key regulators of transcription of a variety of genes involved in immune and inflammatory responses, growth, differentiation, development, and cell death (1-3). NF-B was originally identified as a nuclear factor that binds to the enhancer element of the immunoglobulin kappa light chain gene (4). To date, eight members of the NF-B/Rel proteins have been cloned and characterized. They are c-Rel, NF-B1 (p50/p105), NF-B2 (p52/p100), RelA (p65), RelB, and the Drosophila proteins Dorsal, Dif, and Relish (2). These proteins can form homo-or heterodimers through their N-terminal Rel homology domains, which also function in DNA binding and interaction with inhibitor proteins known as IBs. The prototypical, inducible NF-B complex is a heterodimer containing p50 and p65. The C-terminal region of p65 contains a potent transactivation domain that is lacking in p50 (1, 2).In most cells, NF-B is inactive due to its cytoplasmic sequestration through interactions with inhibitor proteins IBs (1, 2). The activation of NF-B by a wide variety of stimuli such as mitogens, cytokines, bacterial lipopolysaccharide, viral infection, double-stranded RNA, and UV light involves the dissociation of NF-B from IB, allowing the nuclear translocation of the transcription factor (1). There are seven members of the IB family identified: IB␣, IB␤, IB␥, Bcl3, p105, p100, and IB⑀ as well as Drosophila IB protein cactus (2, 6, 7), each of which contains multiple copies of the ankyrin repeat.Stimulation of cells with inducers such as TNF␣ leads to rapid phosphorylation, ubiquitination, and degradation of IB␣. NF-B is therefore released and translocates into the nucleus to activate the expression of target genes (2). Early studies implicated IB phosphorylation as a crucial step for NF-B activation, and much attention has been focused on the signal transduction pathway involved with induced phosphorylation of IB. Recently, it was shown that two highly related serine kinases, IKK␣ and I...

“…Dominant negative mutants of these small GTPases were generated by overlap extension as described elsewhere (31). Cdc42N17 was generated by replacing Thr 17 with Asn employing the overlapping primers 5Ј-GTAAAAACTGTCTCCTGATATCCTAC and 5Ј-GATATCAGGAGA-CAGTTTTTACCAACAGCACC, Rac1N17 was generated by replacing Thr 17 with Asn using the primers 5Ј-CTGTAGGTAAAAACTGCCTACT-GATC and 5Ј-TGATCAGTAGGCAGTTTTTACCTACAGCTCCG, and RhoAN17 was generated by replacing Thr 19 with Asn using the primers 5Ј-GTGGAAAGAACTGCTTGCTCATAGTCTTC and 5Ј-ATGAGCAAG-CAGTTCTTTCCACAGGCTCCATC (the underlined base triplet indicates the mutated amino acid). The Raf-1 dominant negative mutant encompassing the first 259 amino acids encoding the regulatory domain (32) was generated by polymerase chain reaction using full-length human Raf-1 (ATCC 41050) as template.…”

Section: Methodsmentioning

confidence: 99%

“…Similarly, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) has been shown to decrease TNF-␣-induced NF-B activity and interleukin-6 expression (16). More recently, tyrosine phosphorylation has been shown to be essential for NF-B activity in bacterial lipopolysaccharide (LPS)-induced monocytic THP1 cells (17). Regulation of NF-B activity by PKC, extracellular signal-regulated kinase 1, p38 MAPK, or tyrosine phosphorylation acts downstream of IB without interfering with NF-B nuclear translocation and DNA binding.…”

mentioning

confidence: 99%

Regulation of NF-κB RelA Phosphorylation and Transcriptional Activity by p21 and Protein Kinase Cζ in Primary Endothelial Cells

Anrather

Csizmadia

Soares

et al. 1999

181

163

The activity of the transcription factor NF-B is thought to be regulated mainly through cytoplasmic retention by IB molecules. Here we present evidence of a second mechanism of regulation acting on NF-B after release from IB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-B through a pathway involving activation of protein kinase C (PKC) and p21 ras . We show that transcriptional activity of RelA is dependent on phosphorylation of the N-terminal Rel homology domain but not the C-terminal transactivation domain. Inhibition of phosphorylation by dominant negative mutants of PKC or p21 ras results in loss of RelA transcriptional activity without interfering with DNA binding. Raf/MEK, small GTPases, phosphatidylinositol 3-kinase, and stress-activated protein kinase pathways are not involved in this mechanism of regulation.The NF-B/Rel family of dimeric transcription factors is involved in the immediate early transcription, i.e. independent of protein synthesis, of a large array of genes induced by mitogenic or pathogen-associated stimuli. In its active form, NF-B is a nuclear homo-or heterodimeric complex of a number of different Rel family members. The canonical and most abundant form of NF-B is composed of a 50-kDa (p50, or NFB1) and a 65-kDa (p65, or RelA) subunit. Both subunits can form homodimers as well as heterodimers with other members of the Rel family i.e. c-Rel (Rel), p52 (NFB2), and RelB (1). All members of the Rel family exhibit extensive sequence similarity in their N-terminal region referred to as the Rel homology domain (RHD) 1 responsible for DNA binding and formation of Rel dimers. Only RelA, Rel, and RelB carry a transcription activating domain, and thus only dimers containing one of these proteins activate the transcription of NF-B-dependent genes efficiently. With respect to transcription activation, the RelA subunit appears to have the highest activity.In most unstimulated cells, NF-B is constitutively retained in the cytoplasm by inhibitory proteins of the IB family, namely IB␣, IB␤, IB␥, p100, p105, and IB⑀ (2). Formation of NF-B⅐IB complexes masks the nuclear localization signal sequence present in NF-B molecules and thus prevents their nuclear translocation. One of the key events in the activation of NF-B is the liberation of functional NF-B dimers from IB, which results in the translocation of NF-B to the nucleus. Cytoplasmic release of NF-B dimers involves site-specific phosphorylation of IB by kinases of the IB signalosome (3-6), ubiquitination (7), and subsequent proteolytic degradation by the 26 S proteasome pathway (8). Upon nuclear import and binding to specific decameric recognition motifs, which are reflected by the consensus GGGRNNYYCC (where R represents A or G and Y represents C or T), NF-B dimers function as transcriptional activators. IB␣ (9), IB␤, and p105 (10) have been implicated in the inhibition of DNA binding of NF-B complexes. However, there have been several reports showing that NF-B transcriptional activity can be blocked withou...