Activation of the nuclear factor-κB (NF-κB) signal transduction pathway occurs in response to a wide range of stimuli and results in nuclear accumulation of NF-κB transcription factors causing changes in the expression of target genes involved in innate and adaptive immunity, inflammation, cell survival, hematopoiesis, and lymphoid development (www.nf-kb.org
). Identification of human disease mutations in genes encoding NF-κB signaling components and the varied phenotypes seen in NF-κB knockout mouse models have highlighted the importance of precise regulation of the NF-κB pathway (Courtois and Gilmore, 2006
; Gerondakis et al., 2006
). In this review, we describe alternative splicing events that occur in genes involved in mouse and human NF-κB signal transduction pathways, and discuss the functional significance these splice variants may have for regulation of NF-κB.
Initiation of the NF-κB signaling cascade often begins with cellular recognition of extracellular signals. A large number of ligand-receptor interactions can lead to activation of NF-κB. Two of the most studied receptor families involved in recognition of NF-κB activating signals are the tumor necrosis factor receptor (TNFR) family and the Toll/interleukin-1 (IL-1) (TIR) family ().
Shown are the TNFR and TLR/IL-1R (TIR) signaling pathways leading to activation of NF-κB
Tumor necrosis factor (TNF) is a cytokine employed by hematopeitic cells for cell-cell communication and immune responses. TNF is generated by many different cell types, most notably macrophages and monocytes in response to cellular stresses including infection and inflammation. There are two TNF receptors: TNFR1 (p55) which is expressed in a broad range of cell types and the more cell type-specific TNFR2 (p75). The TNFR1 signaling pathway to NF-κB () is known in more detail than the TNFR2 pathway (Bradley, 2008
; Wertz and Dixit, 2008
). Interaction of TNF with TNFR1 leads to receptor trimerization. Upon trimerization, TNFR1 undergoes a conformational change enabling its cytoplasmic domain to interact with the TNFRSF1A-associated via death domain (TRADD) adaptor protein. In the classical NF-κB signaling pathway, TRADD recruits TNFR-associated factor (TRAF) proteins via TRAF’s C-terminal domain to the receptor complex. TRAF then ubiquitinates the TNF receptor interacting protein (RIP1), which enables the inhibitor of κB kinase (IKK) complex to be recruited to the TNFR complex through binding of the IKK subunit NEMO to ubiquitinated RIP. Activated IKK then phosphorylates inhibitor of κB (IκB), signaling IκB for degradation, which enables NF-κB nuclear localization and activation of pro-survival target genes. The pathway is turned off through the action of deqbiquitinases such as A20 and CYLD, and by activation of the IκBα gene by NF-κB.
The Toll/interleukin-1 (IL-1) receptor family (TIR) consists of Toll-like receptors (TLRs) and IL-1 receptors (IL-1Rs). While the extracellular sequences of the Toll-like and IL-1R receptors differ, their cytoplasmic domains are related by the presence of TIR domains (Hayden et al., 2006
). Like TNF, IL-1 is a pro-inflammatory cytokine produced by monocytes and macrophages in response to cell stress and infection. Signaling by IL-1 involves binding to the IL-1R and heterodimerization of IL-1R with the IL-1 receptor accessory protein (IL-1RAcP) (Huang et al., 1997
). Signaling through TLRs occurs via recognition of a wide range of ligands that contain pathogen-associated molecular patterns (PAMPs), thus providing a means for specific TLR recognition of both gram-negative and gram-positive bacteria, fungi, and viruses. For example, lipopolysaccharide (LPS) is a component of gram-negative bacterial membranes that signals pro-inflammatory cellular responses through TLR4.
Upon ligand binding and multimerization of the IL-1R and TLR proteins, their cytoplasmic TIR domains recruit the same protein components. In the most common pathway, adaptor proteins including myeloid differentiation primary response gene (88) (MYD88), TIR-domain-containing adapter-inducing interferon-β (TRIF), and TIR domain-containing adaptor protein (TIRAP) are recruited to the receptor complex via the receptor’s TIR domains. MyD88 associates with interleukin-1 receptor-associated kinase 1 (IRAK1) through the adaptor Tollip. IRAK4 then phosphorylates IRAK1, triggering IRAK1 autophosphorylation. Phosphorylated IRAK1 dissociates from the receptor complex and binds TRAF6. The IRAK1/TRAF6 complex activates the TAK1/TAB1 complex, which in turn activates IKK and thus, induces NF-κB. Nuclear NF-κB transcription factors then regulate target genes involved in cellular defense against the inducing immunological or inflammatory challenge.
The IKK complex contains the two kinase subunits, IKKα and IKKβ,and the regulatory NEMO (aka IKKγ) subunit. In the classical NF-κB signaling pathway, the activated IKKβ subunit phosphorylates the IκB protein, signaling it for ubiquitination and proteasomal degradation. The alternative NF-κB pathway proceeds via IKKα-mediated phosphorylation of C-terminal IκB-like sequences in p100. The phosphorylated C-terminal sequences of p100 are then processed by the proteasome, yielding the mature N-terminal p52 DNA-binding subunit. In both pathways, NF-κB dimers, now liberated from the inhibitory effects of IκB, then enter the nucleus and regulate transcription of specific target genes.
In addition to the primary IKK/IκB-mediated regulation of NF-κB, the activities of proteins in these signaling cascades undergo a multitude of post-translational modifications including phosphorylation, ubiquitination, sumoylation, S-nitrosylation, acetylation, and cysteine oxidization that can further impact NF-κB signaling (Perkins, 2006
). Alternative splicing is a critical cellular process that can generate a large array of mRNA transcripts and protein isoforms from a limited number of genes. Several studies have indicated that alternative splicing of human genes is pervasive, and estimates are that 30–75% of all human genes undergo alternative splicing (Mironov et al., 1999
; Brett et al., 2000
; Kan et al., 2001
; Modrek et al., 2001
; Johnson et al., 2003
). Alternative splicing can provide a means of changing protein functionality by deletion or insertion of protein components. Not surprisingly, these alternatively spliced protein isoforms often show protein-protein interactions, subcellular localization, stability, DNA binding, and enzymatic properties that differ from their normally transcribed/translated counterparts (López et al., 1995
; Stamm et al., 2004). Furthermore, changes in protein function due to alternative splicing have been implicated in a number of human diseases including cystic fibrosis, hemophilia, breast cancer, and prostate cancer (Cáceres et al., 2002
; Brinkman, 2004
; Skotheim and Nees, 2007
). In this review, we describe several alternative splicing events that may regulate NF-κB signaling.