In addition to the activation of canonical NF-kB subunits, activation of SYK pathway by Dectin-1 leads to the induction of the non-canonical NF-kB pathway, which mediates the nuclear translocation of RELB-p52 dimers through the successive activation of NF-kB-inducing kinase (NIK) and IkB kinase-alpha (IKKa) (Geijtenbeek & Gringhuis 2009, Gringhuis et al. 2009). Noncanonical activity tends to build more slowly and remain sustained several hours longer than does the activation of canonical NF-kB. The noncanonical NF-kB pathway is characterized by the post-translational processing of NFKB2 (Nuclear factor NF-kappa-B) p100 subunit to the mature p52 subunit. This subsequently leads to nuclear translocation of p52:RELB (Transcription factor RelB) complexes to induce cytokine expression of some genes (C-C motif chemokine 17 (CCL17) and CCL22) and transcriptional repression of others (IL12B) (Gringhuis et al. 2009, Geijtenbeek & Gringhuis 2009, Plato et al. 2013).
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NFKB2 (also known as p100) is a member of the NF-kB family of transcription factors. It is synthesised as large precursor with an N-terminal RHD (Rel homology domain) and a C-terminal series of ankyrin repeats that masks the nuclear localization signal of NFKB2/p100 localising it to the cytosol. In resting cells, p100 is associated with RELB (Transcription factor RelB) in the cytosol. Upon cell stimulation, the IkB-like C terminus of p100 is proteolyzed, resulting in RELB-p52 dimers that translocate to the nucleus (Senftleben et al. 2001, Hayden & Ghosh 2004). IKKA (I kappa-B kinase alpha) does not associate directly with p100 but in the presence of NIK (NF-kB-inducing kinase), IKKA stably binds to p100. Serine residues 866 and 870 of p100 are essential for the recruitment of IKKA to p100 by NIK. This interaction is required for p100 phosphorylation and subsequent processing by IKKA (Xiao et al. 2001, 2004).
The catalytic activity of NF-kB-inducing kinase (NIK) is regulated by structural conformation rather than by phosphorylation events. Under normal conditions NIK may be present in an autoinhibited state in which its constitutively active kinase domain is shielded by the N-terminal inhibitory element and upon receptor induction NIK kinase domain adopts an active conformation, in agreement with its catalytic activity. This catalytically competent conformation is maintained by an N-terminal extension prior to the kinase domain rather than through a phosphorylation event (Liu et al. 2012). NIK, also known as MAP3K14 (MAPK kinase kinase 14), is a serine/threonine kinase in the MAP3K family (Malinin et al. 1997). In unstimulated cells NIK associates with a complex composed of TNF receptor-associated factor 2 (TRAF2), TRAF3 and cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2. This molecular interaction with TRAF-cIAP complex appears to target NIK for ubiquitination and proteasomal degradation. In response to receptor stimulation, TRAF2 or TRAF3, or both are targeted for proteasomal degradation by cIAP-mediated ubiquitination, which triggers the release and stabilization of NIK (Razani et al. 2010).
Accumulated NF-kB-inducing kinase (NIK) activates I kappa-B kinase alpha (IKKA) by directly phosphorylating the Ser176 and Ser180 with in the activation loop of IKKA. This phoshorylation is required for IKKA activity (Ling et al. 1998). Besides activating IKKA, NIK also serves as a docking molecule recruiting IKKA to p100 (Xiao et al. 2004).
Phosphorylated C-terminal serines 866, 870 and 872 in NFKB2 creates binding site for beta-TRCP (beta-transducin repeat-containing protein), the receptor subunit of a SCF-type of E3 ubiquitin ligase, SCF beta-TRCP (Liang et al. 2006). The SKP1-CUL1-F-box (SCF) ubiquitin E3 ligase superfamily is the largest family of cullin-RING ligases, with interchangeable F-box proteins orchestrating the trafficking proteins for ubiquitination and degradation (Weathington & Mallampalli 2013). Beta-TRCP is an F-box protein that contains two domains, an F-box motif that binds SKP1 and allows assembly into SKP1-CUL1 complexes and a second protein-protein interaction domain that interacts with phosphorylated serines in NFKB2 (Bai et al. 1996, Skowyra et al. 1997, Patton et al. 1998).
Ubiquitination of p100 is very specific. Lysine residue K855 has been identified as the anchoring site for ubiquitin and required for signaling mediated processing of p100 to p52. In the presence of SCF-beta-TRCP E3 ligase the ubiquitin (Ub) conjugated to E2 (E2-Ub thioester) is attached to p100 at K855 (Amir et al. 2004). Several rounds of ubiquitin conjugation can produce long chains of ubiquitin moieties (polyubiquitylation), the first of which is covalently bound to p100. At this point the polyubiquitylated p100 is committed to association with, and unfolding and processing by, the 26S proteasome (Pickart & Cohen 2004). Efficient ubiquitination of phosphorylated p100 by SCF-beta-TRCP E3 ligase also requires the presence of the components of the NEDD8 pathway: UBA3 (NEDD8-activating enzyme E1 catalytic subunit), UBC12 (NEDD8-conjugating enzyme Ubc12 (E2)), NEDD8 (Neural precursor cell expressed developmentally down-regulated protein 8). NEDD8 binds and promotes a conformational change in CUL1 that may result in efficient formation of an E2-E3 complex, thus stimulating SCF complexes activity (Kawakami et al. 2001, Morimoto et al. 2000, Read et al. 2000).
After being recruited into the NIK (NFkB-inducing kinase) complex, activated IKKA (I kappaB kinase alpha) phosphorylates serine residues 99, 108, 115, 123, 866, 870 and 872 located in both N- and C-terminal regions of NFKB2/p100. The phosphorylation of these specific serines is the prerequisite for ubiquitination and subsequent processing of p100. The C-terminal serine residues create a binding site for beta-TRCP (beta-transducinrepeat-containing protein), a ubiquitin E3 ligase (Xiao et al. 2001 & 2004, Liang et al. 2006).
Once polyubiquitinated, the precursor p100 undergoes 26S proteasome mediated processing to form the mature p52 NF-kB subuunit. Different from complete degradation of other IkB proteins, the proteasome-mediated degradation of p100 only leads to loss of their C-terminal ankyrin repeat regions, leaving intact N-termini, p52 respectively (Amir et al. 2004).
Following 26S-proteasomal processing, NFKB2 p52:RELB dimer is translocated from cytosol into the nucleus where it stimulates expression of target genes (Lin & Karin 2003). Dectin-1 induced RELB-p52 triggers the transcription of chemokines C-C motif chemokine 17 (CCL17) and CCL22 and repression of interleukin 12B (IL12B) transcription (Gringhuis et al. 2009).
Signal-induced NIK accumulation and activation is likely an essential step for triggering the downstream signalling events in non-canonical NF-kB pathway. Like other MAP3Ks NIK may also be triggered by its phosphorylation. It has been suggested that threonine (T559) phosphorylation of NIK is required for its activity. T559 phosphorylation of NIK is likely mediated by autophosphorylation, which could be triggered through NIK accumulation (Lin et al. 1998).
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DataNodes
NIK:p-176,S180-IKKA
dimer:p-7S-p100:SCF-beta-TRCPNIK:p-S176,180-IKKA
dimer:p-7S-p100:RELBNIK:p-S176,180-IKKA
dimer:p100:RELBNIK:p-S176,180-IKKA
dimerAnnotated Interactions
NIK:p-176,S180-IKKA
dimer:p-7S-p100:SCF-beta-TRCPNIK:p-176,S180-IKKA
dimer:p-7S-p100:SCF-beta-TRCPNIK:p-S176,180-IKKA
dimer:p-7S-p100:RELBNIK:p-S176,180-IKKA
dimer:p-7S-p100:RELBNIK:p-S176,180-IKKA
dimer:p100:RELBNIK:p-S176,180-IKKA
dimer:p100:RELBNIK:p-S176,180-IKKA
dimerNIK:p-S176,180-IKKA
dimerNIK:p-S176,180-IKKA
dimerEfficient ubiquitination of phosphorylated p100 by SCF-beta-TRCP E3 ligase also requires the presence of the components of the NEDD8 pathway: UBA3 (NEDD8-activating enzyme E1 catalytic subunit), UBC12 (NEDD8-conjugating enzyme Ubc12 (E2)), NEDD8 (Neural precursor cell expressed developmentally down-regulated protein 8). NEDD8 binds and promotes a conformational change in CUL1 that may result in efficient formation of an E2-E3 complex, thus stimulating SCF complexes activity (Kawakami et al. 2001, Morimoto et al. 2000, Read et al. 2000).