CHUK

A key step in NF-kB activation is the stimulation and translocation of PKC theta. The critical element that effects PKC theta activation is PI3K. This enzyme complex translocates to the plasma membrane by interacting with phospho-tyrosines on CD28 via its two SH2 domains located in p85 subunit. The p110 subunit of PI3K phosphorylates the inositol ring of PIP2 to generate PIP3 (steps 17-18). PIP3 may also be dephosphorylated by the phosphatase SHIP to generate PI-3,4-P2.

PIP3 and PI-3,4-P2 acts as binding sites to the PH domain of PKB/Akt and PDK1 (steps 19, 21 and 22). PKB is activated in response to PI3K stimulation by PDK1 (step 23). PDK1 has an essential role in regulating the activation of PKC theta and recruitment of CBM complex to the immune synapse. PKC theta is a member of novel class (DAG dependent, Ca++ independent) of PKC and the only member known to translocate to this synapse. Prior to TCR stimulation PKC theta exists in an inactive closed conformation. Upon release of DAG, it binds to PKC theta via the C1 domain and undergoes phosphorylation on tyrosine 90 by Lck to attain an open conformation. PKC theta is further phosphorylated by PDK1 on threonine 538. This step is critical for PKC activity (steps 24-26).

CARMA1 translocates to the plasma membrane following the interaction of its SH3 domain with the 'PxxP' motif on PDK1. CARMA1 is phosphorylated by PKC-theta on residue S552, leading to the oligomerization of CARMA1. This complex acts as a scaffold, recruiting Bcl10 to the synapse by interacting with their CARD domains.

Bcl10 undergoes phosphorylation mediated by the enzyme RIP2. Activated Bcl10 then mediates the ubiquitination of NEMO by recruiting MALT1 and TRAF6. MALT1 binds to Bcl10 with its Ig-like domains and undergoes oligomerization. TRAF6 binds to the oligomerized MALT1 and also undergoes oligomerization.

Oligomerized TRAF6 acts as a ubiquitin-protein ligase, catalyzing auto-K63-linked polyubiquitination (steps 27-33). This K-63 ubiquitinated TRAF6 activates TAK1 kinase bound to TAB2 and also ubiquitinates NEMO/IKK-gamma in the IKK complex. TAK1 undergoes autophosphorylation on residues T184 and T187 and gets activated. Activated TAK1 kinase phosphorylates IKK-beta on residues S177 and S181 in the activation loop and activates the IKK kinase activity. IKK-beta phosphorylates the IkB-alpha bound to the NF-kB heterodimer, on residues S19 and S23 and directs IkB-beta to 26S proteasome degradation (step 34-38 & 40).

The NF-kB heterodimer with a free NTS sequence finally migrates to the nucleus to regulate gene transcription (step 39)

The binding of TNF-alpha to TNFR1 leads to recruitment of the adapter protein TNFR1-associated death domain (TRADD) and of receptor‑interacting protein 1 (RIPK1). TRADD subsequently recruits also TNF receptor-associated factor 2 (TRAF2). RIPK1 is promptly K63-polyubiquitinated which results in the recruitment of the TAB2:TAK1 complex and the IkB kinase (IKK) complex to TNFR1. The activated IKK complex mediates phosphorylation of the inhibitor of NFkappaB (IkB), which targets IkB for ubiquitination and subsequent degradation. Released NFkappaB induces the expression of a variety of genes including inflammation-related genes and anti-apoptotic genes encoding proteins such as inhibitor of apoptosis proteins cIAP1/2, Bcl-2, Bcl-xL or cellular FLICE-like inhibitory protein (FLIP) (Blonska M et al. 2005; Ea CK et al. 2006; Wu CJ et al. 2006; Chen C et al. 2000; Manna SK et al. 2000; Kreuz S et al. 2001; Micheau O et al. 2001). NFkB-mediated inhibition of cell death also involves attenuating TNF-induced activation of c-Jun activating kinase (JNK). Whereas transient activation of JNK upon TNF treatment is associated with cellular survival, prolonged JNK activation contributes to cell death. However, as caspases activate JNK quite efficiently, JNKs are also regularly stimulated in course of apoptosis without being essential for cell death (Wicovsky A et al. 2007). AP1-mediated gene induction results from activation of JNK via TRAF2 (not shown here) (Tsou HK et al. 2012). While pro-survival signaling is initiated and regulated via the activated TNFR1 receptor complex at the cell membrane, cell death signals are induced by internalization-associated fashion upon the release of RIPK1 from the membrane complex (Micheau O and Tschopp J 2003; Schneider-Brachert W et al. 2004; Tchikov V et al. 2011).

TNFR1-mediated transcriptional activity of NFkB is both antiapoptotic and highly proinflammatory and thus must be tightly regulated to prevent constitutive activation that leads to persistent inflammation and cancer (Ward C et al. 1999; Fujihara S et al. 2002; Pekalski J et al. 2013; Kankaanranta H et al. 2014; Shukla S and Gupta S 2004; Jackson-Bernitsas DG et al. 2007; Zhang JY et al. 2007). Multiple mechanisms normally ensure the proper control of NFkappaB activation including two negative feedback loops mediated by NFkappaB inducible inhibitors, IkB-alpha (NFKBIA) and ubiquitin-editing protein A20 (He KL & Ting AT 2002; Wertz IE et al. 2004; Vereecke L et al. 2009; Pekalski J et al. 2013)

IKBKG is encoded by an X-linked gene. Null alleles of the gene are lethal in hemizygous males, whereas hypomorphic alleles typically result in the impaired NFkB signaling in patients with a broad spectrum of clinical phenotypes in terms of both developmental defects and immunodeficiency (Döffinger R et al. 2001; Hanson EP et al. 2008). Several categories of mutations affecting IKBKG have been reported in humans (Döffinger R et al. 2001; Vinolo E et al. 2006; Fusko F et al. 2008). The first category of these mutations consists of hypomorphic mutations typically involving the zinc finger domain and nearby C-terminal regions and causing hypohidrotic ectodermal dysplasia with immune deficiency (HED-ID) in males (Jain A et al. 2001; Shifera AS 2010). The second category consists of amorphic mutations causing incontinentia pigmenti (IP) in females and, generally, prenatal death in males (Aradhya S et al. 2001; Fusco F et al. 2004). The third category is composed of hypomorphic mutations involving the stop codon causing anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), osteopetrosis and lymphedema (OL-EDA-ID) in males (Döffinger R et al. 2001). Also some patients with a defective IKBKG gene can develop immunodeficiency without ectodermal dysplasia (Orange JS et al. 2004). This module describes several EDA-ID-associated hypomorphic IKBKG mutations that have been reported to affect inflammatory responses initiated by toll like receptors (TLR)

In the absence of extra-cellular signals, cytosolic MAP3K8 (TPL2) is held inactive in the complex with ABIN2 (TNIP2) and NFkB p105 (NFKB1) (Beinke S et al., 2003; Waterfield MR et al., 2003; Lang V et al., 2004). This interaction stabilizes MAP3K8 (TPL2) but also prevents MAP3K8 and NFkB from activating their downstream signaling cascades by inhibiting the kinase activity of MAP3K8 and the proteolysis of NFkB precursor protein p105. Upon activation of MAP3K8 by various stimuli (such as LPS, TNF-alpha, and IL-1 beta), IKBKB phosphorylates NFkB p105 (NFKB1) at Ser927 and Ser932, which trigger p105 proteasomal degradation and releases MAP3K8 from the complex (Beinke S et al., 2003, 2004; Roget K et al., 2012). Simultaneously, MAP3K8 is activated by auto- and/or transphosphorylation (Gantke T et al. 2011; Yang HT et al. 2012). The released active MAP3K8 phosphorylates its substrates, MAP2Ks. The free MAP3K8, however, is also unstable and is targeted for proteasome-mediated degradation, thus restricting prolonged activation of MAP3K8 (TPL2) and its downstream signaling pathways (Waterfield MR et al. 2003; Cho J et al., 2005). Furthermore, partially degraded NFkB p105 (NFKB1) into p50 can dimerize with other NFkB family members to regulate the transcription of target genes.

MAP3K8 activity is thought to regulate the dynamics of transcription factors that control an expression of diverse genes involved in growth, differentiation, and inflammation. Suppressing the MAP3K8 kinase activity with selective inhibitors, such as C8-chloronaphthyridine-3-carbonitrile, caused a significant reduction in TNFalpha production in LPS- and IL-1beta-induced both primary human monocytes and human blood (Hall JP et al. 2007). Similar results have been reported for mouse LPS-stimulated RAW264.7 cells (Hirata K et al. 2010). Moreover, LPS-stimulated macrophages derived from Map3k8 knockout mice secreted lower levels of pro-inflammatory cytokines such as TNFalpha, Cox2, Pge2 and CXCL1 (Dumitru CD et al. 2000; Eliopoulos AG et al. 2002). Additionally, bone marrow-derived dendritic cells (BMDCs) and macrophages from Map3k8 knockout mice showed significantly lower expression of IL-1beta in response to LPS, poly IC and LPS/MDP (Mielke et al., 2009). However, several other studies seem to contradict these findings and Map3k8 deficiency in mice has been also reported to enhance pro-inflammatory profiles. Map3k8 deficiency in LPS-stimulated macrophages was associated with an increase in nitric oxide synthase 2 (NOS2) expression (López-Peláez et al., 2011). Similarly, expression of IRAK-M, whose function is to compete with IL-1R-associated kinase (IRAK) family of kinases, was decreased in Map3k8-/- macrophages while levels of TNF and IL6 were elevated (Zacharioudaki et al., 2009). Moreover, significantly higher inflammation level was observed in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated Map3k8-/- mouse skin compared to WT skin (DeCicco-Skinner K. et al., 2011). Additionally, MAP3K8 activity is associated with NFkB inflammatory pathway. High levels of active p65 NFkB were observed in the nucleus of Map3k8 -/- mouse keratinocytes that dramatically increased within 15-30 minutes of TPA treatment. Similarly, increased p65 NFkB was observed in Map3k8-deficient BMDC both basally and after stimulation with LPS when compared to wild type controls (Mielke et al., 2009). The data opposes the findings that Map3k8-deficient mouse embryo fibroblasts and human Jurkat T cells with kinase domain-deficient protein have a reduction in NFkB activation but only when certain stimuli are administered (Lin et al., 1999; Das S et al., 2005). Thus, it is possible that whether MAP3K8 serves more of a pro-inflammatory or anti-inflammatory role may depend on cell- or tissue type and on stimuli (LPS vs. TPA, etc.) (Mielke et al., 2009; DeCicco-Skinner K. et al., 2012).

MAP3K8 has been also studied in the context of carcinogenesis, however the physiological role of MAP3K8 in the etiology of human cancers is also convoluted (Vougioukalaki M et al., 2011; DeCicco-Skinner K. et al., 2012)

IRAK1 has also been shown to be itself Lys63-polyubiquitinated (probably by Pellino proteins, which have E3 ligase activity). Mutation of the ubiquitination sites on IRAK1 prevented interaction with the NEMO subunit of IKK complex and subsequent IL-1/TLR-induced NF-kappa-B activation [Conze et al 2008]. These data suggest that kinase activity of IRAK1 is not essential for its ability to activate NF-kappa-B, while its Lys63-polyubuquitination allows IRAK1 to bind NEMO thus facilitating association of TRAF6 and TAK1 complex with IKK complex followed by induction of NF-kappa-B.

Upon IL-1/TLR stimulation IRAK1 protein can undergo covalent modifications including phosphorylation [Kollewe et al 2004], ubiquitination [Conze DB et al 2008] and sumoylation [Huang et al 2004]. Depending upon the nature of its modification, IRAK1 may perform distinct functions including activation of IRF5/7 [Uematsu et al 2005, Schoenemeyer et al 2005], NF-kappa-B [Song et al 2006], and Stat1/3 [Huang et al 2004, Nguyen et al 2003]

IRAK1 has also been shown to be itself Lys63-polyubiquitinated (probably by Pellino proteins, which have E3 ligase activity). Mutation of the ubiquitination sites on IRAK1 prevented interaction with the NEMO subunit of IKK complex and subsequent IL-1/TLR-induced NF-kappa-B activation [Conze et al 2008]. These data suggest that kinase activity of IRAK1 is not essential for its ability to activate NF-kappa-B, while its Lys63-polyubuquitination allows IRAK1 to bind NEMO thus facilitating association of TRAF6 and TAK1 complex with IKK complex followed by induction of NF-kappa-B.

Upon IL-1/TLR stimulation IRAK1 protein can undergo covalent modifications including phosphorylation [Kollewe et al 2004], ubiquitination [Conze DB et al 2008] and sumoylation [Huang et al 2004]. Depending upon the nature of its modification, IRAK1 may perform distinct functions including activation of IRF5/7 [Uematsu et al 2005, Schoenemeyer et al 2005], NF-kappa-B [Song et al 2006], and Stat1/3 [Huang et al 2004, Nguyen et al 2003]