Signal Transduction
Scaffold proteins implicated in the p38/JNK pathway.
P38 and JNK kinases are bound to the scaffold protein JIP (four variants, JIP1–4).52 JIP1–3 act to potentiate JNK signalling through the assembly of a signalling cassette comprising members of
the mixed-lineage protein kinase group MAP3K, MKK 7 (MAP2K), and JNK.53,54 They also attach to the TPR domain of the light chain of the microtubule motor protein kinesin and through this they localize the JNK signalling cassette to growth cones of neurons.55,56 JIP4 is somewhat different. It shares with JIP1–3 its interaction with JNK and with kinesin, but it has
a role as an activator of p38-mitogen activated protein kinase by a mechanism that requires MKK3 and MKK6.57 The precise role of JIP in the TAK1-mediated activation of MEK3 and MEK4 remains to be solved.
JNK1 (c-Jun N-terminal kinase) enters the nucleus to phosphorylate the transcription factors ATF2 (a member of the ATF/CREB family of transcription factors)51 and c-Jun. Together they constitute the AP-1 complex (activator protein-1, which is comprised of different combinations of the transcription factors ATF, c-fos, fra1, fra2, c-Jun, or junB). Numerous genes carry AP-1 responsive elements in their promoter regions. P38 remains in the cytosol and acts to enhance inflammatory cytokine production through phosphorylation and activation of MK2 (a member of the MAP kinaseactivated protein kinases) (Figure 15, page 340) that acts to enhance the expression and secretion of inflammatory cytokines through stabilization of the relevant mRNAs.
From TRAF6 to activation of IRF-3
The TRAF6 signalling complex also causes dimerization of the interferonregulatory factor IRF-3 which is then transferred into the nucleus.
The TRAM, TRIF pathway
Here we concentrate on the role of TRAF3 as an adaptor that recruits a kinase complex to the TLR4 receptor (Figure 15.10). First it binds TANK which is associated with the kinases IKK and TBK1, homologues of IKK .59 Recruitment of TBK1 causes phosphorylation of IFR3 which, as a homodimer (or heterodimer IRF3/IRF7), translocates to the nucleus to bind DNA at the ISRE-responsive element (see page 525 onwards). TBK1 also associates with
CBP, inducing expression of IFN- . Transcriptional activity of IFR3 requires the cooperation of NF- B. Other genes expressed through this pathway are IL-10 and the chemokine RANTES. Absence of IFR3 prevents the production of IFNand IL-10 without reducing the expression of the pro-inflammatory cytokines TNF- , IL-1 , or IL-6. Indeed, their production is actually enhanced, possibly due to failure of the negative control normally exerted by IL-10.60
The IRF family of transcription factors
The interferon regulatory factors (IRFs), first identified in the promoter region of the human interferongene,61 operate in both innate and in adaptive immunity. They recognize a consensus DNA sequence known as the IFNstimulated response element (ISRE) and are functionally active as homoor heterodimers. Actually, their application is widespread, with many genes containing the ISRE consensus sequence and they are activated by several
receptors, including TLR4.62 The mammalian IRF family comprises nine members (IRF1–9). Except for IRF1 and IRF2, their C-termini (IAD domain) resemble the Smad family of transcription factors (figure 8, page 609), suggesting interactions with other regulatory transcription factors or with the general machinery of
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The Toll-like Receptor 4 and Signalling through Ubiquitylation
FIG 15.10 TRAM/TRIF-mediated receptor signal complex formation and the activation of IRF3.
(a) TLR4 recruits TRAM and TRIF through their TIR domains. TRIF is bound to TRAF3 (an E3-ligase) which in turn, binds the adaptor TANK coupled to IKK and TBK1 (1). The formed complex causes auto-phosphorylation of IKK , followed by phosphorylation and activation of TBK1 (2). Activated TBK1
phosphorylates IFR3 (3) which now unfolds, thereby exposing its DNA binding domain and its dimerization and transactivation domains (4). Dimerized IFR3 enters the nucleus, binds to ISRE and to CBP (5). This leads to induction of expression of IFNand IL-10. (b) Domain architecture of components of the IFR3 activation pathway. Activation phosphorylation sites of IRF are at the C-terminal in and just outside the IRF association domain that binds the transcription factor CBP. TBK1 is different from IKK with respect to its activation segment, containing only a single serine phosphorylation site. TRAF3, like TRAF6, is an E3-ligase, with a RING domain (R) that binds the E2-conjugating enzyme. As illustrated here, it merely acts as an adaptor. Its role as ubiquitylating enzyme in the TLR4 pathway remains unclear.
gene transcription (see Figure 15.10).63 Different IRFs play different roles, well exemplified by the phenotypes of knockout mice. Loss of IRF7 renders mice vulnerable to viral infection, correlating with a decrease in serum interferon levels.64 Loss of IRF5 has little effect on IFNproduction but the animals are resistant to the lethal effect of systemically applied non-methylated DNA or LPS and this correlates with a reduction of serum IL-6, TNF- , and IL-12.58
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