TAB1 modulates IL-1α mediated cytokine secretion but is dispensable for TAK1 activation
Introduction
The stress–response mitogen-activated protein kinases (MAPK) p38 and JNK, together with NFκB, play critical roles in the induction and maintenance of cellular inflammatory and immune responses. Their activation by growth factors, cytokines, invading pathogens and environmental stress factors triggers transcription of proinflammatory genes and promotes cellular proliferation and differentiation [1], [2]. Because IL-1 and TNFα stimulated signalling cascades result in both p38, JNK and NFκB activation, intensive efforts have sought to identify the signalling components involved and to establish the sequence of events leading to activation.
The TGF-β-activated kinase 1 (TAK1) is an evolutionary conserved MAPKKK, which has been recognized as a potential branching point and key regulator of MAPK and NFκB signalling pathways. TAK1 reportedly interacts with the IκB kinase (IKK) complex, which phosphorylates the inhibitory subunit IκB, thereby targeting it for degradation, resulting in the unmasking of the NFκB nuclear localisation signal [3], [4], [5]. Further, a number of studies have demonstrated that TAK1 interacts with MKK3, MKK4, MKK6 and MKK7 to stimulate p38 and JNK phosphorylation [6], [7], [8]. This causes p38 and JNK to translocate to the nuclear compartment, permitting JNK to phosphorylate c-Jun, comprising part of the AP-1 transcription factor complex, and causing p38 to activate a subset of transcription factors [9]. Overexpression experiments and in vitro studies have clarified several aspects of the IL-1 and TNFα induced signalling cascades critical for TAK1 activation. Binding of IL-1 to its cognate receptor (IL-1R) results in recruitment of MyD88 and Tollip, which serve as adaptors for IL-1R associated kinase 1 (IRAK1) and IRAK4. Subsequent IRAK1 hyperphosphorylation promotes interaction with TNF-R-associated factor 6 (TRAF6), resulting in TRAF6 oligomerization, which causes the IRAK1/TRAF6 complex to dissociate from the receptor and interact with membrane associated TAK1 and TAK1 binding protein 1 (TAB1) [10]. This interaction is dependent on TAB2, which is complexed to TAK1 and binds TRAF6 polyubiquitinated by the Ubc13/Uev1A ubiquitin conjugating enzyme complex [4], [11], [12]. The TRAF6–TAK1–TAB1–TAB2 complex translocates into the cytosolic compartment for further activation, which is, in part, dependent on TAB2 polyubiquitination catalyzed by TRAF6 ubiquitin ligase E3 activity. TNFα engagement with its cognate receptor (TNF-R) induces TNF-R trimerization, which facilitates recruitment and binding of adapter proteins, including TNF-R1-associated death domain (TRADD), TRAF2, TRAF5 and receptor-interacting protein 1 (RIP1), to the membrane associated receptor complex. Ubc13/Uev1A and TRAF2 dependent polyubiquitination of RIP1 serves as a recruitment signal for TAB2, which links TAK1 to the signalling complex where it may cooperate with MEKK3 to stimulate downstream signalling pathways [13].
Numerous reports have demonstrated that the adaptor molecule TAB1 is essential for TAK1 activity and facilitates phosphorylation of Thr184, Thr187 and Ser192 residues within the TAK1 activation loop [14], [15], [16]. A truncated TAB1 variant (amino acid residues 437–504) was demonstrated to be sufficient for full TAK1 activation [14]. A recently published crystal structure of a TAK1–TAB1 chimeric protein revealed the presence of a novel binding pocket in the TAK1 kinase domain, supporting the hypothesis that only the TAB1 C-terminal region interacts with TAK1 and is responsible for subsequent kinase activation [17]. However, the notion that TAB1 functions as a TAK1 activator is derived exclusively from TAK1 and TAB1 overexpression studies [14], [16], [18], [19]. The fact that TAK1 remains inactive in resting wild-type cells despite residing in a complex with TAB1 may either suggest the presence of a negative regulator insufficient to prevent TAK1 activation in TAB1/TAK1 transfected cells, or, alternatively, may imply a dispensable role for TAB1 in TAK1 regulation. The latter interpretation was recently supported by studies on immortalized embryonic fibroblasts from TAB1 knockout mice, which displayed normal IL-1 and TNFα stimulated NFκB and JNK activation [20]. To further evaluate the possible physiological role for TAB1 in TAK1 activation, we investigated the effect of TAB1 gene silencing on nuclear accumulation of the NFκB p65/RelA transcription factor and phosphorylated forms of c-Jun and p38 in intact cells as a measure of NFκB and MAPK signal transduction. These studies were extended to measurements of levels of secreted cytokines from cells transfected with either TAB1 or TAK1 small interfering RNA (siRNA) to examine the potential impact of protein knockdown on functional readouts. In summary, the present study strongly supports the involvement of TAK1 in IL-1 and TNFα mediated inflammatory cellular responses, but argues against a non-redundant role for TAB1 in TAK1 dependent p38, JNK and NFκB activation following IL-1 and TNFα stimulation. However, TAB1 knockdown was associated with a significant decrease in IL-1 mediated IL-6, IL-8 and granulocyte macrophage colony-stimulating factor (GM-CSF) release while TNFα stimulated cytokine production was unchanged. This may indicate a differential effect of TAB1 on proinflammatory signalling pathways, which may be independent of TAK1 activation.
Section snippets
Materials
Rabbit anti-phospho-p38 (Thr180/Tyr182) antibody, rabbit anti-p38 antibody and rabbit anti-c-Jun (60A8) antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-phospho-c-Jun (Ser63) antibody, goat anti-NFκB p65 (C20) antibody, rabbit anti-NFκB p65 (C20) antibody and rabbit anti-TAK (M-579) antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) while mouse anti-actin antibody was from Chemicon (Temecula, CA, USA). Hoechst 33342 and all Alexa-conjugated
TAK1 is essential for NFκB/MAPK activation and cytokine secretion
In order to establish whether TAK1 is implicated in IL-1α or TNFα stimulated phospho-c-Jun, phospho-p38 and NFκB p65 nuclear accumulation, we employed a gene silencing approach. HeLa cells were transfected with TAK1 siRNA or a non-targeting (NT) siRNA pool for 48 h before high content analysis. Densitometric analysis of TAK1 western blots revealed approximately 80% knockdown of TAK1 protein levels in TAK1 siRNA transfected cells (Fig. 1A). Importantly, c-Jun, p38 or NFκB p65 protein levels were
Discussion
Because signalling molecules typically participate in several separate signal transduction networks, scaffolding proteins and adaptor components are required to ensure an efficient, selective and regionalized signalling flow [23]. In addition to functioning as docking sites for upstream and downstream signalling constituents, phosphorylation or ubiquitination reactions may allow these otherwise inert anchor molecules to stimulate effector enzyme activity through conformational changes and hence
Conclusion
In conclusion, our investigations challenge the view that TAB1 is simply an activator of TAK1 kinase activity. Instead, TAB1 may modulate inflammatory gene transcription downstream from stimulation of JNK, p38 and NFκB p65 nuclear translocation. However, further studies are warranted to firmly establish how TAB1 regulates proinflammatory responses and whether this is governed independently of TAK1 associated signalling networks.
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