Phosphorylation of Serine 68 in the IκB Kinase (IKK)-binding Domain of NEMO Interferes with the Structure of the IKK Complex and Tumor Necrosis Factor-α-induced NF-κB Activity

Abstract

The transcription factor NF-B plays a crucial role in the initiation of innate and adaptive immune responses, in inflammation and tumorigenesis (1-3). In its inactive state NF-B is bound to small cytoplasmic proteins, the IB proteins. Stimulation with a wide variety of agonists, for example pro-inflammatory cytokines like TNF-␣, 2 bacterial components like lipopolysaccharide or by antigen receptors, funnels in the activation of a multisubunit IB-kinase complex, which phosphorylates the IB proteins at two specific serine residues. This phosphorylation marks the IB proteins for proteasomal degradation setting NF-B free, which then translocates into the nucleus and supports the expression of various pro-inflammatory or anti-apoptotic gene products. The IB kinase (IKK) complex is composed of two catalytically active subunits, termed IKK␣ (IKK1) and IKK␤ (IKK2), as well as NEMO/IKK␥, a subunit with regulatory and adaptor functions (4, 5). Analysis of mice deficient for either IKK␣ or IKK␤ suggested that the IKK␤ subunit is the major IKK regulating the canonical NF-B pathway, and the IKK␣ subunit is crucial for a second, alternative NF-B pathway leading to the activation of RelB-p52 heterodimers (6, 7). In contrast to the canonical NF-B pathway, which depends on the presence of NEMO, the alternative pathway is NEMO-independent but requires the protein kinase NIK. However, recent data suggest that, instead of a clear assignment of IKK␣ and IKK␤ to the alternative and the canonical NF-B pathway, respectively, both kinases contribute to the activation of NF-B by the canonical pathway with only gradual differences (8,9). Although a precise model regarding the molecular mechanism underlying IKK activation is still missing, it became clear that various post-translational modifications at the different IKK subunits are involved in this process. Besides the phosphorylation of IKK␣ and IKK␤ at two serine residues in their T-loop, the ubiquitination and occasionally the phosphorylation at a specific serine residue of NEMO seem to be required (10 -12). This serine residue at position 85 of NEMO has been identified as a protein kinase C␣, and more recently as an ATM target-site crucial for the IKK activation induced by genotoxic stress (12, 13). In addition, overexpression of the TNF-␣-receptor I or the human T-cell lymphotrophic virus I Tax protein induces the IKK␤-mediated NEMO phosphorylation at several

Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347

“…Therefore, we believe that there exists an equilibrium of NEMO monomer-to-dimer, which may vary in different cell types depending on the redox state of the cytosol in a given cell. Furthermore, our results enable us to speculate that formation of the disulfide-bonded NEMO dimer can be modulated by phosphorylation of Ser68 [20].…”

Annexin-1 interacts with NEMO and RIP1 to constitutively activate IKK complex and NF-κB: implication in breast cancer metastasis

“…NEMO contains two coiled-coil motifs, a leucine zipper, a C-terminal zinc-finger domain (Ea et al, 2006). These two domains are necessary for the correct assembly of the IKK complex (Palkowitsch et al, 2008), and for the recruitment of upstream molecules, whose interaction with NEMO is essential for the IKK-mediated NF-kB activation. In addition to the physical interaction of NEMO with upstream molecules, a novel mechanism of IKK recruitment and activation is linked to the ability of NEMO to recognize polyubiquitinated signaling intermediates, such as RIP (Ea et al, 2006).…”

“…It is possible that besides binding to IKK component, ANXA1 has the ability to interact with components of several other signaling pathways, and thereby, acting as a crosstalk messenger. NEMO is an integral component of IKK complex, and phosphorylation of NEMO leads to a conformational change, which allows the access of phosphatases that subsequently decrease IKK activity (Palkowitsch et al, 2008). It is quite possible that in cells overexpressing ANXA1, phosphatases may not have access to NEMO, and NF-kB activation remains indefinitely active.…”

“…CXCR4 expression increases progressively with increasing malignant potential, and higher expression is associated with lymph node positivity, hormone receptor negativity, Her2 overexpression and poor survival in breast cancer (Cabioglu et al, 2005;Mehta et al, 2007), and that CXCR4 can act as a biomarker for metastasis (Cabioglu et al, 2005). Interestingly, CXCR4 has been shown to be regulated by NF-kB (Palkowitsch et al, 2008) and p53 (Mehta et al, 2007). Overexpression of DN-IkBa in breast cancer cells resulted in reduced expression of CXCR4, and a corresponding loss of SDF1a-mediated migration in vitro (Helbig et al, 2003).…”

Tyrosine phosphatase inhibition triggers sustained canonical serine-dependent NFκB activation via Src-dependent blockade of PP2A

“…After ruling out a number of putative Src substrates to interfere with IB degradation [28][29][30][31][32][33][34] Ser/Thr phosphatase PP2A was found to be the critical component. PP2A is known to modulate NFB activity [47].…”