Phorbol Esters and Cytokines Regulate the Expression of theNEMO-related Protein, a Molecule Involved in a NF-κB-independent Pathway

Abstract

The NF-B signaling pathway plays a crucial role in the immune, inflammatory, and apoptotic responses. Recently, we identified the NF-B Essential Modulator (NEMO) as an essential component of this pathway. NEMO is a structural and regulatory subunit of the high molecular kinase complex (IKK) responsible for the phosphorylation of NF-B inhibitors. Data base searching led to the isolation of a cDNA encoding a protein we called NRP (NEMO-related protein), which shows a strong homology to NEMO. Here we show that NRP is present in a novel high molecular weight complex, that contains none of the known members of the IKK complex. Consistently, we could not observe any effect of NRP on NF-B signaling. Nonetheless, we could demonstrate that treatment with phorbol esters induces NRP phosphorylation and decreases its half-life. This phosphorylation event could only be inhibited by K-252a and stauroporin. We also show that de novo expression of NRP can be induced by interferon and tumor necrosis factor ␣ and that these two stimuli have a synergistic effect on NRP expression. In addition, we observed that endogenous NRP is associated with the Golgi apparatus. Analogous to NEMO, we find that NRP is associated in a complex with two kinases, suggesting that NRP could play a similar role in another signaling pathway.The transcription factor NF-B plays a pivotal role in many cellular processes such as immune responses, inflammation, and apoptosis (1, 2). NF-B is composed of homo-and heterodimers of various members of the NF-B/Rel family (3, 4) and is retained in an inactive form in the cytoplasm by an inhibitory protein belonging to the IB family, mainly represented by IB␣, IB␤, and IB⑀ (5-7).In response to diverse stimuli, including IL-1, 1 LPS, TNF␣, or PMA, as well as several viral proteins, active NF-B translocates to the nucleus as a result of the complete proteolytic degradation of the IB proteins. This mechanism has been best studied for the inhibitor IB␣ and demonstrated to involve phosphorylation on two specific serine residues (8 -13) followed by polyubiquitination and degradation by the 26 S proteasome (14). More recently a specific serine protein kinase activity responsible for IB␣ phosphorylation has been identified as a large cytoplasmic complex (600 -800 kDa) containing two catalytic subunits (IKK1/␣ and IKK2/␤) (15-19). IKK␣ and IKK␤ are related molecules of 85 and 87 kDa, respectively, and share 50% sequence similarity. Both proteins contain NH 2 -terminal kinase domains, leucine zipper, and helix-loop-helix motifs (16,19). In vitro phosphorylation studies have shown that both kinases can phosphorylate IB␣ on serines 32 and 36, but IKK␤ is more active in this regard.Recently, we have cloned, by complementation of an NF-B unresponsive cell line, a third component of the IKK complex (20), that we called NEMO (NF-B Essential Modulator). NEMO is a 48-kDa glutamine-rich protein, which lacks a catalytic subunit, but contains two coiled-coil motifs, a leucine zipper, and a COOH-terminal zinc finger (20). NEMO is a regula...

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Activation of NF-κB by the Human T Cell Leukemia Virus Type I Tax Oncoprotein Is Associated with Ubiquitin-dependent Relocalization of IκB Kinase

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Toward an integrative view of Optineurin functions

“…As for NEMO, Optn is detected in a high molecular complex, probably in association with other interacting proteins. 5 Optn is ubiquitous, and its expression can be induced by TNFα and interferons, probably as a result of NFκB activation, since Optn possesses a functional NFκB binding site in its promoter. 1,5,6 Despite its homology with NEMO, numerous studies indicate that Optn is involved in multiple fundamental cellular functions, signaling pathways and host defense mechanisms that are not related to NFκB.…”

“…3 Optn exhibits strong homology with the NEMO (NFκB essential modulator) at both the primary sequence and domain organization levels. 5 Sequence alignment of the two proteins revealed that Optn shares 53% similarity with NEMO except for the presence of an "insert" of 166 amino acids located in the N-terminal region and containing a putative leucine zipper (LZ) (Fig. 1).…”