NF-kappaB essential modulator (NEMO), the regulatory subunit of the IkappaB kinase (IKK) that activates NF-kappaB, is essential for NF-kappaB activation. NEMO was recently found to contain a region that preferentially binds Lys (K)63-linked but not K48-linked polyubiquitin (polyUb) chains, and the ability of NEMO to bind to K63-linked polyUb RIP (receptor-interacting protein) is necessary for efficient tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. Optineurin is a homolog of NEMO, and mutations in the optineurin gene are found in a subset of patients with glaucoma, a neurodegenerative disease involving the loss of retinal ganglion cells. Although optineurin shares considerable homology with NEMO, in resting cells, it is not present in the high-molecular-weight complex containing IKKalpha and IKKbeta, and optineurin cannot substitute for NEMO in lipopolysaccharide (LPS)-induced NF-kappaB activation. On the other hand, the overexpression of optineurin blocks the protective effect of E3-14.7K on cell death caused by the overexpression of TNFalpha receptor 1 (TNFR1). Here we show that optineurin has a K63-linked polyUb-binding region similar to that of NEMO, and like NEMO, it bound K63- but not K48-linked polyUb. Optineurin competitively antagonized NEMO's binding to polyUb RIP, and its overexpression inhibited TNFalpha-induced NF-kappaB activation. This competition occurs at physiologic protein levels because microRNA silencing of optineurin resulted in markedly enhanced TNFalpha-induced NF-kappaB activity. These results reveal a physiologic role for optineurin in dampening TNFalpha signaling, and this role might provide an explanation for its association with glaucoma.

Specificity of phosphorylation is critical to signal transduction. Recent emphasis on colocalization of substrate and kinase has eclipsed emphasis on peptide specificity, i.e., kinase preference for particular amino acids surrounding the phosphorylation site. We describe an approach to determining peptide specificity by using positional scanning of biotinylated oriented peptide libraries and insights emerging from those determinations. We accurately determine preference (or disfavor) for residues at a given substrate position (such as P؉2) by comparison of in vitro phosphorylation of peptide libraries differing by a single residue at that position. By analysis of all positions near the phosphorylation site, positionspecific scoring matrices are generated and used both to understand the basis of specificity and to predict phosphorylation. PKC-␦ and -predictions have been validated rigorously by comparisons with measured phosphorylation. The results demonstrate specificity and sensitivity (80 -90%) much better than the previous predictive method. These predictions can be accessed at http:͞͞mpr. nci.nih.gov. The accuracy of the specificity determination allows identification of an important difference in peptide specificity between these closely related kinases; Ile͞Leu at the P؊1 position is disfavored by PKC-but not PKC-␦. Our findings and visual representation of peptide specificity highlight the importance of disfavored residues. Finally, analysis of 124 experimentally determined PKC sites from the literature demonstrates a very strong role of peptide specificity in many of those sites. Thus, position-specific scoring matrices generated by this method provide a foundation for quantitative analyses of kinase specificity and improved predictions of previously determined physiologically relevant phosphorylation sites.

Optineurin is a widely-expressed polyubiquitin (polyUb)-binding protein that has been implicated in regulating cell signaling via its NEMO-homologous C-terminal Ub-binding region. Its functions are controversial, with in vitro studies finding that optineurin suppressed TNF-mediated NF-κB activation and virus-induced activation of IRF3, whereas bone marrow-derived macrophages (BMDM) from mice carrying an optineurin Ub-binding point mutation had normal TLR-mediated NF-κB activation and diminished IRF3 activation. We have generated a mouse model in which the entire Ub-binding C-terminal region is deleted (Optn470T). Akin to C-terminal optineurin mutations found in patients with certain neurodegenerative diseases, Optn470T was expressed at substantially lower levels than the native protein, allowing assessment not only of the lack of Ub-binding but also of protein insufficiency. Embryonic lethality with incomplete penetrance was observed for 129 x C57BL/6 Optn470T/470T mice, but after further backcrossing to C57BL/6, offspring viability was restored. Moreover, the mice that survived were indistinguishable from wild type littermates and had normal immune cell distributions. Activation of NF-κB in Optn470T BMDM and BM-derived dendritic cells (BMDC) with TNF or via TLR4, T cells via the TCR, and B cells with LPS or anti-CD40 was normal. In contrast, optineurin and/or its Ub-binding function was necessary for optimal TBK1 and IRF3 activation, and both Optn470T BMDM and BMDC had diminished IFN-β production upon LPS stimulation. Importantly, Optn470T mice produced less IFN-β upon LPS challenge. Therefore, endogenous optineurin is dispensable for NF-κB activation but necessary for optimal IRF3 activation in immune cells.