Vinay Tergaonkar

A Fourth IκB Protein within the NF-κB Signaling Module

An assay method incorporating at least two different chemiluminescent compounds for detection and/or quantitation of at least two substances in a test sample is described. The synthesis of chemiluminescent reagents or conjugates for use in such methods as well as kits incorporating such reagents are also disclosed. The assays have particular application in the field of clinical diagnostics.

IkappaB kinase-independent IkappaBalpha degradation pathway: functional NF-kappaB activity and implications for cancer therapy.

ABSTRACT Antiapoptotic activity of NF-κB in tumors contributes to acquisition of resistance to chemotherapy. Degradation of IκB is a seminal step in activation of NF-κB. The IκB kinases, IKK1 and IKK2, have been implicated in both IκB degradation and subsequent modifications of NFκB. Using mouse embryo fibroblasts (MEFs) devoid of both IKK1 and IKK2 genes (IKK1/2−/−), we document a novel IκB degradation mechanism. We show that this degradation induced by a chemotherapeutic agent, doxorubicin (DoxR), does not require the classical serine 32 and 36 phosphorylation or the PEST domain of IκBα. Degradation of IκBα is partially blocked by phosphatidylinositol 3-kinase inhibitor LY294002 and is mediated by the proteasome. Free NF-κB generated by DoxR-induced IκB degradation in IKK1/2−/− cells is able to activate chromatin based NF-κB reporter gene and expression of the endogenous target gene, IκBα. These results also imply that modification of NF-κB by IKK1 or IKK2 either prior or subsequent to its release from IκB is not essential for NF-κB-mediated gene expression at least in response to DNA damage. In addition, DoxR-induced cell death in IKK1/2−/− MEFs is enhanced by simultaneous inhibition of NF-κB activation by blocking the proteasome activity. These results reveal an additional pathway of activating NF-κB during the course of anticancer therapy and provide a mechanistic basis for the observation that proteasome inhibitors could be used as adjuvants in chemotherapy.

Distinct roles of IκB proteins in regulating constitutive NF-κB activity

The inhibitor of NF-κB (IκB) family of proteins is believed to regulate NF-κB activity by cytoplasmic sequestration. We show that in cells depleted of IκBα, IκBβ and IκBε proteins, a small fraction of p65 binds DNA and leads to constitutive activation of NF-κB target genes, even without stimulation, whereas most of the p65 remains cytoplasmic. These results indicate that although IκBα, IκBβ and IκBε proteins could be dispensable for cytoplasmic retention of NF-κB, they are essential for preventing NF-κB-dependent gene expression in the basal state. We also show that in the absence of IκBα, IκBβ and IκBε proteins, cytoplasmic retention of NF-κB by other cellular proteins renders the pathway unresponsive to activation.

Zebrafish IκB Kinase 1 Negatively Regulates NF-κB Activity

The IkappaB kinase (IKK) activity is critical for processing IkappaB inhibitory proteins and activating the NF-kappaB signaling, which is involved in a series of physiological and developmental steps in vertebrates. The IKK activity resides in two catalytic subunits, IKK1 and IKK2, and two regulatory subunits, NEMO and ELKS. IKK2 is the major cytokine-responsive IkappaB kinase because depletion of IKK1 does not interfere with the IKK activity. In fact, IKK1-/- mice display morphological abnormalities that are independent of its kinase activity and NF-kappaB activation. Hence, using zebrafish (Danio rerio) as a model, we examined the evolutionary role of IKK1 in modulating NF-kappaB. Ikk1-/- zebrafish embryos present head and tail malformations and, surprisingly, show upregulation of NF-kappaB-responsive genes and increased NF-kappaB-dependent apoptosis. Overexpression of ikk1 leads to midline structure defects that resemble NF-kappaB blockage in vivo. Zebrafish Ikk1 forms complexes with NEMO that represses NF-kappaB in vertebrate cells. Indeed, truncation of its NEMO binding domain (NBD) restores NF-kappaB-dependent transcriptional activity and, consequently, the ikk1-overexpressing phenotype. Here, we report that Ikk1 negatively regulates NF-kappaB by sequestering NEMO from active IKK complexes, indicating that IKK1 can function as a repressor of NF-kappaB.

A role for IκB kinase 2 in bipolar spindle assembly

IκB kinase 2 (IKK2 or IKKβ) is a component of the IKK complex that coordinates the cellular response to a diverse set of extracellular stimuli, including cytokines, microbial infection, and stress. In response to an external stimulus, the complex is activated, resulting in the phosphorylation and subsequent proteasome-mediated degradation of IκB proteins. This event triggers the nuclear import of the NF-κB transcription factor, which activates the transcription of genes that regulate a variety of fundamental biological processes, including immune response, cell survival, and development. Here, we define an essential role for IKK2 in normal mitotic progression and the maintenance of spindle bipolarity. Chemical and genetic perturbation of IKK2 promotes the formation of multipolar spindles and chromosome missegregation. Depletion of IKK2 results in the deregulation of Aurora A protein stability and coincident hyperactivation of a putative Aurora A substrate, the mitotic motor KIF11. These data support a function for IKK2 as an antagonist of Aurora A signaling during mitosis. Additionally, our results indicate a direct role for IKK2 in the maintenance of genome stability and underscore the potential for oncogenic consequences in targeting this kinase for therapeutic intervention.

Inhibitors of NF-κB Activity

A part from being a paradigm for understanding cellular signaling, the NF-κB pathway has been thoroughly investigated over the last two decades due to its involvement in a number of human diseases. In the post genomic era, improved knowledge and novel technologies have contributed immensely to the discovery of several hitherto unknown cellular processes that regulate NF-κB. Identification of covalent modifications of many NF-κB pathway components, both in the cytoplasm and the nucleus has shed light on novel mechanisms that regulate NF-κB activity. Similarly, study of a number of cellular and viral proteins that regulate this pathway has added to our understanding of the molecular mechanisms and molecular targets in the NF-κB pathway for drug development.