Cytokine-induced activation of the IkappaB kinases (IKK) IKK-alpha and IKK-beta is a key step involved in the activation of the NF-kappaB pathway. Gene-disruption studies of the murine IKK genes have shown that IKK-beta, but not IKK-alpha, is critical for cytokine-induced IkappaB degradation. Nevertheless, mouse embryo fibroblasts deficient in IKK-alpha are defective in the induction of NF-kappaB-dependent transcription. These observations raised the question of whether IKK-alpha might regulate a previously undescribed step to activate the NF-kappaB pathway that is independent of its previously described cytoplasmic role in the phosphorylation of IkappaBalpha. Here we show that IKK-alpha functions in the nucleus to activate the expression of NF-kappaB-responsive genes after stimulation with cytokines. IKK-alpha interacts with CREB-binding protein and in conjunction with Rel A is recruited to NF-kappaB-responsive promoters and mediates the cytokine-induced phosphorylation and subsequent acetylation of specific residues in histone H3. These results define a new nuclear role of IKK-alpha in modifying histone function that is critical for the activation of NF-kappaB-directed gene expression.

SPT5 and its binding partner SPT4 function in both positively and negatively regulating transcriptional elongation. The demonstration that SPT5 and RNA polymerase II are targets for phosphorylation by CDK9/cyclin T1 indicates that posttranslational modifications of these factors are important in regulating the elongation process. In this study, we utilized a biochemical approach to demonstrate that SPT5 was specifically associated with the protein arginine methyltransferases PRMT1 and PRMT5 and that SPT5 methylation regulated its interaction with RNA polymerase II. Specific arginine residues in SPT5 that are methylated by these enzymes were identified and demonstrated to be important in regulating its promoter association and subsequent effects on transcriptional elongation. These results suggest that methylation of SPT5 is an important posttranslational modification that is involved in regulating its transcriptional elongation properties in response to viral and cellular factors.

The NF-B pathway is important in the control of the immune and inflammatory response. One of the critical events in the activation of this pathway is the stimulation of the IB kinases (IKKs) by cytokines such as tumor necrosis factor-␣ and interleukin-1. Although the mechanisms that modulate IKK activation have been studied in detail, much less is known about the processes that down-regulate its activity following cytokine treatment. In this study, we utilized biochemical fractionation and mass spectrometry to demonstrate that protein phosphatase 2C␤ (PP2C␤) can associate with the IKK complex. PP2C␤ association with the IKK complex led to the dephosphorylation of IKK␤ and decreased its kinase activity. The binding of PP2C␤ to IKK␤ was decreased at early times post-tumor necrosis factor-␣ treatment and was restored at later times following treatment with this cytokine. Experiments utilizing siRNA directed against PP2C␤ demonstrated an in vivo role for this phosphatase in decreasing IKK activity at late times following cytokine treatment. These studies are consistent with the ability of PP2C␤ to down-regulate cytokine-induced NF-B activation by altering IKK activity.The NF-B pathway is a critical regulator of the cellular response to a variety of stimuli including cytokines such as TNF␣ 1 and interleukin-1, bacterial and viral infection, and double-stranded RNA (1-7). Cytokines lead to a rapid increase in the activity of the IB kinases, and this is followed by a subsequent decrease in the activity of these kinases, suggesting both positive and negative regulation of the NF-B pathway. A better understanding of the NF-B pathway will be important in defining how these factors modulate the host immune and inflammatory response and prevent apoptosis (1-7).The NF-B transcription factors, p105/50, p100/52, p65, cRel, and RelB, contain a Rel homology domain that mediates their dimerization and DNA binding properties (2). These proteins are sequestered in the cytoplasm of most cells, where they are bound to a family of inhibitory proteins known as IB (1, 3). Treatment of cells with cytokines, including TNF␣ and interleukin-1, stimulates the activity of IB kinases that phosphorylate IB on amino-terminal serine residues, leading to its ubiquitination and degradation by the proteasome (3-7). This process results in the nuclear translocation of the NF-B proteins where they bind to the promoter elements of a variety of genes involved in the control of the immune and inflammatory response (1-7).Activation of the IB kinases is a critical process in regulating the NF-B pathway (7-12). These kinases, designated IKK␣ and IKK␤, are components of a 600 -900-kDa complex (7-12), which also includes a scaffold protein IKK␥/NEMO (13-16) and the chaperone proteins Hsp90 and Cdc37 (17). In addition to binding to IKK␣ and IKK␤, IKK␥/NEMO has been demonstrated to bind to a variety of other proteins that have been reported to be involved in the regulation of the NF-B pathway, including RIP, A20, CIKS, and the HTLV-I Tax protein (18 -22).Althou...

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The IB kinase (IKK) complex consists of the catalytic subunits IKK␣ and IKK␤ and a regulatory subunit, IKK␥/NEMO. Even though IKK␣ and IKK␤ share significant sequence similarity, they have distinct biological roles. It has been demonstrated that IKKs are involved in regulating the proliferation of both normal and tumor cells, although the mechanisms by which they function in this process remain to be better defined. In this study, we demonstrate that IKK␣, but not IKK␤, is important for estrogen-induced cell cycle progression by regulating the transcription of the E2F1 gene as well as other E2F1-responsive genes, including thymidine kinase 1, proliferating cell nuclear antigen, cyclin E, and cdc25A. The role of IKK␣ in regulating E2F1 was not the result of reduced levels of cyclin D1, as overexpression of this gene could not overcome the effects of IKK␣ knock-down. Furthermore, estrogen treatment increased the association of endogenous IKK␣ and E2F1, and this interaction occurred on promoters bound by E2F1. IKK␣ also potentiated the ability of p300/CBP-associated factor to acetylate E2F1. Taken together, these data suggest a novel mechanism by which IKK␣ can influence estrogen-mediated cell cycle progression through its regulation of E2F1.The mammalian cell cycle is controlled by a series of highly regulated processes, and its dysregulation is frequently associated with growth abnormalities including the development of cancer (reviewed in Refs. 1-3). The Rb/E2F pathway, which governs the G 1 to S phase transition, is one of the most important pathways that regulate the cell cycle. A major function of Rb is to sequester E2F family transcription factors and repress E2F-regulated promoters (4). The Rb family of proteins, which includes Rb, p107, and p130, forms different complexes with the E2F family members (5, 6). In the early G 1 phase, the activated cyclin D-CDK4 complex phosphorylates Rb, leading to its degradation and the release of E2F. This process in turn activates the expression of cyclin E and other genes required for DNA replication (1). Cyclin E then binds to CDK2 to further phosphorylate Rb, thus forming a positive feedback loop to promote the entry of cells into the S phase. Abnormalities in this pathway and in the p53 tumor suppressor pathway are seen in almost all human tumors (4, 7).The E2F family, which is critical for cell cycle progression from the late G 1 into S phase, comprises seven members, designated E2F1-7, and can be further classified into three subfamilies based on sequence homology and function: E2F1-3, E2F4 -5, and E2F6 -7 (5, 6, 8).Although E2F1-3 are positive regulators of gene expression, E2F4 -5 are transcriptional repressors when bound to Rb family proteins, and E2F6 functions as a transcriptional repressor given the fact that it lacks a transactivation domain (1, 5). The E2F family members form complexes with the DP proteins (DP-1 and DP-2) to activate the transcription of genes important for DNA replication (such as thymidine kinase 1 (TK1), 3 proliferating cell nuclear antig...

The IKK complex includes two catalytic components, IKKalpha and IKKbeta, in addition to the scaffold protein IKKgamma/NEMO. Even though IKKalpha and IKKbeta share significant sequence homology, they have distinct biological roles with IKKbeta regulates the classical pathway of NF-kappaB activation and IKKalpha regulates the alternative pathways. In addition, it has been shown that the IKKs regulate the proliferation of both normal and tumor cells; however, the mechanisms by which the IKKs regulate the cell cycle remain to be further defined. Here, we demonstrate that IKKalpha, but not IKKbeta, has role in regulating the M phase of the cell cycle. IKKalpha siRNA knock -down resulted in increased numbers of cells in the G(2)/M phase of the cell cycle as compared to control and IKKbeta siRNA transfected HeLa cells. This effect was associated with upregulation of cyclin B1 and Plk1 protein levels and increased histone H3 phosphorylation, consistent with a potential role of IKKalpha in the regulation of M phase regulatory factors. IKKalpha was found to be associated with Aurora A in the centrosome and regulate Aurora A phosphorylation at threonine residue 288, a site which is important in modulating its kinase activity. Taken together, these data provide the evidence that IKKalpha regulates the M phase of the cell cycle by modulating Aurora A phosphorylation and activation leading to the regulation of the M phase of the cell cycle.

The IB kinase (IKK) complex includes the catalytic components IKK␣ and IKK␤ in addition to the scaffold protein IKK␥/NEMO. Increases in the activity of the IKK complex result in the phosphorylation and subsequent degradation of IB and the activation of the NF-B pathway. Recent data indicate that the constitutive activation of the NF-B pathway by the human T-cell lymphotrophic virus, type I, Tax protein leads to enhanced phosphorylation of IKK␥/NEMO by IKK␤. To address further the significance of IKK␤-mediated phosphorylation of IKK␥/NEMO, we determined the sites in IKK␥/ NEMO that were phosphorylated by IKK␤, and we assayed whether IKK␥/NEMO phosphorylation was involved in modulating IKK␤ activity. IKK␥/NEMO is rapidly phosphorylated following treatment of cells with stimuli such as tumor necrosis factor-␣ and interleukin-1 that activate the NF-B pathway. By using both in vitro and in vivo assays, IKK␤ was found to phosphorylate IKK␥/NEMO predominantly in its carboxyl terminus on serine residue 369 in addition to sites in the central region of this protein. Surprisingly, mutation of these carboxyl-terminal serine residues increased the ability of IKK␥/NEMO to stimulate IKK␤ kinase activity. These results indicate that the differential phosphorylation of IKK␥/NEMO by IKK␤ and perhaps other kinases may be important in regulating IKK activity.The NF-B pathway is a critical regulator of the cellular response to a variety of stimuli including the cytokines, TNF␣ 1 and IL-1, bacterial and viral infection, double-stranded RNA, and the human T-cell leukemia virus transactivator protein Tax (1-4). The ability to activate rapidly and subsequently silence the NF-B pathway in response to a variety of extracellular stimuli suggests that both positive and negative regulation is involved in its control. The further characterization of the mechanisms that regulate this pathway will be important for better understanding how NF-B is involved in the control of the host immune and inflammatory responses.The members of the NF-B family of transcription factors, which include p105/50, p100/52, p65, c-Rel, and RelB, contain a Rel homology domain that mediates their heterodimerization and homodimerization properties and DNA-binding properties (2). These proteins are sequestered in the cytoplasm of most cells where they are bound to a family of inhibitory proteins known as IB (1, 3). Treatment of cells with a variety of stimuli including the cytokines TNF␣ and IL-1 stimulate the activity of kinases that phosphorylate IB on amino-terminal serine residues resulting in its ubiquitination and degradation by the proteasome (3-6). This process leads to the nuclear translocation of the NF-B proteins, which then bind to consensus DNA sequences located upstream of a variety of cellular genes that are involved in the control of the immune and the inflammatory response and prevent apoptosis (1-3, 5-7).Activation of the IB kinases, which phosphorylate the IB proteins on the amino-terminal serine residues to result in their degradation, is a critical...

We have studied the alkaline unfolding of bovine liver catalase and its dependence on ionic strength by enzymic activity measurements and a combination of optical methods like circular dichroism, fluorescence and absorption spectroscopies. Under conditions of high pH (11.5) and low ionic strength, the native tetrameric enzyme dissociates into monomers with complete loss of enzymic activity and a significant loss of A-helical content. Increase in ionic strength by addition of salts like potassium chloride and sodium sulphate resulted in folding of alkaline-unfolded enzyme by association of monomers to tetramer but with significantly different structural properties compared to native enzyme. The salt-induced tetrameric intermediate is characterized by a significant exposure of the buried hydrophobic clusters and significantly reduced A-helical content compared to the native enzyme. The refolding/reconstitution studies showed that the salt-induced partially folded tetrameric intermediate shows significantly higher efficiency of refolding/reconstitution as compared to alkaline-denatured catalase in the absence of salts. These studies suggest that folding of multimeric enzymes proceeds probably through the hydrophobic collapse of partially folded multimeric intermediate with exposed hydrophobic clusters.Keywords : bovine liver catalase; alkaline unfolding; monomer ; salts ; refolding.Catalase is a highly active ubiquitous enzyme which occurs The extent of unfolding of denatured states of proteins under different conditions has long been of interest because of the pos-in almost all aerobically respiring organisms and in part serves sible relevance of their conformations to the protein folding to protect the cells from the toxic effects of hydrogen peroxide. pathways. It has been demonstrated that residual structural pref-Bovine liver catalase, molecular mass 240 kDa, contains four erences, ranging from local clusters of side chains to highly or-identical 57-kDa subunits each equipped with a high-spin Fe(III) dered side chains to highly ordered subdomains, persist in dena-protoporphyrin IX [18]. We have investigated the changes in the tured states of proteins [1Ϫ6]. Hence, there has been a growing structural and functional properties associated with the alkaline recognition of the importance of the compact denatured and par-denaturation of bovine liver catalase and also studied its depentially folded states of proteins, as characterization of these struc-dence on ionic strength. tures and the factors involved in their stability would provide important insight into the interactions responsible for their formation as well as their role in protein folding. EXPERIMENTAL PROCEDURES pH is known to influence the stability of a protein by alteringMaterials. Crystalline bovine liver catalase was prepared acthe net charge on the protein. Many proteins denature at extreme cording to the earlier reported method [19]. The purity of the pH because of the presence of destabilizing repulsive interacprotein was checked by SDS/PAGE foll...