Search

Two related kinases, IkappaB kinase alpha (IKKalpha) and IKKbeta, phosphorylate the IkappaB proteins, leading to their degradation and the subsequent activation of gene expression by NF-kappaB. IKKbeta has a much higher level of kinase activity for the IkappaB proteins than does IKKalpha and is more critical than IKKalpha in modulating tumor necrosis factor alpha activation of the NF-kappaB pathway. These results indicate an important role for IKKbeta in activating the NF-kappaB pathway but leave open the question of the role of IKKalpha in regulating this pathway. In the current study, we demonstrate that IKKalpha directly phosphorylates IKKbeta. Moreover, IKKalpha either directly or indirectly enhances IKKbeta kinase activity for IkappaBalpha. Finally, transfection studies to analyze NF-kappaB-directed gene expression suggest that IKKalpha is upstream of IKKbeta in activating the NF-kappaB pathway. These results indicate that IKKalpha, in addition to its previously described ability to phosphorylate IkappaBalpha, can increase the ability of IKKbeta to phosphorylate IkappaBalpha.

The IkappaB kinases IKKalpha and IKKbeta are critical in activating the NF-kappaB pathway. Although these proteins have a similar structure that includes kinase, leucine zipper, and helix-loop-helix domains, they exhibit marked differences in their kinase activity and functional properties. For example, IKKbeta has a 10-20-fold higher level of kinase activity for IkappaBalpha than does IKKalpha. Furthermore, disruption of the murine IKKbeta gene, but not the IKKalpha gene, results in severe defects in activating the NF-kappaB pathway. Mice lacking IKKbeta succumb to severe hepatic apoptosis because of failure to activate the NF-kappaB pathway, whereas mice deficient in IKKalpha exhibit skin and skeletal abnormalities and an embryonic lethal phenotype. To better characterize differences in the functional properties of these kinases, hybrid IKK proteins were constructed by domain swapping, and their kinase activity was assayed. These studies demonstrated that differences in the IKKalpha and IKKbeta helix-loop-helix domains are primarily responsible for differences in their kinase activity. In contrast, their kinase and leucine zipper domains exhibited relatively conserved function. These studies further define the properties of IKKalpha and IKKbeta, which are involved in their unique regulatory roles.

Sulindac is a non-steroidal anti-inflammatory agent that is related both structurally and pharmacologically to indomethacin. In addition to its anti-inflammatory properties, sulindac has been demonstrated to have a role in the prevention of colon cancer. Both its growth inhibitory and anti-inflammatory properties are due at least in part to its ability to decrease prostaglandin synthesis by inhibiting the activity of cyclooxygenases. Recently, we demonstrated that both aspirin and sodium salicylate, but not indomethacin, inhibited the activity of an IkappaB kinase beta (IKKbeta) that is required to activate the nuclear factor-kappaB (NF-kappaB) pathway. In this study, we show that sulindac and its metabolites sulindac sulfide and sulindac sulfone can also inhibit the NF-kappaB pathway in both colon cancer and other cell lines. Similar to our previous results with aspirin, this inhibition is due to sulindac-mediated decreases in IKKbeta kinase activity. Concentrations of sulindac that inhibit IKKbeta activity also reduce the proliferation of colon cancer cells. These results suggest that the growth inhibitory and anti-inflammatory properties of sulindac may be regulated in part by inhibition of kinases that regulate the NF-kappaB pathway.

Two cytokine-inducible kinases, IKKalpha and IKKbeta, are components of a 700-kDa kinase complex that specifically phosphorylates IkappaB. Phosphorylation of IkappaB by IKK leads to its ubiquitination and subsequent degradation, resulting in the nuclear translocation of NF-kappaB. The oncogenic protein Tax, encoded by human T-cell leukemia virus type-1 (HTLV-1), stimulates IKK activity to result in constitutive nuclear levels of NF-kappaB. In an attempt to gain insights into the mechanism by which Tax mediates constitutive activation of the NF-kappaB pathway, we analyzed the chromatographic distribution of IKK proteins using cellular extracts prepared from three T lymphocytes either lacking or containing Tax. IKK kinase activity and the distribution of proteins in the IKK complex were characterized. In extracts prepared from cells containing Tax, the activity of both IKKalpha and IKKbeta present in the 700-kDa IKK complex were increased. Surprisingly, cell lines expressing Tax also contained an additional peak of IKKbeta, but not IKKalpha activity, that migrated at 300 kDa rather than at 700 kDa. We noted that extracts containing Tax had extremely low levels of IkappaBbeta, but not IkappaBalpha, and contained predominantly a truncated form of the MAP3K MEKK1. These results suggest that Tax may target several components of the NF-kappaB pathway leading to constitutive activation of this important regulator of cellular gene expression.

The transcriptional regulation of the human immunodeficiency virus (HIV) type I involves the interaction of both viral and cellular proteins. The viral protein tat is important in increasing the amount of viral steady‐state mRNA and may also play a role in regulating the translational efficiency of viral mRNA. To identify distinct functional domains of tat, oligonucleotide‐directed mutagenesis of the tat gene was performed. Point mutations of cysteine residues in three of the four Cys‐X‐X‐Cys sequences in the tat protein resulted in a marked decrease in transcriptional activation of the HIV long terminal repeat. Point mutations which altered the basic C‐domain of the protein also resulted in decreases in transcriptional activity, as did a series of mutations that repositioned either the N or C termini of the protein. Conservative mutations of other amino acids in the cysteine‐rich or basic regions and in a series of proline residues in the N terminus of the molecule resulted in minimal changes in tat activation. These results suggest that several domains of tat protein are involved in transcriptional activation with the cysteine‐rich domain being required for complete activity of the tat protein.

The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP‐1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide‐directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat‐activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem‐loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide‐directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat‐activation. This indicated a significant role for the stem‐loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat‐activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted this stem and loop mutation relative to the HIV LTR mRNA start site resulted in even larger decreases in tat‐activation. This suggests that multiple determinants, including protein binding, the loop sequence, and RNA or DNA secondary structure, are important in tat‐activation and suggests that tat may interact with cellular proteins binding to DNA to increase HIV gene expression.

The human immunodeficiency virus (HIV) is a human retrovirus which is the etiologic agent of the acquired immunodeficiency syndrome. To study the cellular factors involved in the transcriptional regulation of this virus, we performed DNase I footprinting of the viral LTR using partially purified HeLa cell extracts. Five regions of the viral LTR appear critical for DNA binding of cellular proteins. These include the negative regulatory, enhancer, SP1, TATA and untranslated regions. Deletion mutagenesis of these binding domains has significant effects on the basal level of transcription and the ability to be induced by the viral tat protein. Mutations of either the negative regulatory or untranslated regions affect factor binding to the enhancer region. In addition, oligonucleotides complementary to several of the binding domains specifically compete for factor binding. These results suggest that interactions between several distinct cellular proteins are required for HIV transcriptional regulation.

Transcription of the human immunodeficiency virus type 1 (HIV‐1) is regulated by viral proteins and cellular factors that bind to the viral long terminal repeat (LTR). At least five regions of the HIV LTR serve as binding sites for HeLa cellular proteins. One region containing two copies of the sequence GGGACTTTCC functions as an enhancer element for HIV transcriptional regulation. Another region between −17 and +44 known as the TAR region contains two copies of the sequence CTCTCTGG and is also important in tat‐induced activation of the HIV LTR. HeLa cell extracts were used to purify cellular proteins binding to portions of the enhancer region (EBP‐1) and the TAR region (UBP‐1) by a combination of conventional and DNA affinity chromatography. Several species of proteins of between 55 and 60 kd were found to bind to specific sequences in the enhancer region and these proteins also bound to a portion of the NF‐kappa B binding site in the immunoglobulin kappa enhancer. Two proteins of between 61 and 63 kd were the major species found to bind to specific sequences in the TAR region and fractions containing these proteins also bind to the TATA region. Both UBP‐1 and EBP‐1 exhibited specific binding as demonstrated by both UV cross‐linking and DNase I footprinting. Mutations of either the enhancer or TAR regulatory regions prevented binding of these purified factors. These results demonstrate the binding of highly purified cellular proteins to important transcriptional regulatory regions in the HIV LTR.

Transcription from all early adenovirus promoters is stimulated by a 289 amino acid phosphoprotein encoded in the pre-early transcription unit E1A. To determine if this protein could act on a nonviral gene placed on the viral chromosome, adenovirus recombinants were constructed in which the rat preproinsulin I gene, including its promoter region, was substituted in both orientations for E1A. Preproinsulin mRNA synthesis from these recombinants was greatly stimulated after infection of line 293 cells, which constitutively express E1A protein, compared to HeLa cells, which do not. Expression of the preproinsulin gene was also greatly stimulated when HeLa cells were coinfected with the recombinants and wild-type adenovirus or a mutant defective in a second E1A protein, but much less so by coinfection with a mutant defective in the 289 amino acid phosphoprotein. Much of the E1A-induced preproinsulin mRNA had a 5' end at the same position as the preproinsulin mRNA isolated from insulinoma cells, but a considerable fraction had 5' ends mapping heterogeneously within several hundred nucleotides of this site. Preproinsulin mRNA was also detected in 293 cells but not HeLa or HEK cells after transfection of a plasmid containing the preproinsulin gene with no adenovirus sequence. This indicates that there is no cis-acting adenovirus sequence required for E1A protein stimulation of preproinsulin transcription. Infection of rat cells with adenovirus did not induce detectable mRNA synthesis from the endogenous preproinsulin I gene. These results demonstrate that the E1A protein can induce expression of a nonviral gene when it is newly introduced into mammalian cells by viral infection or transfection, but it does not induce the endogenous cellular gene.

Nuclease hypersensitive sites exist in vivo in the chromatin of the integrated human immunodeficiency virus (HIV)-1 proviral genome, in the 5'-long terminal repeat (LTR) within the promoter/enhancer region near Sp1 and NFkappaB binding sites. Previous studies from the Kadonaga and Jones laboratories have shown that Sp1 and NFkappaB can establish hypersensitive sites in a truncated form of this LTR when added before in vitro chromatin assembly with Drosophila extracts, thus facilitating subsequent transcriptional activation of a linked reporter gene upon the association of additional factors (Pazin, M. J., Sheridan, P. L., Cannon, K., Cao, Z., Keck, J. G., Kadanaga, J. T., and Jones, K. A. (1996) Genes & Dev. 10, 37-49). Here we assess the role of a full-length LTR and 1 kilobase pair of downstream flanking HIV sequences in chromatin remodeling when these transcription factors are added after chromatin assembly. Using Xenopus laevis oocyte extracts to assemble chromatin in vitro, we have confirmed that Sp1 and NFkappaB can indeed induce sites hypersensitive to DNase I, micrococcal nuclease, or restriction enzymes on either side of factor binding sites in chromatin but not naked DNA. We extend these earlier studies by demonstrating that the process is ATP-dependent when the factors are added after chromatin assembly and that histone H1, AP1, TBP, or Tat had no effect on hypersensitive site formation. Furthermore, we have found that nucleosomes upstream of NFkappaB sites are rotationally positioned prior to factor binding and that their translational frame is registered after binding NFkappaB. On the other hand, binding of Sp1 positions adjacent downstream nucleosome(s). We term this polar repositioning because each factor aligns nucleosomes only on one side of its binding sites. Mutational analysis and oligonucleotide competition each demonstrated that this remodeling required Sp1 and NFkappaB binding sites.

A positive regulatory element in the interleukin-2 (IL-2) promoter, designated the antigen receptor response element-2, is essential for the induction of IL-2 gene expression upon the binding of an inducible multiprotein complex of proteins known as nuclear factor of activated T cells. In the current study, we demonstrated that the winged-helix transcription factor IL-2 enhancer binding factor (ILF) is constitutively expressed in both resting and activated Jurkat cells and binds to two adjacent sequence motifs immediately downstream of the binding site for NFAT. One of these elements has a high degree of homology with consensus binding sites for a variety of winged-helix DNA binding proteins, and the second site functions to modulate ILF binding. Mutagenesis of each of the two sequence elements required for ILF binding decreased IL-2 promoter activity when assayed in transfection assays. Although ILF bound constitutively to the IL-2 promoter, it was not detected as a component of the NFAT complex. These results suggest that important regulatory sequences in the IL-2 promoter are bound by ILF and that this binding may be involved in the control of IL-2 gene expression.

The regulation of human T-cell leukemia virus type 1 (HTLV-1) gene expression is dependent on three cis-acting elements, known as the 21-bp repeats, in the long terminal repeat. Each of the 21-bp repeats contains a nonpalindromic cyclic AMP response element (CRE) sequence which is capable of binding members of the ATF/CREB family of transcription factors. The HTLV-1 transactivator protein Tax is able to markedly stimulate the in vitro binding of CREB to the CRE sites present in each of the 21-bp repeats but not to CRE sites present in cellular promoters. The ability to Tax to stimulate CREB binding to different CRE sites correlates with the ability of Tax to activate gene expression from these sites. We wished to determine how sequence differences between the somatostatin CRE and the 21-bp repeat were involved in this different response to Tax. Scatchard analysis indicated that CREB bound to the somatostatin CRE with a single class of high-affinity binding while CREB bound to the 21-bp repeats with a biphasic binding pattern, indicating the presence of both low- and high-affinity binding. Tax increased the affinity of CREB binding but not that of another ATF/CREB protein, CREB2, to the 21-bp repeat. However, Tax did not increase affinity of binding of CREB to the somatostatin CRE. To determine the mechanism by which Tax increased dCREB binding affinity, immobilized oligonucleotides corresponding to either the 21-bp repeat or the somatostatin CRE were used to demonstrate that Tax formed a highly specific complex with CREB on the 21-bp repeat but not on the somatostatin CRE. These results indicate that formation of a complex between Tax and CREB results in specific high-affinity binding of this ternary complex to the HTLV-1 21 bp repeats.

The HIV-1 (human immunodeficiency virus type 1) and HIV-2 Tat proteins increase the level of transcription from their corresponding long terminal repeats. Tat activates transcription likely by interaction with components of the transcriptional initiation and elongation complexes during different stages of the transcription reaction. In the current study, two approaches were used to address the sites at which Tat becomes stably associated with the HIV transcription complex. First, we isolated column purified HIV-1 and HIV-2 transcription complexes that were competent for in vitro transcription and found that wild-type but not mutant Tat protein was specifically associated with this complex. An intact HIV TATA element and the presence of functional TATA-binding protein were necessary for Tat association. In contrast, the HIV-1 and HIV-2 TAR bulge sequences which serve as binding sites for Tat were not required for its association with the HIV preinitiation complex. A second complementary approach using immobilized HIV-1 and HIV-2 templates also demonstrated a functional association of Tat with HIV-1 and HIV-2 preinitiation complexes. Wild-type but not mutant Tat proteins associated with transcription complexes assembled on immobilized HIV-1 and HIV-2 templates and the association of Tat correlated with increases in the level of in vitro transcription. These results indicate that Tat can associate with HIV-1 and HIV-2 transcription complexes prior to the initiation of transcription by RNA polymerase II.

Gene expression from the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is mediated by three cis-acting regulatory elements known as 21-base pair (bp) repeats in addition to the transactivator protein Tax. Each of the 21-bp repeats contain nucleotide sequences which are homologous to a cAMP response element (CRE) which bind members of the ATF/CREB family of transcription factors. In this study, we investigated whether CREB alone or in the presence of Tax was able to induce DNA structural changes when bound to CRE sites in the HTLV-I 21 bp, the cellular somatostatin promoter, or a hybrid CRE construct comprised of both the somatostatin and 21-bp repeat sequences. Circular permutation analysis indicated that CREB was able to induce DNA flexure upon binding to each of these elements. However, phasing analysis, which is a more sensitive method to determine the degree and orientation of directed DNA bending, demonstrated that CREB induced DNA bending of the HTLV-I 21-bp repeat and the hybrid CRE but not the somatostatin CRE. The addition of Tax did not change CREB-mediated bending of the 21-bp repeat or the hybrid CRE although it markedly increased the amount of CREB bound to each of these DNA elements. These results indicate that sequence motifs flanking the CRE in the 21-bp repeat are critical for inducing DNA structural changes and that these changes are likely important in mediating Tax activation of the HTLV-I LTR.

A double-stranded RNA structure transcribed from the HIV-1 long terminal repeat known as TAR is critical for increasing gene expression in response to the transactivator protein Tat. Two cellular factors, RNA polymerase II and TRP-185, bind specifically to TAR RNA, but require the presence of cellular proteins known as cofactors which by themselves are unable to bind to TAR RNA. In an attempt to determine the mechanism by which these cofactors stimulate binding to TAR RNA, we purified these factors from HeLa nuclear extract and amino acid microsequence analysis performed. Three proteins were identified in the cofactor fraction including two previously described proteins, elongation factor 1alpha (EF-1alpha) and the polypyrimidine tract-binding protein (PTB), and a novel protein designated the stimulator of TAR RNA-binding proteins (SRB). SRB has a high degree of homology with a variety of cellular proteins known as chaperonins. Recombinant EF-1alpha, PTB, and SRB produced from vaccinia expression vectors stimulated the binding of RNA polymerase II and TRP-185 to TAR RNA in gel retardation analysis. These studies define a group of cellular factors that function in concert to stimulate the binding of TRP-185 and RNA polymerase II to HIV-1 TAR RNA.

Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) are human retroviruses which normally infect T-lymphoid cells. HTLV-I infection is associated with adult T-cell leukemia-lymphoma, and HTLV-II is associated with an indolent form of hairy-cell leukemia. To identify potential transcriptional regulatory elements of these two related human retroviruses, we performed DNase I footprinting of both the HTLV-I and HTLV-II long terminal repeats (LTRs) by using extracts prepared from uninfected T cells, HTLV-I and HTLV-II transformed T cells, and HeLa cells. Five regions of the HTLV-I LTR and three regions of the HTLV-II LTR showed protection by DNase I footprinting. All three of the 21-base-pair repeats previously shown to be important in HTLV transcriptional regulation were protected in the HTLV-I LTR, whereas only one of these repeats was protected in the HTLV-II LTR. Several regions exhibited altered protection in extracts prepared from lymphoid cells as compared with HeLa cells, but there were minimal differences in the protection patterns between HTLV-infected and uninfected lymphoid extracts. A number of HTLV-I and HTLV-II LTR fragments which contained regions showing protection in DNase I footprinting were able to function as inducible enhancer elements in transient CAT gene expression assays in the presence of the HTLV-II tat protein. The alterations in the pattern of the cellular proteins which bind to the HTLV-I and HTLV-II LTRs may in part be responsible for differences in the transcriptional regulation of these two related viruses.

Adenovirus early region 1A (E1A) encodes two acidic phosphoproteins which are required for transactivation of viral transcription, efficient viral DNA replication in phase G0-arrested human cells, and oncogenic transformation of rodent cells. Biochemical analysis of in vivo 32P-labeled adenovirus type 2 E1A proteins purified with monoclonal antibodies demonstrated that these proteins were phosphorylated at multiple serine residues. Two-dimensional phosphotryptic peptide maps of wild-type and mutant E1A proteins were used to locate a major site of E1A protein phosphorylation at serine-219 of the large E1A protein. Although this serine fell within a consensus sequence for phosphorylation by the cyclic AMP-dependent protein kinases, experiments with mutant CHO cells defective in these enzymes indicated that it was not. Oligonucleotide-directed mutagenesis was used to substitute an alanine for serine-219. This mutation prevented phosphorylation at this site. Nonetheless, the mutant was indistinguishable from the wild type for early gene transactivation, replication on G0-arrested WI-38 cells, and transformation of cloned rat embryo fibroblast cells.

Proteins encoded in regions EIA and EIB of human adenoviruses cause transformation of rodent cells. One protein from EIA also stimulates transcription of other early regions at early times in a productive infection. In the past, direct analysis of these proteins synthesized in vivo has been difficult because of the low levels produced in both transformed cells and productively infected cells. We present a simple method which leads to expression of EIA and EIB mRNAs and proteins at 30-fold greater levels than those observed during the early phase of a standard productive infection. Under these conditions, these proteins are among the most prominent translation products of infected cells. This allowed direct visualization of EIA and EIB proteins on two-dimensional gels of pulse-labeled total cell protein. Experiments with EIA and EIB mutants confirm that the identified proteins are indeed encoded in these regions. Two EIA proteins are observed, one translated from each of the major early EIA mRNAs. Both of these EIA proteins are phosphorylated.