Rapid lipopolysaccharide-induced differentiation of CD14(+) monocytes into CD83(+) dendritic cells is modulated under serum-free conditions by exogenously added IFN-gamma and endogenously produced IL-10.

We showed previously that about half of purified CD14(+) peripheral blood monocytes cultured under serum-free conditions and treated with GM-CSF and bacterial LPS rapidly (2 - 4 day) differentiate into CD83(+) dendritic cells (DC). The remaining cells retain the CD14(+)/CD83(-) monocyte/macrophage phenotype. In order to identify factors that influence whether monocytes differentiate into DC or remain on the monocyte/macrophage developmental pathway, we evaluated the effects of exogenously added IFN-gamma and endogenously produced IL-10 on the proportion and function of CD14(+) monocytes that adopt DC characteristics in response to LPS. IFN-gamma priming dramatically increased the proportion of monocytes that adopted stable DC characteristics in response to LPS, improved their T cell allosensitizing capacity, and enhanced levels of secreted IL-12 heterodimer. IFN-gamma priming also suppressed the production of IL-10, a cytokine known to have inhibitory effects on DC differentiation. When monocytes were treated with LPS plus IL-10-neutralizing antibodies, dramatically enhanced DC differentiation, IL-12 secretion, and T cell allosensitizing capacity were observed, mimicking in many respects the effects of IFN-gamma priming. IFN-gamma primed cells still displayed appreciable sensitivity to exogenously added IL-10, suggesting that attenuated IL-10 secretion is partially responsible for the enhancing effects of IFN-gamma. These studies therefore identify IFN-gamma as a DC differentiation co-factor for CD14(+) monocytes, and IL-10 as an autocrine/paracrine inhibitor of DC differentiation, linking these agents for the first time as mutually opposed regulators that govern whether CD14(+) cells differentiate into DC upon contact with LPS or remain on the monocyte/macrophage developmental pathway.

CD14+ monocytes as dendritic cell precursors: diverse maturation-inducing pathways lead to common activation of NF-kappab/RelB.

Dendritic cells are extremely potent antigen-presenting cells that are primarily responsible for the sensitization of naïve T cells to protein antigen in vivo. For this reason, dendritic cells are the focus of intense study. Despite this interest, relatively little information is available on the signal transduction pathways that regulate the development and activity of these cells. The last several years, however, have seen a steady accumulation of data regarding methods to cultivate large numbers of DC, the characterization of attendant signals that drive DC development from various precursor cells, and the induction of nuclear transcription factors that presumably direct alterations in gene expression that regulate aspects of DC development. In this review, we briefly summarize some of these findings, with emphasis on monocyte-derived dendritic cells and a discussion of two distinct types of signaling pathways that appear to regulate the final maturation of DC: one pathway calcium-dependent and cyclosporine A-sensitive, the other pathway CsA-insensitive. Although evidence suggests these signaling pathways are quite divergent in their upstream components, they both appear to activate NF-kappaB nuclear factors, particularly RelB.

Bacterial lipopolysaccharide, TNF-alpha, and calcium ionophore under serum-free conditions promote rapid dendritic cell-like differentiation in CD14+ monocytes through distinct pathways that activate NK-kappa B.

To facilitate the study of signaling pathways involved in myeloid dendritic cell (DC) differentiation, we have developed a serum-free culture system in which human CD14+ peripheral blood monocytes differentiate rapidly in response to bacterial LPS, TNF-alpha, or calcium ionophore (CI). Within 48-96 h, depending on the inducing agent, the cells acquire many immunophenotypical, morphological, functional, and molecular properties of DC. However, there are significant differences in the signaling pathways used by these agents, because 1) LPS-induced, but not CI-induced, DC differentiation required TNF-alpha production; and 2) cyclosporin A inhibited differentiation induced by CI, but not that induced by LPS. Nevertheless, all three inducing agents activated members of the NF-kappaB family of transcription factors, including RelB, suggesting that despite differences in upstream elements, the signaling pathways all involve NF-kappaB. In this report we also demonstrate and offer an explanation for two observed forms of the RelB protein and show that RelB can be induced in myeloid cells, either directly or indirectly, through a calcium-dependent and cyclosporin A-sensitive pathway.

Role of NF-kappaB in p53-mediated programmed cell death.

The tumour suppressor p53 inhibits cell growth through activation of cell-cycle arrest and apoptosis, and most cancers have either mutation within the p53 gene or defects in the ability to induce p53. Activation or re-introduction of p53 induces apoptosis in many tumour cells and may provide effective cancer therapy. One of the key proteins that modulates the apoptotic response is NF-kappaB, a transcription factor that can protect or contribute to apoptosis. Here we show that induction of p53 causes an activation of NF-kappaB that correlates with the ability of p53 to induce apoptosis. Inhibition or loss of NF-kappaB activity abrogated p53-induced apoptosis, indicating that NF-kappaB is essential in p53-mediated cell death. Activation of NF-kappaB by p53 was distinct from that mediated by tumour-necrosis factor-alpha and involved MEK1 and the activation of pp90rsk. Inhibition of MEK1 blocked activation of NF-kappaB by p53 and completely abrogated p53-induced cell death. We conclude that inhibition of NF-kappaB in tumours that retain wild-type p53 may diminish, rather than augment, a therapeutic response.

Similarities and differences between human and murine TNF promoters in their response to lipopolysaccharide.

Transcription of the TNF gene is rapidly and transiently induced by LPS in cells of monocyte/macrophage lineage. Previous data suggested that multiple NF-kappaB/Rel binding sites play a role in the transcriptional response to LPS of the murine gene. However, the relevance of homologous sites in the human TNF gene remained a matter of controversy, partly because the high affinity NF-kappaB/Rel site located at -510 in the murine promoter is not conserved in humans. Here we used two sets of similarly designed human and mouse TNF promoter deletion constructs and overexpression of IkappaB in the murine macrophage cell line ANA-1 to show remarkable similarity in the pattern of the transcriptional response to LPS, further demonstrating the functional role of the distal promoter region located between -600 and -650. This region was characterized by mutagenesis of protein binding sites, including two relatively low affinity NF-kappaB/Rel sites, #2 and 2a. Mutation in each of the NF-kappaB sites resulted in 2- to 3-fold lower transcriptional activity in response to LPS. In contrast to LPS activation, the response to PMA was substantially lower in magnitude and required only the proximal promoter region. In summary, the functional topography of human and murine promoters when assayed in the same system has some marked similarities. Our observations support the notion that full LPS response of TNF gene requires both NF-kappaB and non-NF-kappaB nuclear proteins. Our data also suggest that the functional activity of a given kappaB site depends on the entire DNA sequence context in the promoter region.

E2F-1 potentiates cell death by blocking antiapoptotic signaling pathways.

The E2F family of transcription factors plays an essential role in promoting cell cycle progression, and one member of the family, E2F-1, is also capable of inducing apoptosis. We show here that E2F-1 can induce apoptosis by a death receptor-dependent mechanism, by downregulating TRAF2 protein levels and inhibiting activation of antiapoptotic signals including NF-kappa B. In this way, E2F-1 expression can lead to the sensitization of cells to apoptosis by a number of agents independently of p53. Deregulation of E2F-1 activity occurs in the majority of human tumors, and the ability of E2F-1 to inhibit antiapoptotic signaling may contribute to the enhanced sensitivity of transformed cells to chemotherapeutic agents.

The N-terminal domain of IkappaB alpha masks the nuclear localization signal(s) of p50 and c-Rel homodimers.

Members of the Rel/NF-kappaB family of transcription factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding, dimerization, and binding to inhibitor. At the C-terminal end of the RHD, each protein has a nuclear localization signal (NLS). The crystal structures of the p50 and RelA family members show that the RHD consists of two regions: an N-terminal section which contains some of the DNA contacts and a C-terminal section which contains the remaining DNA contacts and controls dimerization. In unstimulated cells, the homo- or heterodimeric Rel/NF-kappaB proteins are cytoplasmic by virtue of binding to an inhibitor protein (IkappaB) which somehow masks the NLS of each member of the dimer. The IkappaB proteins consist of an ankyrin-repeat-containing domain that is required for binding to dimers and N- and C-terminal domains that are dispensable for binding to most dimers. In this study, we examined the interaction between IkappaB alpha and Rel family homodimers by mutational analysis. We show that (i) the dimerization regions of p50, RelA, and c-Rel are sufficient for binding to IkappaB alpha, (ii) the NLSs of RelA and c-Rel are not required for binding to IkappaB alpha but do stabilize the interaction, (iii) the NLS of p50 is required for binding to IkappaB alpha, (iv) only certain residues within the p50 NLS are required for binding, and (v) in a p50-IkappaB alpha complex or a c-Rel-IkappaB alpha complex, the N terminus of IkappaB alpha either directly or indirectly masks one or both of the dimer NLSs.

Reconstitution of the NF-kappa B system in Saccharomyces cerevisiae for isolation of effectors by phenotype modulation.

NF-kappa B is a ubiquitous transcription factor that contributes to the induction of many genes playing a central role in immune and inflammatory responses. The NF-kappa B proteins are subject to multiple regulatory influences including post-translational modifications such as phosphorylation and proteolytic processing. A very important component of this regulation is the control of their subcellular localization: cytoplasmic retention of NF-kappa B is achieved through interaction with I kappa B molecules. In response to extracellular signals, these molecules undergo degradation, NF-kappa B translocates to the nucleus and activates its target genes. To investigate novel proteins involved in this dynamic response, we have reconstituted the NF-kappa B/I kappa Beta system in the yeast Saccharomyces cerevisiae. We have successively introduced p65, the main transcriptional activator of the NF-kappa B family, which leads to the activation of two reporter genes controlled by kappa B sites, and the I kappa B alpha inhibitory protein, which abolishes this activation. By transforming such a yeast strain with a cDNA library we have performed a genetic screen for cDNAs encoding proteins capable of either dissociating the p65/I kappa B alpha complex or directly transactivating the expression of the reporter genes. The efficiency of our screen was demonstrated by the isolation of a cDNA encoding the p105 precursor of the p50 subunit of NF-kappa B. We also used this system to test stimuli known to activate signalling pathways in yeast, in order to investigate whether the related mammalian cascades might be involved in NF-kappa B activation. We showed that yeast endogenous kinase cascades activated by pheromone, hypo- or hyperosmotic shock cannot modulate NF-kappa B activity in our system, and that the p38 human MAP kinase does not act directly on the p65/I kappa B alpha complex.

Expression of NFAT-family proteins in normal human T cells.

NFAT proteins constitute a family of transcription factors involved in mediating signal transduction. Using a panel of specific antisera in immunoprecipitation assays, we found that NFATp (135 kDa) is constitutively expressed in normal human T cells, while synthesis of NFATc (predominant form of 86 kDa) is induced by ionomycin treatment. NFAT4/x was very weakly expressed in unstimulated cells, and its level did not increase upon treatment with activating agents. NFAT3 protein was not observed under any conditions. Higher-molecular-weight species of NFATc (of 110 and 140 kDa) were also detected. In addition, translation of NFATc mRNA apparently initiates at two different AUG codons, giving rise to proteins that differ in size by 36 amino acids. Additional size heterogeneity of both NFATc and NFATp results from phosphorylation. In contrast to ionomycin treatment, exposure of cells to phorbol myristate acetate (PMA) plus anti-CD28 did not induce NFATc, indicating that under these conditions, interleukin-2 synthesis by these cells is apparently independent of NFATc. In DNA binding assays, both PMA plus anti-CD28 and PMA plus ionomycin resulted in nuclear NFAT. Surprisingly, the PMA-ionomycin-induced synthesis of NFATc that was detected by immunoprecipitation was not mirrored in the DNA binding assays: nearly all of the activity was due to NFATp. This is the first study of expression of all family members at the protein level in normal human T cells.

I kappa B epsilon, a novel member of the I kappa B family, controls RelA and cRel NF-kappa B activity.

We have isolated a human cDNA which encodes a novel I kappa B family member using a yeast two-hybrid screen for proteins able to interact with the p52 subunit of the transcription factor NF-kappa B. The protein is found in many cell types and its expression is up-regulated following NF-kappa B activation and during myelopoiesis. Consistent with its proposed role as an I kappa B molecule, I kappa B-epsilon is able to inhibit NF-kappa B-directed transactivation via cytoplasmic retention of rel proteins. I kappa B-epsilon translation initiates from an internal ATG codon to give rise to a protein of 45 kDa, which exists as multiple phosphorylated isoforms in resting cells. Unlike the other inhibitors, it is found almost exclusively in complexes containing RelA and/or cRel. Upon activation, I kappa B-epsilon protein is degraded with slow kinetics by a proteasome-dependent mechanism. Similarly to I kappa B-alpha and I kappa B, I kappa B-epsilon contains multiple ankyrin repeats and two conserved serines which are necessary for signal-induced degradation of the molecule. A unique lysine residue located N-terminal of the serines appears to be not strictly required for degradation. Unlike I kappa B- alpha and I kappa B-beta, I kappa B-epsilon does not contain a C-terminal PEST-like sequence. I kappa B-epsilon would, therefore, appear to regulate a late, transient activation of a subset of genes, regulated by RelA/cRel NF-kappa B complexes, distinct from those regulated by other I kappa B proteins.

IkappaBalpha deficiency results in a sustained NF-kappaB response and severe widespread dermatitis in mice.

The ubiquitous transcription factor NF-kappaB is an essential component in signal transduction pathways, in inflammation, and in the immune response. NF-kappaB is maintained in an inactive state in the cytoplasm by protein-protein interaction with IkappaBalpha. Upon stimulation, rapid degradation of IkappaBalpha allows nuclear translocation of NF-kappaB. To study the importance of IkappaBalpha in signal transduction, IkappaBalpha-deficient mice were derived by gene targeting. Cultured fibroblasts derived from IkappaBalpha-deficient embryos exhibit levels of NF-kappaB1, NF-kappaB2, RelA, c-Rel, and IkappaBbeta similar to those of wild-type fibroblasts. A failure to increase nuclear levels of NF-kappaB indicates that cytoplasmic retention of NF-kappaB may be compensated for by other IkappaB proteins. Treatment of wild-type cells with tumor necrosis factor alpha (TNF-alpha) resulted in rapid, transient nuclear localization of NF-kappaB. IkappaBalpha-deficient fibroblasts are also TNF-alpha responsive, but nuclear localization of NF-kappaB is prolonged, thus demonstrating that a major irreplaceable function Of IkappaBalpha is termination of the NF-kappaB response. Consistent with these observations, and with IkappaBalpha and NF-kappaB's role in regulating inflammatory and immune responses, is the normal development Of IkappaBalpha-deficient mice. However, growth ceases 3 days after birth and death usually occurs at 7 to 10 days of age. An increased percentage of monocytes/macrophages was detected in spleen cells taken from 5-, 7-, and 9-day-old pups. Death is accompanied by severe widespread dermatitis and increased levels of TNF-alpha mRNA in the skin.

Activation of nuclear factor of activated T cells in a cyclosporin A-resistant pathway.

The mechanism of action of the immunosuppressive drug cyclosporin A (CsA) is the inactivation of the Ca2+/calmodulin-dependent serine-threonine phosphatase calcineurin by the drug-immunophilin complex. Inactive calcineurin is unable to activate the nuclear factor of activated T cells (NFAT), a transcription factor required for expression of the interleukin 2 (IL-2) gene. IL-2 production by CsA-treated cells is therefore dramatically reduced. We demonstrate here, however, that NFAT can be activated, and significant levels of IL-2 can be produced by the CsA-resistant CD28-signaling pathway. In transient transfection assays, both multicopy NFAT- and IL-2 promoter-beta-galactosidase reporter gene constructs could be activated by phorbol 12-myristate 13-acetate (PMA)/alpha-CD28 stimulation, and this activation was resistant to CsA. Electrophoretic mobility shift assay showed the induction of a CsA-resistant NFAT complex in the nuclear extracts of peripheral blood T cells stimulated with PMA plus alphaCD28. Peripheral blood T cells stimulated with PMA/alphaCD28 produced IL-2 in the presence of CsA. Collectively, these data suggest that NFAT can be activated and IL-2 can be produced in a calcineurin independent manner.

Identification of an I kappa B alpha-associated protein kinase in a human monocytic cell line and determination of its phosphorylation sites on I kappa B alpha.

Nuclear factor kappa B (NF-kappa B) is stored in the cytoplasm as an inactive form through interaction with I kappa B. Stimulation of cells leads to a rapid phosphorylation of I kappa B alpha, which is presumed to be important for the subsequent degradation. We have recently reported the establishment of a lipopolysaccharide (LPS)-dependent cell-free activation system of NF-kappa B in association with the induction of I kappa B alpha phosphorylation. In this study, we have identified a kinase in cell extracts from the LPS-stimulated human monocytic cell line, THP-1, that specifically binds and phosphorylates I kappa B alpha. LPS stimulation transiently enhanced the I kappa B alpha-bound kinase activity in THP-1 cells. Mutational analyses of I kappa B alpha and competition experiments with the synthetic peptides identified major phosphorylation sites by the bound kinase as Ser and Thr residues in the C-terminal acidic domain of I kappa B alpha. Moreover, we show that the peptide, corresponding to the C-terminal acidic domain of I kappa B alpha, blocked the LPS-induced NF-kappa B activation as well as inducible phosphorylation of endogenous I kappa B alpha in a cell-free system using THP-1 cells. These results suggested that the bound kinase is involved in the signaling pathway of LPS by inducing the phosphorylation of the C-terminal region of I kappa B alpha and subsequent dissociation of the NF-kappa B.I kappa B alpha complex.

Conserved kappa B element located downstream of the tumor necrosis factor alpha gene: distinct NF-kappa B binding pattern and enhancer activity in LPS activated murine macrophages.

Transcriptional activation of various genes by lipopolysaccharide (LPS) is known to be mediated, at least in part, by the NF-kappa B/Rel family of transcription factors. We have identified a novel kappa B element located immediately downstream of the TNF-alpha gene that is conserved together with its flanking sequences across species lines and can act as an LPS-responsive enhancer for reporter gene constructs driven by the minimal TNF promoter. In extracts from activated murine macrophages and macrophage cell lines this element binds several non-canonical NF-kappa B/Rel complexes, in addition to p50 (NFKB1) homodimer and p50-p65 (NKFB1-RelA) heterodimer. Combination of high-resolution electrophoretic mobility shift assays (EMSA) with monospecific antibodies and u.v.-cross-linking indicates that the prominent slow migrating complex III contain p65 homodimer and c-Rel. The appearance of complex III in EMSA parallels the translocation of p65 and c-Rel into the nucleus and occurs shortly after LPS induction. Transfection experiments with reporter constructs driven by this kappa B element indicate strong inducibility by LPS and p65, moderate inducibility by c-Rel and repression by p50. Functional activity of sandwich TNF-CAT-TNF constructs further suggests that LPS-inducible transcriptional activation of the TNF gene in murine macrophages may be partly mediated by a downstream enhancer.

The PEST-like sequence of I kappa B alpha is responsible for inhibition of DNA binding but not for cytoplasmic retention of c-Rel or RelA homodimers.

In most cells, proteins belonging to the Rel/NF-kappa B family of transcription factors are held in inactive form in the cytoplasm by an inhibitor protein, I kappa B alpha. Stimulation of the cells leads to degradation of the inhibitor and transit of active DNA-binding Rel/NF-kappa B dimers to the nucleus. I kappa B alpha is also able to inhibit DNA binding by Rel/NF-kappa B dimers in vitro, suggesting that it may perform the same function in cells when the activating signal is no longer present. Structurally, the human I kappa B alpha molecule can be divided into three sections: a 70-amino-acid N terminus with no known function, a 205-residue midsection composed of six ankyrin-like repeats, and a very acidic 42-amino-acid C terminus that resembles a PEST sequence. In this study we examined how the structural elements of the I kappa B alpha protein correlate with its functional capabilities both in vitro and in vivo. Using a battery of I kappa B alpha mutants, we show that (i) a dimer binds a single I kappa B alpha molecule, (ii) the acidic C-terminal region of I kappa B alpha is not required for protein-protein binding and does not mask the nuclear localization signal of the dimer, (iii) the same C-terminal region is required for inhibition of DNA binding, and (iv) this inhibition may be accomplished by direct interaction between the PEST-like region and the DNA-binding region of one of the subunits of the dimer.

Homodimer of p50 (NF kappa B1) does not introduce a substantial directed bend into DNA according to three different experimental assays.

Transcription factors can distort the conformation of the DNA double helix upon binding to their target sites. Previously, studies utilizing circular permutation--electrophoretic mobility shift assay suggested that the homodimer of p50 (NF kappa B1), canonical NF-kappa B (p65-p50), as well as several non-canonical NF-kappa B/Rel complexes, may induce substantial DNA bending at the binding site. Here we have applied three additional experimental approaches, helical phasing analysis, minicircle binding and cyclization kinetics, and conclude that the homodimer of p50 introduces virtually no directed bend into the consensus kappa B sequences GGGACTTTCC or GGGAATTCCC.

Effect of CD28 signal transduction on c-Rel in human peripheral blood T cells.

Optimal T-cell activation requires both an antigen-specific signal delivered through the T-cell receptor and a costimulatory signal which can be delivered through the CD28 molecule. CD28 costimulation induces the expression of multiple lymphokines, including interleukin 2 (IL-2). Because the c-Rel transcription factor bound to and activated the CD28 response element within the IL-2 promoter, we focused our study on the mechanism of CD28-mediated regulation of c-Rel in human peripheral blood T cells. We showed that CD28 costimulation accelerated the kinetics of nuclear translocation of c-Rel (and its phosphorylated form), p50 (NFKB1), and p65 (RelA). The enhanced nuclear translocation of c-Rel correlated with the stimulation of Il-2 production and T-cell proliferation by several distinct anti-CD28 monoclonal antibodies. This is explained at least in part by the long-term downregulation of I kappa B alpha following CD28 signalling as opposed to phorbol myristate acetate alone. Furthermore, we showed that the c-Rel-containing CD28-responsive complex is enhanced by, but not specific to, CD28 costimulation. Our results indicate that c-Rel is one of the transcription factors targeted by CD28 signalling.

Alterations in NF kappa B/Rel family proteins in splenic T-cells from tumor-bearing mice and reversal following therapy.

It has recently been shown that T-cell signal transduction molecules are altered in tumor-bearing mice. We have examined the expression of NF kappa B/Rel family proteins in T-cells from mice bearing Renca, a murine renal carcinoma. T-cells from Renca-bearing mice expressed undetectable levels of nuclear c-Rel, NF kappa B p65, and p50; however, two shorter forms of p50 (p48 and p46), truncated at the NH2-terminus, were present exclusively in the nucleus and were able to bind DNA. These T-cells have reduced expression of gamma-interferon mRNA. In mice successfully treated with flavone 8-acetic acid and recombinant human interleukin 2, the T-cells expressed normal levels of all three nuclear NF kappa B/Rel proteins. These results suggest that alterations in transcription factors may accompany changes in signal transduction molecules in T-cells from tumor-bearing animals; however, the changes are reversed with successful biological therapy.

The v-abl tyrosine kinase negatively regulates NF-kappa B/Rel factors and blocks kappa gene transcription in pre-B lymphocytes.

Transformation of B-lineage precursors by the Abelson murine leukemia virus appears to arrest development at the pre-B stage. Abelson-transformed pre-B cell lines generally retain transcriptionally inactive, unrearranged immunoglobulin kappa alleles. We demonstrate that nontransformed pre-B cells expanded from the mouse bone marrow efficiently transcribe unrearranged kappa alleles. In addition, they contain activated complexes of the NF-kappa B/Rel transcription factor family, in contrast with their Abelson-transformed counterparts. Using conditionally transformed pre-B cell lines, we show that the v-abl viral transforming protein, a tyrosine kinase, blocks germ-line kappa gene transcription and negatively regulates NF-kappa B/Rel activity. An active v-abl kinase specifically inhibits the NF-kappa B/Rel-dependent kappa intron enhancer, which is implicated in promoting both transcription and rearrangement of the kappa locus. v-abl inhibits the activated state of NF-kappa B/Rel complexes in a pre-B cell via a post-translational mechanism that results in increased stability of the inhibitory subunit I kappa B alpha. This analysis suggests a molecular pathway by which v-abl inhibits kappa locus transcription and rearrangement.

Expression of v-src in T cells correlates with nuclear expression of NF-kappa B.

NF-kappa B is a rapidly inducible transcriptional activator that responds to a variety of signals and influences the expression of many genes involved in the immune response. Protein tyrosine kinases transmit signals from cytokine and immune receptors. Very little information exists linking these two important classes of signaling molecules. We now demonstrate that v-src expression correlates with nuclear expression of a kappa B binding complex similar to that induced by phorbol ester and ionomycin, as detected by electrophoretic mobility shift assay using a variety of kappa B sites. This complex was blocked by the tyrosine kinase inhibitor, herbimycin A. The v-src-induced complex comprised the p50 and p65 components of NF-kappa B, as determined by supershift and immunoblot analysis. As a functional correlate of this finding, transient co-transfection of HIV-1 LTR reporter constructs in a different T cell line demonstrated that v-src activated this promoter in a kappa B-dependent manner. We found that transactivation of the HIV-1 LTR by v-src was more sensitive to mutations of the proximal, rather than the distal, kappa B element. The implications for T cell receptor signaling and HIV-1 gene expression are considered.