Novel NEMO/IkappaB kinase and NF-kappa B target genes at the pre-B to immature B cell transition.

The IkappaB kinase (IKK) signaling complex is responsible for activating NF-kappaB-dependent gene expression programs. Even though NF-kappaB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-kappaB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cells versus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKgamma/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-kappaB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118, Pea-15/MAT1, CD82, CD40L, Wnt10a, Notch 1, R-ras, Rgs-16, PAC-1, ISG15, and CD36) and five repressed (CCR2, VpreB, lambda5, SLPI, and CMAP/Cystatin7) genes, respectively, were bona fide NF-kappaB targets by virtue of their response to a transdominant IkappaBalphaSR (super repressor). MyD118 and ISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-kappaB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors.

Functional isoforms of IkappaB kinase alpha (IKKalpha) lacking leucine zipper and helix-loop-helix domains reveal that IKKalpha and IKKbeta have different activation requirements.

The activity of the NF-kappaB family of transcription factors is regulated principally by phosphorylation and subsequent degradation of their inhibitory IkappaB subunits. Site-specific serine phosphorylation of IkappaBs by two IkappaB kinases (IKKalpha [also known as CHUK] and IKKbeta) targets them for proteolysis. IKKalpha and -beta have a unique structure, with an amino-terminal serine-threonine kinase catalytic domain and carboxy-proximal helix-loop-helix (HLH) and leucine zipper-like (LZip) amphipathic alpha-helical domains. Here, we describe the properties of two novel cellular isoforms of IKKalpha: IKKalpha-DeltaH and IKKalpha-DeltaLH. IKKalpha-DeltaH and IKKalpha-DeltaLH are differentially spliced isoforms of the IKKalpha mRNA lacking its HLH domain and both its LZip and HLH domains, respectively. IKKalpha is the major RNA species in most murine cells and tissues, except for activated T lymphocytes and the brain, where the alternatively spliced isoforms predominate. Remarkably, IKKalpha-DeltaH and IKKalpha-DeltaLH, like IKKalpha, respond to tumor necrosis factor alpha stimulation to potentiate NF-kappaB activation in HEK293 cells. A mutant, catalytically inactive form of IKKalpha blocked IKKalpha-, IKKalpha-DeltaH-, and IKKalpha-DeltaLH-mediated NF-kappaB activation. Akin to IKKalpha, its carboxy-terminally truncated isoforms associated with the upstream activator NIK (NF-kappaB-inducing kinase). In contrast to IKKalpha, IKKalpha-DeltaLH failed to associate with either itself, IKKalpha, IKKbeta, or NEMO-IKKgamma-IKKAP1, while IKKalpha-DeltaH complexed with IKKbeta and IKKalpha but not with NEMO. Interestingly, each IKKalpha isoform rescued HEK293 cells from the inhibitory effects of a dominant-negative NEMO mutant, while IKKalpha could not. IKKalpha-DeltaCm, a recombinant mutant of IKKalpha structurally akin to IKKalpha-DeltaLH, was equally functional in these assays, but in sharp contrast, IKKbeta-DeltaCm, a structurally analogous mutant of IKKbeta, was inactive. Our results demonstrate that the functional roles of seemingly analogous domains in IKKalpha and IKKbeta need not be equivalent and can also exhibit different contextual dependencies. The existence of cytokine-inducible IKKalpha-DeltaH and IKKalpha-DeltaLH isoforms illustrates potential modes of NF-kappaB activation, which are not subject to the same in vivo regulatory constraints as either IKKalpha or IKKbeta.

Activation and regulation of the IkappaB kinase in human B cells by CD40 signaling.

This study demonstrates that the engagement of CD40 results in the activation of the recently described IkappaB kinase (IKK) in a human B cell line. The kinase appears to reside within the cell in a cytosolic signalsome complex consisting of IKK, IkappaB, and an MKP-1-like molecule. While the binding of CD154 to CD40 induces the assembly of a CD40-TRAF receptor complex, IKK is not recruited to this complex. Nonetheless, a functional link between TRAF2 and IKK activity in B cells is demonstrated by the fact that overexpression of TRAF2 constitutively induces IKK activity, NF-kappaB luciferase and Fas expression. Synergy in the activation of IKK and NF-kappaB-dependent gene expression was observed by the simultaneous engagement of the B cell receptor and CD40, establishing an early means for cross-talk between these two B cell activation pathways. This study discusses the sequential biochemical events that transpire upon CD40 engagement by its ligand in human B cells.

Recombinant IkappaB kinases alpha and beta are direct kinases of Ikappa Balpha.

Activation of the transcription factor NF-kappaB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IkappaB. A large multiprotein complex, the IkappaB kinase (IKK), catalyzes the phosphorylation of IkappaB. The two kinase components of the IKK complex, IKKalpha and IKKbeta, were overexpressed in insect cells and purified to homogeneity. Both purified IKKalpha and IKKbeta specifically catalyzed the phosphorylation of the regulatory serine residues of Ikappa Balpha. Hence, IKKalpha and IKKbeta were functional catalytic subunits of the IKK complex. Purified IKKalpha and IKKbeta also preferentially phosphorylated serine as opposed to threonine residues of Ikappa Balpha, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKalpha and IKKbeta revealed that the kinase activity of IKKbeta on Ikappa Balpha is 50-60-fold higher than that of IKKalpha. The primary difference between the two activities is the Km for Ikappa Balpha. The kinetics of both IKKalpha and IKKbeta followed a sequential Bi Bi mechanism. No synergistic effects on Ikappa Balpha phosphorylation were detected between IKKalpha and IKKbeta. Thus, in vitro, IKKalpha and IKKbeta are two independent kinases of Ikappa Balpha.

Molecular determinants of NF-kappaB-inducing kinase action.

NF-kappaB corresponds to an inducible eukaryotic transcription factor complex that is negatively regulated in resting cells by its physical assembly with a family of cytoplasmic ankyrin-rich inhibitors termed IkappaB. Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), induces nuclear NF-kappaB expression. TNF-alpha signaling involves the recruitment of at least three proteins (TRADD, RIP, and TRAF2) to the type 1 TNF-alpha receptor tail, leading to the sequential activation of the downstream NF-kappaB-inducing kinase (NIK) and IkappaB-specific kinases (IKKalpha and IKKbeta). When activated, IKKalpha and IKKbeta directly phosphorylate the two N-terminal regulatory serines within IkappaB alpha, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome. This process liberates the NF-kappaB complex, allowing it to translocate to the nucleus. In studies of NIK, we found that Thr-559 located within the activation loop of its kinase domain regulates NIK action. Alanine substitution of Thr-559 but not other serine or threonine residues within the activation loop abolishes its activity and its ability to phosphorylate and activate IKKalpha. Such a NIK-T559A mutant also dominantly interferes with TNF-alpha induction of NF-kappaB. We also found that ectopically expressed NIK both spontaneously forms oligomers and displays a high level of constitutive activity. Analysis of a series of NIK deletion mutants indicates that multiple subregions of the kinase participate in the formation of these NIK-NIK oligomers. NIK also physically assembles with downstream IKKalpha; however, this interaction is mediated through a discrete C-terminal domain within NIK located between amino acids 735 and 947. When expressed alone, this C-terminal NIK fragment functions as a potent inhibitor of TNF-alpha-mediated induction of NF-kappaB and alone is sufficient to disrupt the physical association of NIK and IKKalpha. Together, these findings provide new insights into the molecular basis for TNF-alpha signaling, suggesting an important role for heterotypic and possibly homotypic interactions of NIK in this response.

Efficient recombination of a switch substrate retrovector in CD40-activated B lymphocytes: implications for the control of CH gene switch recombination.

Maturing B lymphocytes possess a recombination activity that switches the class of heavy chain Ig. The nature of the recombination activity, its molecular requirements and regulation remain elusive questions about B lymphocyte biology and development. Class switch recombination is controlled by cytokine response elements that are required to differentially activate CH gene transcription before their subsequent recombination. Here, we show that cultures of purified murine and human B cells, stimulated only by CD40 receptor engagement, possess a potent switch recombination activity. CD40 ligand-stimulated murine and human B lymphocytes were infected with recombinant retroviruses containing Smu and S gamma 2b sequences. Chromosomally integrated switch substrate retrovectors (SSRs), harboring constitutively transcribed S sequences, underwent extensive recombinations restricted to their S sequences with structural features akin to endogenous switching. SSR recombination commenced 4 days postinfection (5 days poststimulation) with extensive switch sequence recombination over the next 2 to 3 days. In contrast, endogenous S gamma 2b and S gamma 1 sequences did not undergo appreciable switch recombination upon CD40 signaling alone. As expected, IL-4 induced endogenous Smu to S gamma 1 switching, while endogenous Smu to S gamma 2b fusions remained undetectable. Surprisingly, IL-4 enhanced the onset of SSR recombination in CD40-stimulated murine B cells, with S-S products appearing only 2 days postinfection and reaching a maximum within 2 to 3 days. The efficiency of switch recombination with SSRs resembles that seen for endogenous C(H) class switching.

Generation of switch hybrid DNA between Ig heavy chain-mu and downstream switch regions in B lymphocytes.

Ig heavy chain isotype switching in B lymphocytes is known to be preceded by transcription of a portion of the particular heavy chain gene segment that is targeted for recombination. Here, we describe an active role for these transcripts in the switch recombination process. Using an in vitro assay that exposes an artificial switch-mu (Smu) minisubstrate to switch region transcripts in the presence of nuclear extracts from switching cells, we demonstrate that free 3' ends of the Smu sequence are extended onto switch region transcripts by reverse transcription. The activity was induced in splenic B lymphocytes upon activation with LPS or CD40 ligand. This in vitro process is thought to be relevant to in vivo class switching for two reasons: 1) although only one-third of the Smu minisubstrate actually contains Smu sequence, all crossovers between switch regions occurred in the Smu portion; and 2) treatment of B lymphocytes with IL-4, which enriches for switching to S gamma 1, increases the ratio of Smu-S gamma 1 to Smu-S gamma 3 hybrids by 16% after LPS treatment and by 37% after CD40 ligand activation, implicating this S mu-primed reverse transcription of switch region transcripts as a novel mechanism of regulating the specificity of isotype switching. Further evidence for an active role of switch region transcripts was obtained by expressing S alpha RNA in trans in the Bcl1B1 B lymphoma line. Endogenous S mu-S alpha switch circles were detected in Bcl1B1 cells expressing exogenous S alpha RNA but not in mock-transfected cells.

Human T-cell leukemia virus type 1 Tax induction of NF-kappaB involves activation of the IkappaB kinase alpha (IKKalpha) and IKKbeta cellular kinases.

Tax corresponds to a 40-kDa transforming protein from the pathogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) that activates nuclear expression of the NF-kappaB/Rel family of transcription factors by an unknown mechanism. Tax expression promotes N-terminal phosphorylation and degradation of IkappaB alpha, a principal cytoplasmic inhibitor of NF-kappaB. Our studies now demonstrate that HTLV-1 Tax activates the recently identified cellular kinases IkappaB kinase alpha (IKKalpha) and IKKbeta, which normally phosphorylate IkappaB alpha on both of its N-terminal regulatory serines in response to tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) stimulation. In contrast, a mutant of Tax termed M22, which does not induce NF-kappaB, fails to activate either IKKalpha or IKKbeta. Furthermore, endogenous IKK enzymatic activity was significantly elevated in HTLV-1-infected and Tax-expressing T-cell lines. Transfection of kinase-deficient mutants of IKKalpha and IKKbeta into either human Jurkat T or 293 cells also inhibits NF-kappaB-dependent reporter gene expression induced by Tax. Similarly, a kinase-deficient mutant of NIK (NF-kappaB-inducing kinase), which represents an upstream kinase in the TNF-alpha and IL-1 signaling pathways leading to IKKalpha and IKKbeta activation, blocks Tax induction of NF-kappaB. However, plasma membrane-proximal elements in these proinflammatory cytokine pathways are apparently not involved since dominant negative mutants of the TRAF2 and TRAF6 adaptors, which effectively block signaling through the cytoplasmic tails of the TNF-alpha and IL-1 receptors, respectively, do not inhibit Tax induction of NF-kappaB. Together, these studies demonstrate that HTLV-1 Tax exploits a distal part of the proinflammatory cytokine signaling cascade leading to induction of NF-kappaB. The pathological alteration of this cytokine pathway leading to NF-kappaB activation by Tax may play a central role in HTLV-1-mediated transformation of human T cells, clinically manifested as the adult T-cell leukemia.

Stimulation of murine B lymphocytes induces a DNA exonuclease whose activity on switch-mu DNA is specifically inhibited by other germ-line switch region RNAs.

The Ig heavy chain class switch in B lymphocytes involves a unique genetic recombination that fuses specific regions within the Ig locus and deletes intervening sequences. Here we describe a novel exonuclease activity in nuclear lysates of B cells in an in vitro assay. This activity was induced in B lymphocytes after treatment with either LPSs or CD40 ligand/anti-delta-dextran, both of which induce switch recombination, and considerably less activity was detected in untreated or anti-delta-dextran-treated B cells, Con A-stimulated spleen cells, liver cells, or a number of cell lines. The exonuclease activity was dependent on divalent cations, and both 3' and 5' labels were efficiently removed from DNA substrates. The presence of RNase A, but not RNase H, inhibited exonucleolytic digestion, suggesting that a ribonucleoprotein is responsible for the exonucleolysis. The DNA digestion appears to be nonspecific, since DNA substrates with either switch-mu or unrelated sequence were hydrolyzed with comparable efficiency. Germ-line switch region transcripts (Ig gamma1, Ig gamma3, and Ig alpha) strongly inhibited the exonucleolysis of switch-mu DNA but not that of unrelated control DNA, while switch antisense RNA or tRNA were much less effective inhibitors.

Antibody class switch recombinase activity is B cell stage specific and functions stochastically in the absence of 'targeted accessibility' control.

Chromosomally integrated retroviral switch (S) substrates have been developed to reveal switch recombinase-like activities (SRLA) in pre-B and mature B cell lines. Switch substrate retrovectors (SSR) contain a long-terminal repeat-driven neomycin (Neo) gene for proviral chromosomal maintenance (pre- and post-S recombination) and a CMV promoter-driven, chimeric hygromycin-thymidine kinase (Hytk) gene (flanked by S mu and S gamma 2b recombination targets) to select for (ganciclovir) and against (hygromycin B) S region recombination. The retro-substrates' strong, constitutive promoters ensure that variations in cellular switch recombinase activities are independent of S region accessibility control. By initially selecting for proviral integrants in hygromycin followed by shifting into neomycin + ganciclovir to select for S sequence-mediated deletions, switch recombinations can be specifically forestalled in B cell lines whilst most switch-incompetent cells do not survive secondary selection. A qualitative, direct PCR assay reveals that SSR recombinations are stochastic in B cell lines generating a product array akin to natural GH class switching. A semi-quantitative DC-PCR assay detects a significant recombinase activity only in a restricted set of late stage pre-B and mature B cell lines. BCL1B1 mature B cells have the highest level of recombinase activity with 25% or more of proviral integrants accumulating S mu/S gamma 2b substrate recombinations within 10-14 cell generations. The SSR recombinase assay can be performed in a transient fashion wherein extensive, B cell-specific recombination can be visualized within only a few cell divisions post proviral integration. We propose that switch recombinase activity becomes activated during B cell ontogeny independent of or prior to the acquisition of CH locus accessibility and that endogenous S segment targeting to pre-existing recombinase requires a level of accessibility beyond transcriptional activation.

Plasmacytoma-associated neuronal glycoprotein, Pang, maps to mouse chromosome 6 and human chromosome 3.

A new member of the immunoglobulin/fibronectin superfamily of adhesion molecules, Pang (plasmacytoma-associated neuronal glycoprotein), was recently isolated from a plasmacytoma. In previous studies, Pang was found to be normally expressed in the brain and ectopically activated by intracisternal A-type particle long terminal repeats in plasmacytomas. In this study, Pang was initially mapped to mouse Chr 6 by somatic cell hybrid analysis and further positioned on the chromosome between Wnt7a and Pcp1. Southern blot analysis of human-rodent somatic cell hybrids together with predictions from the mouse map location indicate that human PANG is located at 3p26.

v-Abl activates c-myc transcription through the E2F site.

The v-abl oncogene of Abelson murine leukemia virus encodes a deregulated form of the cellular nonreceptor tyrosine kinase. v-Abl activates c-myc transcription, and c-Myc is an essential downstream component in the v-Abl transformation program. To explore the mechanism by which v-Abl activates c-myc transcription, a cotransfection assay was developed. We show that transactivation of a c-myc promoter by v-Abl requires the SH1 (tyrosine kinase) and SH2 domains of v-Abl; the C-terminal domains are not required for transactivation. The assay also identified the E2F site in the c-myc promoter as a v-Abl-responsive element. In addition, multimerized E2F sites were shown to be sufficient to confer v-Abl-dependent activation on a minimal promoter. This is the first identification of a v-Abl response element for transcriptional activation. v-Abl tyrosine kinase-dependent changes in proteins binding the c-myc E2F site were also demonstrated, including induction of a complex containing DP1, p107, cyclin A, and cdk2. Identification of v-Abl-dependent changes in E2F-binding proteins provides an important link between v-Abl, transcription, cell cycle regulation, and control of cellular growth.

A Myc-associated zinc finger protein binding site is one of four important functional regions in the CD4 promoter.

The CD4 promoter plays an important role in the developmental control of CD4 transcription. In this report, we show that the minimal CD4 promoter has four factor binding sites, each of which is required for full function. Using biochemical and mutagenesis analyses, we determined that multiple nuclear factors bind to these independent sites. We determined that an initiator-like sequence present at the cap site and an Ets consensus sequence are required for full promoter function. We also demonstrate that the Myc-associated zinc finger protein (MAZ) appears to be the predominant factor binding to one of these sites. This last site closely resembles the ME1a1 G3AG4AG3 motif previously shown to be a critical element in the P2 promoter of the c-myc gene. We therefore believe that the MAZ transcription factor is also likely to play an important role in the control of developmental expression of the CD4 gene.

CHUK, a conserved helix-loop-helix ubiquitous kinase, maps to human chromosome 10 and mouse chromosome 19.

Helix-loop-helix proteins contain stretches of DNA that encode two amphipathic alpha-helices joined by a loop structure and are involved in protein dimerization and transcriptional regulation essential to a variety of cellular processes. CHUK, a newly described conserved helix-loop-helix ubiquitous kinase, was mapped by somatic cell hybrid analyses to human Chr 10q24-q25. Chuk and a related sequence, Chuk-rs1, were mapped to mouse chromosomes 19 and 16, respectively, by a combination of somatic cell hybrid, recombinant inbred, and backcross analyses.

CHUK, a new member of the helix-loop-helix and leucine zipper families of interacting proteins, contains a serine-threonine kinase catalytic domain.

We have identified a new member of the helix-loop-helix (H-L-H) and leucine zipper gene families via a reverse transcriptase-polymerase chain reaction based strategy. This new gene, CHUK (conserved helix-loop-helix ubiquitous kinase), may represent the founding member of a new class of interacting chimeric proteins. The nucleotide sequence of a near full-length murine CHUK cDNA clone revealed an encoded polypeptide specifying: a carboxyl-terminal H-L-H domain, an amino terminal serine-threonine kinase catalytic domain, and a leucine zipper-like amphipathic alpha-helix juxtaposed in between the H-L-H and kinase domains. CHUK is highly conserved in evolution and ubiquitously expressed in diverse types of established cell lines, whereas it is differentially expressed in normal murine tissues. The structural features of the CHUK polypeptide suggest that its putative kinase activity may be targetted to H-L-H and/or leucine zipper transcription factors. Alternatively, the dual amphipathic a helices may serve to control its intrinsic kinase activity by interactions with other cellular factors. CHUK may provide new insights into the regulated transmission of cytoplasmic signals to specific nuclear factors manifesting rapid alterations in patterns of cellular gene expression.