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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CD28, which is a member of the immunoglobulin superfamily of molecules (IgSF), is a homodimer of two polypeptides containing a single V-like domain with short transmembrane and cytoplasmic regions. It serves as a co-signalling molecule for T cell activation through binding to its cognate counter-receptors CD80 and B70, expressed on antigen presenting cells. In the current study, we investigated the regions of CD28 which are involved in its interactions with CD80 and B70, using site directed mutagenesis, CD28 mAb epitope mapping, receptor based adhesion assays and direct binding of Ig-fusion proteins to cell surface receptors. Truncation or substitution of a stretch of a proline rich "hallmark" sequence, "MYPPPY", abrogates binding to CD80 or B70, while retaining CD28 mAb epitopes and cell surface expression. On an Ig-fold model of the CD28 V-domain, this fully conserved motif localizes to a CDR3-like region. Mutations introduced into other loops, including the CDRI-like and CDR2-like regions, had very little effect on CD80 or B70 binding. Mutations introduced within the predicted beta-strand regions caused loss of receptor expression. Conservative substitution of both the flanking tyrosine residues within the "MYPPPY" motif with phenylalanine, caused loss of binding to B70 but not to CD80. These results show that, although the same overall region on CD28 may be involved in the interactions with CD80 and B70, subtle but important differences distinguish recognition by the two molecules. These finding, along with previous observations on the differential pattern of expression and tissue distribution of CD80 and B70, support the contention that these molecules play distinct roles in the regulation of immune responses in vivo.
Mol Immunol 1996 Feb
PMID:Differential recognition by CD28 of its cognate counter receptors CD80 (B7.1) and B70 (B7.2): analysis by site directed mutagenesis. 864 53

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) upregulates the expression of several cellular genes by activating members of both the NF-kappaB and bZIP families of transcription factors. Recent studies demonstrate that the CD28 response element (CD28RE) of the interleukin 2 (IL-2) promoter is the site upregulated by Tax in stimulated T cells. Although some reports suggest that this site is transactivated by NF-kappaB family members, others disagree, leaving the identity of the transcription factor(s) binding the CD28RE unclear. The studies presented here further characterize the response of the IL-2 promoter and CD28RE to the HTLV-1 Tax protein and demonstrate that the TATA-proximal AP-1 binding site of the IL-2 promoter is also necessary for Tax transactivation in stimulated Jurkat cells. In contrast to its upregulation of the IL-2 promoter which requires T-cell stimulation, Tax transactivates the isolated CD28RE-AP-1 element without stimulation but is greatly synergized by calcium ionophore and phorbol ester. Additionally, transactivation of the IL-2 promoter requires the Tax activation domain involved in upregulation of bZIP-enhanced transcription while the NF-kappaB-activating domain of Tax is dispensable. Interestingly, both domains appear to be necessary for the activation of the isolated CD28RE-AP-1 sequence in the context of a heterologous promoter construct. This strongly suggests that activation of NF-kappaB is insufficient to activate transcription via the CD28RE-AP-1 element of the IL-2 promoter and that a different transcription factor, upregulated via the activation domain of the HTLV-1 Tax protein, may be involved.
Mol Cell Biol 1996 Jul
PMID:Requirements for interleukin 2 promoter transactivation by the Tax protein of human T-cell leukemia virus type 1. 866 73

Telomerase activity is involved in telomere length maintenance. Leukocytes, unlike many human somatic tissues, have detectable telomerase activity. These cells provide a normal human cell type in which to study telomerase. We studied the regulation of telomerase activity and the telomerase RNA component as leukocytes were stimulated to enter the cell cycle. In primary human leukocytes stimulated with phytohemagglutinin, telomerase activity increased > 10-fold as naturally quiescent cells entered the cell cycle. Antibodies to the T cell receptor (TCR)/CD3 complex and the costimulatory CD28 receptor induced telomerase activity in a T cell-enriched population of cells. Rapamycin, an immunosuppressant that blocks TCR/CD3 signal transduction pathways and cdk2 activation, blocked telomerase induction. Hydroxyurea, an inhibitor of S phase, did not block cdk2 kinase activity or telomerase activation. In summary, telomerase is regulated in G1 phase as normal human T cells enter the cell cycle.
Mol Biol Cell 1996 Sep
PMID:Telomerase regulation during entry into the cell cycle in normal human T cells. 888 38

Optimal activation of T cells requires at least two signals delivered by the T-cell receptor complex and costimulatory molecules such as CD28. The CD28 signaling participates in the transcription of the interleukin-2 gene through activation of an enhancer termed the CD28-responsive element (CD28RE). Stimulation of CD28 enhances mitogen-mediated induction of CD28RE-binding proteins including members of the NF-kappaB/Rel transcription factor family, although the underlying mechanism remains elusive. In this report, we show that CD28 costimulation leads to biphasic induction of NF-kappaB/Rel heterodimers, including early-phase induction of p50/RelA and c-Rel/RelA and late-phase induction of p50/c-Rel. Interestingly, activation of these NF-kappaB/Rel complexes by the CD28 signal is associated with the rapid degradation of both IkappaBalpha and IkappaBbeta, two major cytoplasmic inhibitors of NF-kappaB/Rel. Although IkappaBalpha degradation can be induced by phorbol ester alone, degradation of IkappaBbeta is largely dependent on the CD28 costimulatory signal. We further demonstrate that CD28-mediated transactivation of the CD28RE enhancer is potently inhibited by an N-terminal truncation mutant of IkappaBbeta that is incapable of responding to the degradation signals. Together, these results suggest that the CD28 costimulatory signal augments activation of NF-kappaB/Rel by promoting degradation of IkappaBbeta as well as enhancing degradation of IkappaBalpha and that induction of NF-kappaB/Rel serves as an essential step in the signal-mediated activation of the CD28RE enhancer.
Mol Cell Biol 1996 Dec
PMID:CD28 mediates a potent costimulatory signal for rapid degradation of IkappaBbeta which is associated with accelerated activation of various NF-kappaB/Rel heterodimers. 894 28

We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-D-Fos complexes. In contrast, AP-1 and NF-kappaB complexes induced in B7-1- and LFA-3-costimulated T cells showed similar contents. Transient transfection of Jurkat T cells with a construct encoding the IL-2 enhancer-promoter region (position -500 to +60) linked to a luciferase reporter gene revealed that B7-1 costimulation was required to induce strong transcriptional activity. Combined B7-1-LFA-3 costimulation resulted in a synergistic increase in IL-2 transcriptional activity. Multimers of the AP-1, NF-AT, NF-kappaB, and CD28 response elements showed distinct kinetics and activity after LFA-3 and B7-1 costimulation and revealed that B7-1 and LFA-3 converge to superinduce transcriptional activity of the AP-1, NF-AT, and CD28 response elements. Transcriptional studies with an IL-2 enhancer-promoter carrying a mutation in the CD28 response element site revealed that the activity was reduced by 80% after B7-1 and B7-1-LFA-3 costimulation whereas the transcriptional activity induced by LFA-3 was unaffected. Our data strongly suggest a selectivity in induction of nuclear factors by the CD2-LFA-3 and CD28-B7-1 pathways. This selectivity may contribute to regulation of the levels of IL-2 induced by LFA-3 and B7-1 costimulation and favor autocrine and paracrine T-cell responses, respectively.
Mol Cell Biol 1997 Mar
PMID:Costimulation by B7-1 and LFA-3 targets distinct nuclear factors that bind to the interleukin-2 promoter: B7-1 negatively regulates LFA-3-induced NF-AT DNA binding. 903 58

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.
Mol Cell Biol 1997 May
PMID:Expression of NFAT-family proteins in normal human T cells. 911 16

The CD28 costimulatory signal enhances antigen-mediated induction of interleukin-2 (IL-2) gene transcription through activation of an enhancer termed the CD28-responsive element (CD28RE). Although various nuclear proteins have been shown to bind to CD28RE, their in vivo functions in the regulation of this enhancer remain elusive. In this report, we show that CD28RE binds distinct transcription factors in cells treated with different mitogenic stimuli. Stimulation of the T-cell receptor (TCR) complex in the absence of a CD28 costimulatory signal induces a member of the nuclear factor of the activated T cells, NF-ATp; however, this treatment fails to activate the CD28RE enhancer activity. Significant activation of CD28RE was detected when the cells were treated with both the TCR stimulators and an anti-CD28 monoclonal antibody (anti-CD28), which induces the NF-kappaB/Rel enhancer binding proteins in addition to NF-ATp. The costimulatory activity of anti-CD28 can be further enhanced by a phorbol ester. Kinetic analyses demonstrate that activation of endogenous IL-2 gene transcription is correlated with the binding of CD28RE by NF-ATp and different NF-kappaB/Rel species. Transient-transfection studies reveal that expression of either NF-ATp or the p50-RelA NF-kappaB heterodimer leads to the potent transactivation of both the CD28RE enhancer and the intact IL-2 promoter in mitogen-stimulated cells. Remarkably, coexpression of these two families of enhancer-binding proteins in Jurkat T cells results in the transactivation of the CD28RE enhancer even in the absence of any cellular stimuli. Together, these results suggest that activation of IL-2 gene transcription by the TCR- and CD28-mediated signals involves the interaction of CD28RE with NF-ATp and various NF-kappaB/Rel transcription factors.
Mol Cell Biol 1997 May
PMID:Regulation of the interleukin-2 CD28-responsive element by NF-ATp and various NF-kappaB/Rel transcription factors. 911 30

Mutagenesis studies have demonstrated the requirement for the CD28-responsive element (CD28RE) within the interleukin-2 (IL-2) promoter for transcriptional upregulation by CD28. Here, we demonstrate that CD28 responsiveness is conferred by a composite element containing both the CD28RE and the NF-IL-2B AP-1 sites (RE/AP). Mutations at either site within the RE/AP composite element abolish activity. The RE/AP composite element is a site for signal integration within the IL-2 promoter, since its activation is dependent on at least two separate signalling pathways being activated, through the T-cell receptor, CD28, and/or phorbol myristate acetate. Activation is maximal when all three signals occur simultaneously. By using a panel of CD28 cytoplasmic domain mutants, it was found that the transcriptional activation of the RE/AP composite element correlates exactly with the pattern of IL-2 secretion induced by these mutants upon stimulation. Similar to the upregulation of IL-2 secretion, the transcriptional upregulation of the RE/AP composite element by CD28 is FK506 insensitive. The pattern of activation of the RE/AP composite element is different from that observed for either an NFAT or consensus AP-1 site, implying that RE/AP represents a unique element. Using gel shift analysis, we demonstrate that stimulation by CD28 induces the association of the NF-kappaB family member c-Rel to the CD28RE within the RE/AP composite element. The transcriptional upregulation of IL-2 by CD28 appears, therefore, to be mediated through the RE/AP composite element, involving the association of c-Rel with the CD28RE.
Mol Cell Biol 1997 Jul
PMID:CD28 mediates transcriptional upregulation of the interleukin-2 (IL-2) promoter through a composite element containing the CD28RE and NF-IL-2B AP-1 sites. 919 40

Recent evidence implicates PI 3-kinase in TCR signal transduction. The fungal metabolite wortmannin is a specific inhibitor of PI 3-kinase both in vitro and in vivo when used at nanomolar concentrations. Therefore, we examined the effect of wortmannin on stimulation of primary T cells and T cell lines. Wortmannin had a dose-dependent inhibitory effect on TCR-dependent primary T cell proliferation with IC50 in the nanomolar range. Furthermore, activation of T cell lines independently of antigen presenting cells and, therefore of any CD28 co-stimulatory signaling, was also sensitive to wortmannin. As expected, phorbol ester stimulation bypassed PI 3-kinase signal transduction. Importantly, the effect of wortmannin correlated with inhibition of activation of PI 3-kinase in stimulated T cells. The earliest step in T cell activation, tyrosine kinase activation, was not significantly affected by wortmannin. We conclude that a wortmannin-sensitive enzyme, probably PI 3-kinase, acting downstream of tyrosine kinases, but independently of the phorbol ester activated pathway, is necessary for stimulation of T cells via the TCR, and that this requirement is independent of any role of PI 3-kinase in co-stimulation via CD28 coreceptor. PI 3-kinase is most probably involved in generation of 3-phosphorylated lipid products, and is not merely an adaptor.
Mol Immunol 1997 Feb
PMID:Evidence for phosphatidylinositol 3-kinase-dependent T cell antigen receptor (TCR) signal transduction. 922 64

Seven random peptide libraries (two displaying linear peptides and five displaying cysteine-constrained peptides) were constructed as gene III fusion proteins of the bacteriophage fd-tet. These libraries were used to screen a blocking monoclonal antibody raised against B7-1 (CD80), a human cell surface antigen that binds two T cell receptors, CD28 and CTLA-4. After three rounds of screening against the immobilized antibody, 1000-fold enrichment was observed in libraries displaying both linear and cysteine-constrained peptides. DNA sequencing of the enriched phage revealed two distinct consensus sequences: HXG(A/Y)XH and DVCXXGGPGC. Phage expressing these consensus sequences bound to L307.4 but not to an isotype matched antibody, indicating that binding was antibody specific. Synthetic peptides corresponding to both motifs inhibited phage binding to L307.4, indicating that the gene III protein is not required for peptide binding. In addition, the cyclized forms of synthetic peptides containing the DVCXXGGPGC motif were capable of inhibiting L307.4 binding to soluble B7-1/Fc fusion. Moreover, phage expressing only the HXG(A/Y)XH consensus sequence were inhibited from binding to L307.4 by the presence of chelating agents. These results indicate that the framework within which the peptide is presented on the surface of the phage may allow the identification of unique peptide motifs with distinct binding characteristics. These peptide motifs could be used for the design of peptidomimetics with therapeutic applications if they inhibit the binding of B7-1 to its T cell receptors.
Mol Divers 1996 Feb
PMID:Utilization of multiple phage display libraries for the identification of dissimilar peptide motifs that bind to a B7-1 monoclonal antibody. 923 96


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