otherwise fatal overstimulation of the lymphoid system 6-9 . Here we report the identification of a third member of this family of molecules, inducible co-stimulator (ICOS), which is a homodimeric protein of relative molecular mass 55,000-60,000 (M r 55K-60K). Matching CD28 in potency, ICOS enhances all basic Tcell responses to a foreign antigen, namely proliferation, secretion of lymphokines, upregulation of molecules that mediate cell-cell interaction, and effective help for antibody secretion by B cells. Unlike the constitutively expressed CD28, ICOS has to be de novo induced on the T-cell surface, does not upregulate the production of interleukin-2, but superinduces the synthesis of interleukin-10, a B-cell-differentiation factor. In vivo, ICOS is highly expressed on tonsillar T cells, which are closely associated with B cells in the apical light zone of germinal centres, the site of terminal B-cell maturation. Our results indicate that ICOS is another major regulator of the adaptive immune system.We identified ICOS by generating monoclonal antibodies against activated human T cells. The ICOS-specific monoclonal antibody F44 did not react with resting human peripheral-blood T cells, but stained CD4 + and CD8 + T lymphocytes that had been activated by stimulation of the T-cell antigen receptor (TCR) complex (Fig. 1a). No signal was detected on resting or appropriately activated B cells, monocytes, natural killer cells, granulocytes, dendritic cells or platelets (data not shown). Immunoprecipitations using monoclonal Figure 1 Identification, purification and cloning of ICOS. a, Expression of ICOS on human T cells after 36 h of stimulation.Peripheral-blood CD4 + or CD8 + T cells were left untreated or were stimulated with the solid-phase-bound anti-CD3 monoclonal antibody OKT 3 (1:1,000 dilution of ascites), and were analysed by flow cytometry using the fluorescein isothiocyanate (FITC)-labelled monoclonal antibody F44. At 14 h after stimulation, ICOS could not yet be detected on CD8 + T cells, whereas CD4 + T cells expressed levels of ICOS that were similar to those expressed after 36 h (data not shown). The background signal obtained with the isotype-control monoclonal antibody MOPC-21 (Sigma) is shown in black. Stimulation by phorbol-12-myristate-13-acetate (PMA) and ionomycin led to the expression of ICOS on most CD4 + and CD8 + T cells (data not shown). b, Structure of the homodimeric ICOS protein. ICOS protein was immunoprecipitated from surface-iodinated MOLT-4V cells with monoclonal antibody F44 and separated by two-dimensional (nonreducing/reducing) SDS-PAGE. Numbers at the top and right side of the gel are M r values. The 55K-60K protein species on the diagonal corresponds to residual dimeric ICOS, which was not reduced by the in-gel reducing procedure required for the twodimensional SDS-PAGE (a full reduction of this species was routinely observed in onedimensional SDS-PAGE; data not shown). Identical data were obtained with iodinated activated primary T cells (data not shown). c, ICOS mRNA expression...
NLS proteins are transported into the nucleus by the importin alpha/beta heterodimer. Importin alpha binds the NLS, while importin beta mediates translocation through the nuclear pore complex. After translocation, RanGTP, whose predicted concentration is high in the nucleus and low in the cytoplasm, binds importin beta and displaces importin alpha. Importin alpha must then be returned to the cytoplasm, leaving the NLS protein behind. Here, we report that the previously identified CAS protein mediates importin alpha re-export. CAS binds strongly to importin alpha only in the presence of RanGTP, forming an importin alpha/CAS/RanGTP complex. Importin alpha is released from this complex in the cytoplasm by the combined action of RanBP1 and RanGAP1. CAS binds preferentially to NLS-free importin alpha, explaining why import substrates stay in the nucleus.
Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.
The T-cell-specific cell-surface receptors CD28 and CTLA-4 are important regulators of the immune system. CD28 potently enhances those T-cell functions that are essential for an effective antigen-specific immune response, and the homologous CTLA-4 counterbalances the CD28-mediated signals and thus prevents an otherwise fatal overstimulation of the lymphoid system. Here we report the identification of a third member of this family of molecules, inducible co-stimulator (ICOS), which is a homodimeric protein of relative molecular mass 55,000-60,000 (M(r) 55K-60K). Matching CD28 in potency, ICOS enhances all basic T-cell responses to a foreign antigen, namely proliferation, secretion of lymphokines, upregulation of molecules that mediate cell-cell interaction, and effective help for antibody secretion by B cells. Unlike the constitutively expressed CD28, ICOS has to be de novo induced on the T-cell surface, does not upregulate the production of interleukin-2, but superinduces the synthesis of interleukin-10, a B-cell-differentiation factor. In vivo, ICOS is highly expressed on tonsillar T cells, which are closely associated with B cells in the apical light zone of germinal centres, the site of terminal B-cell maturation. Our results indicate that ICOS is another major regulator of the adaptive immune system.
A novel protein complex has been identified in human cells that has a molecular mass of approximately 450 kDa. It consists of at least eight different subunits including JAB1, the Jun activation-domain binding protein 1, and Trip15, the thyroid hormone receptor-interacting protein 15. The purified complex contains COP9 and COP11 protein homologs and is very similar, if not identical, to the plant COP9 complex involved in light-mediated signal transduction. The isolated JAB1-containing particle has kinase activity that phosphorylates IkappaBalpha, the carboxy terminus of p105, and Ser63 and/or Ser73 of the amino-terminal activation domain of c-Jun. The phosphorylation of c-Jun requires the carboxy terminus of the protein containing the DNA binding and dimerization domains. Three subunits of the new complex--Sgn3, Sgn5/JAB1, and Sgn6--exhibit sequence similarities to regulatory components of the 26S proteasome, which could indicate the existence of common substrate binding sites. Immunofluorescence staining reveals that the new complex shows a subcellular distribution similar to that of the 26S proteasome. The functional relationship of the two particles in regulating transcriptional activity is discussed. Considering the putative role of the complex in signal transduction and its widespread occurrence, we suggest the name JAB1-containing signalosome.
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