Regine Kraft

ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28

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 (Mr 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.

Export of Importin α from the Nucleus Is Mediated by a Specific Nuclear Transport Factor

Abstract NLS proteins are transported into the nucleus by the importin α/β heterodimer. Importin α binds the NLS, while importin β 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 β and displaces importin α. Importin α must then be returned to the cytoplasm, leaving the NLS protein behind. Here, we report that the previously identified CAS protein mediates importin α re-export. CAS binds strongly to importin α only in the presence of RanGTP, forming an importin α/CAS/RanGTP complex. Importin α is released from this complex in the cytoplasm by the combined action of RanBP1 and RanGAP1. CAS binds preferentially to NLS-free importin α, explaining why import substrates stay in the nucleus.

The base of the proteasome regulatory particle exhibits chaperone-like activity

Protein substrates of the proteasome must apparently be unfolded and translocated through a narrow channel to gain access to the proteolytic active sites of the enzyme. Protein folding in vivo is mediated by molecular chaperones. Here, to test for chaperone activity of the proteasome, we assay the reactivation of denatured citrate synthase. Both human and yeast proteasomes stimulate the recovery of the native structure of citrate synthase. We map this chaperone-like activity to the base of the regulatory particle of the proteasome, that is, to the ATPase-containing assembly located at the substrate-entry ports of the channel. Denatured but not native citrate synthase is bound by the base complex. Ubiquitination of citrate synthase is not required for its binding or refolding by the base complex of the proteasome. These data suggest a model in which ubiquitin–protein conjugates are initially tethered to the proteasome by specific recognition of their ubiquitin chains; this step is followed by a nonspecific interaction between the base and the target protein, which promotes substrate unfolding and translocation.

Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope

BACKGROUND Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide. RESULTS We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. CONCLUSIONS 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.

COP9 signalosome‐specific phosphorylation targets p53 to degradation by the ubiquitin system

In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin–26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)‐specific phosphorylation targets human p53 to ubiquitin–26S proteasome‐dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N‐terminus with CSN subunit 5/Jab1 as shown by far‐western and pull‐down assays. The CSN‐specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Δp53(145–164), specifically inhibits CSN‐mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6‐dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6‐dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant (PAb 240+) conformation. It induces the cyclin‐dependent inhibitor p21. In HeLa and MCF‐7 cells, inhibition of CSN kinase by curcumin or Δp53(145–164) results in accumulation of endogenous p53.

A novel protein complex involved in signal transduction possessing similarities to 26S proteasome subunits

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 IκBα, 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.—Seeger, M., Kraft, R., Ferrell, K., Bech‐Otschir, D., Dumdey, R., Schade, R., Gordon, C., Naumann, M., Dubiel, W. A novel protein complex involved in signal transduction possessing similarities to 26S proteasome subunits. FASEB J. 12, 469–478 (1998)

Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes.

Transport receptors of the importin β superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co‐operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment‐specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin β family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF‐5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF‐5A appears to be complex and to involve the hypusine modification that is unique to eIF‐5A. We discuss possible cellular roles for nuclear export of eIF‐5A.

Importin 13: a novel mediator of nuclear import and export

Importin β‐related receptors mediate translocation through nuclear pore complexes. Co‐operation with the RanGTPase system allows them to bind and subsequently release their substrates on opposite sides of the nuclear envelope, which in turn ensures a directed nucleocytoplasmic transport. Here we identify a novel family member from higher eukaryotes that functions primarily, but not exclusively, in import. It accounts for nuclear accumulation of the SUMO‐1/sentrin‐conjugating enzyme hUBC9 and mediates import of the RBM8 (Y14) protein, and is therefore referred to as importin 13 (Imp13). Unexpectedly, Imp13 also shows export activity towards the translation initiation factor eIF1A and is thus a case where a single importin β‐like receptor transports different substrates in opposite directions. However, Imp13 operates differently from typical exportins in that the binding of eIF1A to Imp13 is only regulated indirectly by RanGTP, and the cytoplasmic release of eIF1A from Imp13 is triggered by the loading of import substrates onto Imp13.

Importin Provides a Link between Nuclear Protein Import and U snRNA Export

Importin-alpha mediates nuclear protein import by binding nuclear localization signals and importin-beta. We find approximately 30% of SRP1p, the yeast importin-alpha, in a nuclear complex with the Saccharomyces cerevisiae nuclear cap-binding protein complex (CBC). Similarly, a large fraction of Xenopus CBC is associated with importin-alpha in the nucleus. CBC promotes nuclear export of capped U snRNAs and shuttles between nucleus and cytoplasm. The CBC-importin-alpha complex binds specifically to capped RNA, suggesting that CBC might shuttle while bound to importin-alpha. Strikingly, importin-beta binding displaces the RNA from the CBC-importin-alpha complex. Thus, the commitment of CBC for nuclear reentry triggers the release of the export substrate into the cytoplasm. We provide evidence for a mechanism that ensures that importin-mediated RNA release is a specifically cytoplasmic event.

Analysis of mammalian 20S proteasome biogenesis: the maturation of beta-subunits is an ordered two-step mechanism involving autocatalysis.

Maturation of eukaryotic 20S proteasomes involves the processing of beta‐subunits by limited proteolysis. To study the processing mechanism we analysed different point mutations of the beta‐subunit LMP2 in transfected human T2 cells. Here we show that the presence of the intact Gly‐1Thr1 consensus motif and Lys33 are essential for correct processing. Mutation of Thr1, the active site residue in mature subunits, or of Lys33, results in complete inhibition of processing at the consensus site. In addition, proprotein processing in vitro of wild‐type LMP2, incorporated in immature 16S precursor complexes, can be blocked by a proteasome‐specific inhibitor. While the processing of inhibitor‐treated wild‐type proprotein was completely prevented, the site‐directed mutagenesis of LMP2 results in processing intermediates carrying an extension of 8–10 residues preceding Thr1, suggesting an additional cleavage event within the prosequence. Furthermore, exchange of mammalian prosequences interferes with processing efficiency and suggests subunit specificity. Based on our data we propose a model for self‐activation of proteasomal beta‐subunits in which residue Thr1 serves as nucleophile and Lys33 as proton donor/acceptor. We provide evidence that subunit processing of mammalian beta‐subunits proceeds via a novel ordered two‐step mechanism involving autocatalysis.