Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B

Nuclear factor kappa B (NF-kappa B), which was first detected by its binding to the kappa B site in the immunoglobulin kappa-gene enhancer, is important for the regulated expression of the kappa-gene and is partly responsible for the induction in appropriate cells of interleukin-2 (IL-2), IL-2 alpha receptor, beta-interferon and serum amyloid A protein. NF-kappa B is present as a nuclear DNA-binding protein in B lymphocytes and mature macrophages, but is found in the cytoplasm of many cells in a form unable to bind to DNA. The cytoplasmic form is bound to an inhibitor protein, I kappa B, from which it can be released in vitro by deoxycholate and other agents. Activation of cells by various agents, notably the phorbol esters that stimulate protein kinase C (PKC), leads to dissociation in vivo of the NF-kappa B/I kappa B complex and migration of NF-kappa B to the nucleus. Therefore, it acts as a second messenger system, transducing activation signals from the cytoplasm to the nucleus. To elucidate the mechanism of signal transfer, we have used an in vitro system in which addition of purified protein kinases to a partially purified NF-kappa B/I kappa B complex leads to the activation of the DNA-binding activity of NF-kappa B. Using gel retardation assays we found that PKC, cyclic AMP-dependent protein kinase (PKA) and a haem-regulated eIF-2 kinase (HRI) could activate NF-kappa B in vitro, whereas casein kinase II was ineffective. To determine the target for the protein kinases we purified and characterized both NF-kappa B and I kappa B and found that I kappa B is phosphorylated and inactivated in the presence of PKC and HRI but not PKA.     

TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2

The I kappa B kinase (IKK), consisting of the IKK1 and IKK2 catalytic subunits and the NEMO (also known as IKK gamma) regulatory subunit, phosphorylates I kappa B proteins, targeting them for degradation and thus inducing activation of NF-kappa B (reviewed in refs 1, 2). IKK2 and NEMO are necessary for NF-kappa B activation through pro-inflammatory signals. IKK1 seems to be dispensable for this function but controls epidermal differentiation independently of NF-kappa B. Previous studies suggested that NF-kappa B has a function in the growth regulation of epidermal keratinocytes. Mice lacking RelB or I kappa B alpha, as well as both mice and humans with heterozygous NEMO mutations, develop skin lesions. However, the function of NF-kappa B in the epidermis remains unclear. Here we used Cre/loxP-mediated gene targeting to investigate the function of IKK2 specifically in epidermal keratinocytes. IKK2 deficiency inhibits NF-kappa B activation, but does not lead to cell-autonomous hyperproliferation or impaired differentiation of keratinocytes. Mice with epidermis-specific deletion of IKK2 develop a severe inflammatory skin disease, which is caused by a tumour necrosis factor-mediated, alpha beta T-cell-independent inflammatory response that develops in the skin shortly after birth. Our results suggest that the critical function of IKK2-mediated NF-kappa B activity in epidermal keratinocytes is to regulate mechanisms that maintain the immune homeostasis of the skin.     

A novel nuclear export activity in HIV-1 matrix protein required for viral replication

An important aspect of the pathophysiology of human immunodeficiency virus type-1 (HIV-1) infection is the ability of the virus to replicate in non-dividing cells. HIV-1 matrix (MA), the amino-terminal domain of the Pr55 gag polyprotein (Pr55), bears a nuclear localization signal that promotes localization of the viral preintegration complex to the nucleus of non-dividing cells following virus entry. However, late during infection, MA, as part of Pr55, directs unspliced viral RNA to the plasma membrane, the site of virus assembly. How MA can mediate these two opposing targeting functions is not understood. Here we demonstrate that MA has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal, nuclear export is mediated through the conserved Crm1p pathway and functions in both mammalian cells and yeast. A mutation that disrupts the MA nuclear export signal (MA-M4) mislocalizes Pr55 and genomic viral RNA to the nucleus, thereby severely impairing viral replication. Furthermore, we show that MA-M4 can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild-type MA. We conclude that the MA nuclear export signal is required to counteract the MA nuclear localization signal, thus ensuring the cytoplasmic availability of the components required for virion assembly.     

Structure of pre-pro-von Willebrand factor and its expression in heterologous cells

Von Willebrand factor (vWF), a multifunctional haemostatic glycoprotein derived from endothelial cells and megakaryocytes, mediates platelet adhesion to injured subendothelium and binds coagulation factor VIII in the circulation. Native vWF is a disulphide-bonded homopolymer; the monomeric subunits, of apparent relative molecular mass (Mr) 220,000 (220K) are derived from an intracellular precursor estimated at 260-275K. Multimer assembly is preceded by the formation of dimers, linked near their C-termini, which then assemble into filamentous polymers. The importance of the removal of the large vWF pro-polypeptide during multimer assembly, and whether this or other stages of the complex post-translational processing require components specific to endothelial cells or megakaryocytes, is unknown. Here we report an analysis of the complete sequence of pre-pro-vWF and expression of the molecule in heterologous cells. The vWF precursor is composed of several repeated subdomains. When expressed in COS and CHO cells, it is cleaved and assembled into biologically active high relative molecular mass disulphide bonded multimers. This suggests that the information for assembly of this complex molecule resides largely within its primary structure.     

Molecular components of the B-cell antigen receptor complex of the IgM class

The antigen receptors on mature B lymphocytes are membrane-bound immunoglobulins of the IgM and IgD classes whose cross-linking by polyvalent antigens results in B-cell proliferation and differentiation. How these membrane-bound immunoglobulin chains, which lack a cytoplasmic tail, generate a cell activation signal is not at present known. We now show that the IgM molecule is non-covalently associated in the membrane of B cells with two proteins of relative molecular mass 34,000 (Mr 34 K; IgM-alpha) and 39 K (Ig-beta) which form a disulphide-linked heterodimer. Surface expression of IgM seems to require the formation of an appropriate complex between IgM and the heterodimer. A transfection experiment indicates that IgM-alpha is the product of mb-1, a B-cell specific gene encoding a transmembrane protein with sequence homology to proteins of the T-cell antigen receptor-CD3 complex.     

Differential expression of calmodulin-binding proteins in B, T lymphocytes and thymocytes

Changes in intracellular free Ca2+ are involved in the transmembrane signalling of different cells, including lymphocytes. Since calmodulin (CaM) is a primary receptor for Ca2+ (ref. 4), it may mediate the activation of crucial enzymes after antigen-induced increases in cytosolic Ca2+. Using a biotinylated-CaM (Bio-CaM) detection procedure to identify such proteins, we found that a peptide of relative molecular mass 59,000 (59K) was the predominant soluble CaM-binding protein (CaM-BP) in T cells and B lymphocytes from murine spleen; immunoblotting experiments identified it as a subunit of the CaM-dependent phosphatase, 'calcineurin' (CN). Smaller amounts of larger CaM-BPs, thought to be cytoskeletal-binding proteins, were also detected. CaM-BPs were expressed differentially, with B lymphocytes having four times more of the CN-like protein than T lymphocytes, while in thymocytes, a 65K polypeptide was the major CaM-BP. However, limited proteolysis analysis suggested that this thymus-specific peptide may be a precursor of CN. These data suggest that Ca2+-stimulated protein dephosphorylation may be an important and highly regulated function in lymphoid cells.     

Abnormal tyrosine phosphorylation on T-cell receptor in lymphoproliferative disorders

The study of human autoimmune diseases has benefited greatly from analysis of animal models. Mice that are homozygous for either the lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) mutant genes develop a disease characterized by massive lymphadenopathy and autoantibody formation. With age, the lymphoid organs in these mice are replaced with a greatly expanded population of abnormal lymphocytes. Recent work has shown that these cells are likely to be in the T-cell lineage. They rearrange and transcribe the genes for the alpha and beta subunits of the T-cell receptor (TCR) and a third, T-cell receptor-like gene, T gamma. As determined by immunofluorescence with anti-receptor antibodies the cells also express TCR on the cell surface. The murine T-cell receptor consists of the alpha and beta chains, derived from the rearranged alpha and beta genes, in non-covalent association with seven other chains; the delta chain, of relative molecular mass (Mr) 26,000 (26K), the epsilon chain (25K), a glycosylated 21K chain (gp21) which is probably the homologue of the gamma chain of T3 (CD3), a 16K homodimer (zeta) and a 21K dimer (p21). This multichain complex is thought to be the murine analogue of the human T3 complex. After activation of normal T cells by antigen or lectin, p21 is phosphorylated on tyrosine residues and gp21 is phosphorylated on serine residues. In contrast, in the gld and lpr cells, p21 is phosphorylated even in the absence of antigen or lectin, whereas gp21 is not phosphorylated.     

Isolation of cDNA clones encoding the 20K non-glycosylated polypeptide chain of the human T-cell receptor/T3 complex

The antigen receptor on human T lymphocytes consists of two variable immunoglobulin-like glycoproteins, alpha and beta, which occur in association with three invariable T3 membrane proteins. In humans two of these proteins, T3-gamma and T3-delta, are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20,000 (20K), respectively, while the third, T3-epsilon, is a 20K non-glycosylated protein. On the surface of murine T cells, a non-glycosylated protein dimer composed of 17K subunits (T3-zeta) is found associated with the T-cell receptor alpha and beta chains and the three T3-like polypeptide chains. It is generally accepted that major histocompatibility complex-restricted antigen recognition is a function of the alpha-beta heterodimer. This has led to the postulation that the proteins of the T3 complex are involved in the signal transduction that immediately follows antigen recognition via the antigen receptor. Events believed to be involved in early T-cell activation, such as rapid increases in phosphatidylinositol turnover and free intracellular calcium, can be triggered by antibodies directed against either the T3 complex or the clonotypic receptor. We have previously reported our findings on the cloning of the complementary DNA and genomic structure encoding both the human and murine 20K glycoprotein, T3-delta (refs 11-13). We now present our results on the cloning of the cDNA encoding the human 20K non-glycosylated chain, T3-epsilon.     

Tyrosine-containing motif that transduces cell activation signals also determines internalization and antigen presentation via type III receptors for IgG.

Type III receptors for IgG (Fc gamma RII; ref. 1), high-affinity IgE receptors (Fc epsilon RI; ref. 2), as well as the T- and B-cell antigen receptors, consist of multiple components with specialized ligand-binding and signal transduction functions. Fc gamma RII alpha (ligand-binding) and gamma (signal-transducing) subunits are expressed in macrophages, a cell type involved in the uptake of antigen, its processing and the presentation of the resulting peptides to major histocompatibility complex class II-restricted T lymphocytes. Here we show that murine Fc gamma RIII, transfected into Fc gamma R-negative antigen-presenting B-lymphoma cells, mediate rapid ligand internalization and strongly increase the efficiency of antigen presentation when antigen is complexed to IgG. Efficient internalization and antigen presentation via Fc gamma RIII did not require the cytoplasmic domain of the ligand-binding alpha-chain, but did require the gamma-subunit. Using chimaeric molecules, we show that gamma-chain contains a signal for receptor internalization and that the mutation of either of the two tyrosine residues present in its cytoplasmic domain prevents efficient internalization and antigen presentation of immune complexes. Thus, associated chains and their tyrosine-containing motif are not exclusively involved in cell activation, but also determine multimeric receptor internalization.