▪ Abstract The transcription factor NF-κB, more than a decade after its discovery, remains an exciting and active area of study. The involvement of NF-κB in the expression of numerous cytokines and adhesion molecules has supported its role as an evolutionarily conserved coordinating element in the organism's response to situations of infection, stress, and injury. Recently, significant advances have been made in elucidating the details of the pathways through which signals are transmitted to the NF-κB:IκB complex in the cytosol. The field now awaits the discovery and characterization of the kinase responsible for the inducible phosphorylation of IκB proteins. Another exciting development has been the demonstration that in certain situations NF-κB acts as an anti-apoptotic protein; therefore, elucidation of the mechanism by which NF-κB protects against cell death is an important goal. Finally, the generation of knockouts of members of the NF-κB/IκB family has allowed the study of the roles of these protein...

NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses

Communication between cells assumes particular importance in multicellular organisms. The growth, migration and differentiation of cells in the embryo, and their organization into specific tissues, depend on signals transmitted from one cell to another. In the adult, cell signalling orchestrates normal cellular behaviour and responses to wounding and infection. The consequences of breakdowns in this signalling underlie cancer, diabetes and disorders of the immune and cardiovascular systems. Conserved protein domains that act as key regulatory participants in many of these different signalling pathways are highlighted.

Protein modules and signalling networks

https://www.cell.com/cell/pdf/S0092-8674(00)80984-8.pdf

TRADD–TRAF2 and TRADD–FADD Interactions Define Two Distinct TNF Receptor 1 Signal Transduction Pathways

Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2).

CD40 is a cell surface receptor that belongs to the tumor necrosis factor-R (TNF-R) family, and that was first identified and functionally characterized on B lymphocytes. Its critical role in T cell-dependent humoral immune responses was demonstrated by patients with the hyper-IgM syndrome, as well as by gene targeting in mice. However, in recent years it has become clear that CD40 is expressed much more broadly, including expression on monocytes, dendritic cells, endothelial cells, and epithelial cells. In addition, the CD40-ligand (CD40-L/CD154), a member of the TNF family, is also expressed more widely than activated CD4+ T cells only. Therefore it is now thought that CD40-CD40-L interactions play a more general role in immune regulation. Collectively these studies have culminated in pre-clinical and clinical studies that are in progress. This article reviews recent developments in this field of research, with main emphasis on (1) structure and expression of CD40 and its ligand; (2) CD40 signal transduction; (3) in vitro function of CD40 on different cell types; and (4) in vivo functions of CD40/CD40-L interactions.

https://jlb.onlinelibrary.wiley.com/doi/pdf/10.1002/jlb.67.1.2

CD40-CD40 ligand.

CD40 is a receptor on the surface of B lymphocytes, the activation of which leads to B cell survival, growth, and differentiation. A yeast two-hybrid screen identified a gene, CRAF1, encoding a protein that interacts directly with the CD40 cytoplasmic tail through a region of similarity to the tumor necrosis factor-alpha (TNF-alpha) receptor-associated factors. Overexpression of a truncated CRAF1 gene inhibited CD40-mediated up-regulation of CD23. A region of CRAF1 was similar to the TNF-alpha receptor-associated factors TRAF1 and TRAF2 and so defined a shared TRAF-C domain that was necessary and sufficient for CD40 binding and homodimerization. The CRAF1 sequence also predicted a long amphipathic helix, a pattern of five zinc fingers, and a zinc ring finger. It is likely that other members of the TNF receptor superfamily use CRAF-related proteins in their signal transduction processes.

https://science.sciencemag.org/content/sci/267/5203/1494.full.pdf

Involvement of CRAF1, a relative of TRAF, in CD40 signaling

To understand the normal and oncogenic functions of the protein-tyrosine kinase Abl, the yeast two-hybrid system has been used for identifying proteins that interact with it. One interacting protein is Crk-I, an SH3/SH2-containing adapter protein that was originally identified as the oncogenic element in the avian sarcoma virus CT10. Direct interaction between the Crk-I SH3 and Abl at novel, -10 amino acid sites just carboxy-terminal to the Abl kinase domain occurs in vitro and in mammalian cells. There is a nearby site specific for binding another adapter, Nck, and these sites also bind Grb-2. When bound to Abl, Crk-I was phosphorylated on tyrosine. Thus, the SH3-binding sites on Abl serve as substrate recognition sites for the relatively nonspecific kinase of Abl. In Crk-I-transformed cells, Crk-I associates with endogenous c-Abl and is phosphorylated on tyrosine. The association of Crk and Abl suggests that Abl could play a role in v-Crk and Crk-I transformation and that normal Abl function may be partly mediated through bound adapter molecules.

http://genesdev.cshlp.org/content/8/7/783.full.pdf

Abl protein-tyrosine kinase selects the Crk adapter as a substrate using SH3-binding sites.

Oct-2, a POU homeo domain transcription factor, is believed to stimulate B-cell-restricted expression of immunoglobulin genes through binding sites in immunoglobulin gene promoters and enhancers. To determine whether Oct-2 is required for B-cell development or function, or has other developmental roles, the gene was disrupted by homologous recombination. Oct-2^(-/-) mice develop normally but die within hours of birth for undetermined reasons. Mutants contain normal numbers of B-cell precursors but are somewhat deficient in IgM+ B cells. These B cells have a marked defect in their capacity to secrete immunoglobulin upon mitogenic stimulation in vitro. Thus, Oct-2 is not required for the generation of immunoglobulin-bearing B cells but is crucial for their maturation to immunoglobulin-secreting cells and for another undetermined organismal function.

http://genesdev.cshlp.org/content/7/4/570.full.pdf

Oct-2, although not required for early B-cell development, is critical for later B-cell maturation and for postnatal survival.

Bruton's tyrosine kinase (Btk) is a recently described B-cell-specific tyrosine kinase. Mutations in this gene lead to human X chromosome-linked agammaglobulinemia and murine X-linked immunodeficiency. Although genetic evidence strongly suggests that Btk plays a crucial role in B-lymphocyte differentiation and activation, its precise mechanism of action remains unknown, primarily because the proteins that it interacts with have not yet been identified. Here, we show that Btk interacts with Src homology 3 domains of Fyn, Lyn, and Hck, protein-tyrosine kinases that get activated upon stimulation of B- and T-cell receptors. These interactions are mediated by two 10-aa motifs in Btk. An analogous site with the same specificity is also present in Itk, the T-cell-specific homologue of Btk. Our data extend the range of interactions mediated by Src homology 3 domains and provide an indication of a link between Btk and established signaling pathways in B lymphocytes.

https://www.pnas.org/content/pnas/91/17/8152.full.pdf

Binding of Bruton's tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domain-mediated interaction

Genes for immunoglobulins and T-cell receptor are generated by a process known as V(D)J recombination. This process is highly regulated and mediated by the recombination activating proteins RAG-1 and RAG-2. By the use of the two-hybrid protein interaction system, we isolated a human protein that specifically interacts with RAG-1. This protein is the human homologue of the yeast SRP1 (suppressor of a temperature-sensitive RNA polymerase I mutation). The SRP1-1 mutation is an allele-specific dominant suppressor of a temperature-sensitive mutation in the zinc binding domain of the 190-kDa subunit of Saccharomyces cerevisiae RNA polymerase I. The human SRP cDNA clone was used to screen a mouse cDNA library. We obtained a 3.9-kbp cDNA clone encoding the mouse SRP1. The open reading frame of this cDNA encodes a 538-amino acid protein with eight degenerate repeats of 40-45 amino acids each. The mouse and human SRP1 are 98% identical, while the mouse and yeast SRP1 have 48% identity. After cotransfection of the genes encoding RAG-1 and human SRP1 into 293T cells, a stable complex was evident. Deletion analysis indicated that the region of the SRP1 protein interacting with RAG-1 involved four repeats. The domain of RAG-1 that associates with SRP1 mapped N-terminal to the zinc finger domain. Because this region of RAG-1 is not required for recombination and SRP1 appears to be bound to the nuclear envelope, we suggest that this interaction helps to localize RAG-1.

Zheng-Sheng Ye

Papers

Involvement of CRAF1, a relative of TRAF, in CD40 signaling

Abl protein-tyrosine kinase selects the Crk adapter as a substrate using SH3-binding sites.

Oct-2, although not required for early B-cell development, is critical for later B-cell maturation and for postnatal survival.

Binding of Bruton's tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domain-mediated interaction

RAG-1 interacts with the repeated amino acid motif of the human homologue of the yeast protein SRP1.