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.

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.

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.

Genes for immunoglobulins and T-ceil receptor are generated by a process known as V(D)J recombination. This process is highly regulated and mediated by the recombinatlon 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-i. This protein is the human homologue of the yeast SRP1 ( Here we describe the human (h) and mouse (m) homologues of SRP1, we show their interaction with RAG-1 in yeast as well as in mammalian cells, and we map the regions responsible for the interaction in both proteins. The possible role of SRP1 in V(D)J recombination is discussed.MATERIALS AND METHODS Cels and Antibodies. 293T cells (19) were grown in Dulbecco's modified Eagle's medium containing 10o fetal bovine serum, penicillin, and streptomycin. Anti-influenza hemagglutinin (HA) monoclonal antibody (12CA-5) was from Berkeley antibody company. Anti-glutathione S-transferase (GST) antibodies were generated in rabbits and affinity purified. pcDNAI/amp (Invitrogen), p65/GST, and p50-HA expression vectors will be described elsewhere.DNA Constructs. A pEG202RAG-1 vector encoding fulllength RAG-1 was assembled from a BamHI/EcoRV PCR product, an EcoRV/Not I fragment from RAG-1/CDM8, and a Not I/BamHI pEG202 vector (15). Ligation of these three fiagments generated pEG202/RAG-1. The joining segments were sequenced and the expression of the fusion product RAG-1-LexA was tested by transformation in yeast followed by Western blotting. pEG202/RAG-1 was used to screen a human cDNA library. All the subcloning of RAG-1 and hSRP1 homologue was done by production of in-frame PCR firgments or by directly subcloning from the cDNA with short oligonucleotides to leave the protein in-frame. The junctions of all constructs were sequenced.For immunoprecipitation experiments, pcDNAI/amp (Invitrogen) was modified by introducing an oligonucleotide containing an initiation codon in-frame with the HA epitope Abbreviations: GST, glutathione S-transferase; V(D)J, variablediversity-joining region; h-, human; in-, mouse; HA, hemagglutinin; NLS, nuclear localization signal. 7633The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 11734 solely to indicate this fact.

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 inmunodeficiency. 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. (11). To understand how Btk is involved in B-cell development and activation, we have searched for proteins with which Btk might interact. We show that Btk has a specific binding site that interacts with the SH3 domains of the Src-related Fyn, Lyn, and Hck kinases. These interactions occur through two prolinecontaining 10-aa sites in the amino-terminal region of Btk, just carboxyl-terminal to the PH domain. We suggest that Btk is on the pathway of signaling by which fyn, lyn, and hck control cellular events. MATERIALS AND METHODSCloning. To isolate Btk cDNA clones, oligonucleotides corresponding to either the amino terminus or carboxyl terminus of the human Btk protein were made according to the published sequences (5) and used to probe duplicate lifts of A phage plaques on filters. Ten independent doubly positive clones were isolated from -1 x 106 phage plaques. We partially sequenced these 10 cDNA clones and found that they all contained the full-length Btk coding sequence. A DNA fragment containing the amino-terminal portion of a mouse Btk cDNA clone was constructed in the EG202 vector, which contains the LexA DNA binding domain as its amino terminus (12). When this LexA-BtkN bait, along with the reporter plasmid pSH18-34 containing the LexA binding site driving the lacZ gene, was transformed into the yeast strain EGY48 (MATa, trpl, ura3, his, Leu2.plexAop6-leu2), colonies required leucine to grow and did not turn blue on 5-bromo-4-chloro-3-indolyl -D-galactoside (X-Gal) medium. Thus, the bait did not contain intrinsic transactivation potential and was suitable for library screening. Library plasmids (pJG4-5) containing the B42 acidic activation domain and cDNAs derived from HeLa cells were then transformed into the yeast clone carrying both the LexA-BtkN bait and the reporter plasmid pSH18-34. A total of 4.5 x 106 yeast transformants were selected on Ura-, His-, Trp-, and Leu-X-Gal/galactose plates. Approximately 3000 colonies grew in the absence of leucine, but only 90 turned blue. Because the library vector pJG4-5 used here contained a galactoseinducible promotor, colonies of authentic interacting clones should turn blue when galactose but not glucose is used as a carbon source. Colony replicas of the 90 clones were pl...