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Biomedical subjects

D Baltimore

Publications and source records attributed to D Baltimore.

At least 109 records · Page 6Linked to original sources
PubMed1994

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

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.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

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

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.

Amino Acid Sequence↗
PubMed1994

Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice.

We have examined the regulatory role of the individual components of the immunoglobulin antigen receptor in B-cell development by transgenic complementation of Rag-1 deficient (Rag-1-) mice. Complementation with a membrane mu heavy chain (mu HC) gene allows progression of developmentally arrested Rag-1- pro-B-cells to the small pre-B cell stage, whereas the introduction of independently integrated mu HC and kappa light chain (kappa LC) transgenes promotes the appearance of peripheral lymphocytes which, however, remain unresponsive to external stimuli. Complete reconstitution of the B-cell lineage and the emergence of functionally nature Rag-1- peripheral B cells is achieved by the introduction of cointegrated heavy and light chain transgenes encoding an anti-H-2k antibody. This experimental system demonstrates the competence of the mu HC and kappa LC to direct and regulate the sequential stages of B-cell differentiation, defines the time at which negative selection of self-reactive B cells occurs, and shows that elimination of these cells occurs equally well in the absence of Rag-1 as in its presence. These data also support the hypothesis that Rag-1 directly participates in the V(D)J recombination process.

Animals↗
PubMed1994

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

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, approximately 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.

3T3 Cells↗
PubMed1994

Human immunodeficiency virus type 1 mRNA expression in peripheral blood cells predicts disease progression independently of the numbers of CD4+ lymphocytes.

To address the significance of human immunodeficiency virus (HIV) replication in peripheral blood mononuclear cells (PBMCs), we have used reverse transcriptase-initiated PCR to measure HIV-1 mRNA expression in PBMC specimens collected from a cohort of HIV-infected individuals during a long-term prospective study. We found dramatic differences in HIV mRNA expression among individuals with very similar clinical and laboratory indices, and this variation strongly correlated with the future course of the disease. No evidence of viral replication was detected in PBMCs from asymptomatic individuals who, thereafter, had normal levels of CD4+ cells for at least 5 years. Irrespective of whether the CD4+ cell numbers were normal at the time of sampling, abundant expression of HIV-1 mRNA in PBMCs predicted accelerated disease progression within the next 2 years. Thus, independently of what may be the rate of HIV replication in other viral reservoirs, such as lymphatic tissue, the amount of HIV mRNA in PBMCs appears to reflect the subsequent development of HIV disease. We have also used the reverse transcriptase-initiated PCR assay to demonstrate a transient response to 3'-azido-3'-deoxythymidine treatment. Determination of HIV-1 mRNA expression in the PBMCs of infected individuals could, therefore, have significant clinical utility as a prognostic indicator and as a means to guiding and monitoring antiviral therapies.

CD4-Positive T-Lymphocytes↗
PubMed1994

SH2 and SH3 domains as molecular adhesives: the interactions of Crk and Abl.

The Src homology domains SH2 and SH3 are modular components present in many signal transduction proteins. They allow rapid formation of stable protein complexes and may also regulate protein function through intramolecular binding events. SH2 domains recognize phosphotyrosyl residues in a specific sequence context, while SH3 domains recognize a PxxP motif and additional residues that mediate binding specificity.

Adhesiveness↗
PubMed1994

Functional commitment to helper T cell lineage precedes positive selection and is independent of T cell receptor MHC specificity.

Thymocyte differentiation proceeds from double positive CD4+CD8+ to single positive T cells. It has been proposed that this process occurs by an instructive or a stochastic mechanism. In this report, we show that in recombination-deficient mice (RAG-1-I-) constitutive expression of a CD8 transgene allows maturation of CD4+(CD8tg+) cells, which express mature levels of a transgenic class I-restricted T cell receptor, F5. Rescued F5+CD4+(CD8tg+) cells have equivalent levels of T cell receptor expression as CD8end+ cells, respond to cognate antigen and, upon stimulation, they exhibit a phenotype characteristic of CD4+ helper T cells. These data are consistent with a model of differentiation that predicts that thymocytes become functionally committed to a helper or cytotoxic lineage before the final step of positive selection and independently of MHC specificity of their T cell receptor.

Animals↗
PubMed1994

The COOH terminus of the c-Abl tyrosine kinase contains distinct F- and G-actin binding domains with bundling activity.

The myristoylated form of c-Abl protein, as well as the P210bcr/abl protein, have been shown by indirect immunofluorescence to associate with F-actin stress fibers in fibroblasts. Analysis of deletion mutants of c-Abl stably expressed in fibroblasts maps the domain responsible for this interaction to the extreme COOH-terminus of Abl. This domain mediates the association of a heterologous protein with F-actin filaments after microinjection into NIH 3T3 cells, and directly binds to F-actin in a cosedimentation assay. Microinjection and cosedimentation assays localize the actin-binding domain to a 58 amino acid region, including a charged motif at the extreme COOH-terminus that is important for efficient binding. F-actin binding by Abl is calcium independent, and Abl competes with gelsolin for binding to F-actin. In addition to the F-actin binding domain, the COOH-terminus of Abl contains a proline-rich region that mediates binding and sequestration of G-actin, and the Abl F- and G-actin binding domains cooperate to bundle F-actin filaments in vitro. The COOH terminus of Abl thus confers several novel localizing functions upon the protein, including actin binding, nuclear localization, and DNA binding. Abl may modify and receive signals from the F-actin cytoskeleton in vivo, and is an ideal candidate to mediate signal transduction from the cell surface and cytoskeleton to the nucleus.

3T3 Cells↗
PubMed1994

Distinct modes of human immunodeficiency virus type 1 proviral latency revealed by superinfection of nonproductively infected cell lines with recombinant luciferase-encoding viruses.

To study the basis of cellular latency of human immunodeficiency virus (HIV), we have used a recombinant luciferase-encoding HIV (HXB-Luc) to superinfect nonproductively HIV-1-infected human leukemic cell lines. HXB-Luc contains the Photinus pyralis luciferase gene in place of the nef gene and provides a highly sensitive, simple assay for HIV infection and expression. To circumvent any superinfection block in latently infected cells, we also generated viruses pseudotyped with murine leukemia virus amphotropic envelope (HXB-Luc:ampho). The parental uninfected lines, U937 and A3.01, from which the latently infected cell lines U1 and ACH-2, respectively, were derived could be readily infected with pseudotyped or nonpseudotyped reporter viruses. However, superinfection of U1 cells with either HXB-Luc or HXB-Luc:ampho resulted in only low levels of luciferase activity. Like the endogenous provirus, HXB-Luc provirus could be efficiently activated by phorbol ester treatment of HXB-Luc:ampho-superinfected U1 cells. In contrast, superinfection of ACH-2 cells resulted in active expression of the secondarily introduced virus even in unstimulated cells and luciferase production higher than in the parental cell line A3.01. Thus, the proviral latency in U1 cells appears to result from a defect in the cellular environment (a trans effect), whereas the latency in ACH-2 is specific to the integrated provirus and is probably a cis effect due to the site of integration. These results demonstrate distinct modes of proviral latency in these two cell line models and may have implications in our understanding of the regulation and significance of cellular latency in HIV infection.

Base Sequence↗
PubMed1994

Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase.

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

Animals↗
PubMed1994

Sequential induction of NF-kappa B/Rel family proteins during B-cell terminal differentiation.

The NF-kappa B/Rel family of at least five transcription factor polypeptides is thought to function both as a developmental regulator in B cells and as a rapid response system in all cells. To examine this notion in more detail, we determined the protein contents of both the inducible and constitutive NF-kappa B/Rel activities in a pre-B-cell line, 70Z/3, and a mature B-cell line, WEHI 231. NF-kappa B p50/p65 is the major inducible nuclear complex after lipopolysaccharide or phorbol myristate acetate treatment of 70Z/3 cells. The constitutive and inducible complexes in WEHI 231 cells are mainly composed of p50 and Rel. The constitutive or induced activities are all sensitive to I kappa B-alpha, but this inhibitor is very short-lived in WEHI 231 cells, suggesting that the balance between synthesis and degradation of I kappa B-alpha determines whether a particular cell lineage has constitutive activity. A patterned expression of the NF-kappa B/Rel activator proteins emerges from an analysis of other B-lineage cell lines and splenic B cells: mainly p50 and p65 in pre-B (and non-B) cells, a predominance of Rel and p50 in mature B cells, and expression of p52 and RelB in plasmacytoma lines. This ordered pattern of regulators may reflect the requirement for expression of different genes during terminal B-cell differentiation because different combinations of NF-kappa B/Rel family members preferentially activate distinct kappa B sites in reporter constructs.

Animals↗
PubMed1993

Production of high-titer helper-free retroviruses by transient transfection.

The generation of high-titer, helper-free retroviruses by transient transfection has been achieved by using the highly transfectable 293T cell line into which are stably introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packaging cell line BOSC 23 produces infectious retrovirus at > 10(6) infectious units/ml of supernatant within 72 hr after CaPO4-mediated transfection. A stringent assay for replication-competent virus showed that no helper virus was present. The system can produce high titers of retroviral vectors expressing genes that are extremely difficult to propagate at high titer in stable producer lines. This method should facilitate and extend the use of helper-free retroviral gene transfer, as well as be useful for gene therapy.

3T3 Cells↗
PubMed1993

Dispensable sequence motifs in the RAG-1 and RAG-2 genes for plasmid V(D)J recombination.

As a probe of whether RAG-1 and RAG-2 gene products activate other genes or form part of the recombinase itself, certain mutants of the RAG genes were assayed for their ability to activate variable-diversity-joining region [V(D)J] recombination in a plasmid substrate in fibroblasts. The results indicate that the N-terminal one-third of RAG-1, including a zinc-finger-like domain, and an acidic domain of RAG-2 are dispensable for activating V(D)J recombination in a fibroblast, although they contribute quantitatively. In contrast, deletion of the C-terminal segment of RAG-1, which has homology to a topoisomerase-like protein from yeast, abolished recombination activation. These results do not support the hypothesis that the RAG gene products are transcription factors and suggest the possibility that they are parts of the recombination machinery.

3T3 Cells↗