Showing papers by "David Baltimore published in 1995"

Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B.

Abstract: NF-κB, which consists of two polypeptides, p50 (Mr 50K) and p65/RelA (Mr 65K), is thought to be a key regulator of genes involved in responses to infection, inflammation and stress1. Indeed, although developmentally normal, mice deficient in p50 display functional defects in immune responses2. Here we describe the generation of mice deficient in the RelA subunit of NF-κB. Disruption of the relA locus leads to embryonic lethality at 15–16 days of gestation, concomitant with a massive degeneration of the liver by programmed cell death or apoptosis. Embryonic fibroblasts from RelA-deficient mice are defective in the tumour necrosis factor (TNF)-mediated induction of messenger RNAs for IκBα and granulocyte/macrophage colony stimulating factor (GM-CSF), although basal levels of these transcripts are unaltered. These results indicate that RelA controls inducible, but not basal, transcription in NF-κB-regulated pathways.

Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4.

Abstract: Human immunodeficiency virus (HIV) and simian immunodeficiency virus Nef proteins contain a conserved motif with the minimal consensus (PxxP) site for Src homology region 3 (SH3)-mediated protein-protein interactions. Nef PxxP motifs show specific binding to biotinylated SH3 domains of Hck and Lyn, but not to those of other tested Src family kinases or less related proteins. A unique cooperative role of a distant proline is also observed. Endogenous Hck of monocytic U937 cells can be specifically precipitated by matrix-bound HIV-1 Nef, but not by mutant protein lacking PxxP. Intact Nef PxxP motifs are dispensable for Nef-induced CD4 down-regulation, but are required for the higher in vitro replicative potential of Nef+ viruses. Thus, CD4 down-regulation and promotion of viral growth are two distinct functions of Nef, and the latter is mediated via SH3 binding.

Reduction of caveolin and caveolae in oncogenically transformed cells

Abstract: Caveolae are flask-shaped non-clathrin-coated invaginations of the plasma membrane. In addition to the demonstrated roles for caveolae in potocytosis and transcytosis, caveolae may regulate the transduction of signals from the plasma membrane. Transformation of NIH 3T3 cells by various oncogenes leads to reductions in cellular levels of caveolin, a principal component of the protein coat of caveolae. The reduction in caveolin correlates very well with the size of colonies formed by these transformed cells when grown in soft agar. Electron microscopy reveals that caveolae are morphologically absent from these transformed cell lines. These observations suggest that functional alterations in caveolae may play a critical role in oncogenic transformation, perhaps by disrupting contact inhibition in transformed cells.

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

Abstract: 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.

Constitutive NF-kappa B activation, enhanced granulopoiesis, and neonatal lethality in I kappa B alpha-deficient mice.

Abstract: Transcription factors belonging to the NF-kappa B family are controlled by inhibitory I kappa B proteins, mainly I kappa B alpha and I kappa B beta. Apparently normal at birth, I kappa B alpha-/- mice exhibit severe runting, skin defects, and extensive granulopoiesis postnatally, typically dying by 8 days. Hematopoietic tissues from these mice display elevated levels of both nuclear NF-kappa B and mRNAs of some, but not all, genes thought to be regulated by NF-kappa B. NF-kappa B elevation results in these phenotypic abnormalities because mice lacking both I kappa B alpha and the p50 subunit of NF-kappa B show a dramatically delayed onset of abnormalities. In contrast to hematopoietic cells, I kappa B alpha-/- embryonic fibroblasts show minimal constitutive NF-kappa B, as well as normal signal-dependent NF-kappa B activation that is concomitant with I kappa B beta degradation. Our results indicate that I kappa b beta, but not I kappa B alpha, is required for the signal-dependent activation of NF-kappa B in fibroblasts. However, I kappa B alpha is required for the postinduction repression of NF-kappa B in fibroblasts. These results define distinct roles for the two forms of I kappa B and demonstrate the necessity for stringent control of NF-kappa B.

Proline-rich sequences that bind to Src homology 3 domains with individual specificities.

Abstract: To study the binding specificity of Src homology 3 (SH3) domains, we have screened a mouse embryonic expression library for peptide fragments that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhibit differential binding specificity to various SH3 domains. Src-SH3-specific binding uses a sequence of 7 aa of the consensus RPLPXXP, in which the N-terminal arginine is very important. The SH3 domains of the Src-related kinases Fyn, Lyn, and Hck bind to this sequence with the same affinity as that of the Src SH3. In contrast, a quite different proline-rich sequence from the Btk protein kinase binds to the Fyn, Lyn, and Hck SH3 domains, but not to the Src SH3. Specific binding of the Abl SH3 requires a longer, more proline-rich sequence but no arginine. One clone that binds to both Src and Abl SH3 domains through a common site exhibits reversed binding orientation, in that an arginine indispensable for binding to all tested SH3 domains occurs at the C terminus. Another clone contains overlapping yet distinct Src and Abl SH3 binding sites. Binding to the SH3 domains is mediated by a common PXXP amino acid sequence motif present on all ligands, and specificity comes about from other interactions, often ones involving arginine. The rules governing in vivo usage of particular sites by particular SH3 domains are not clear, but one binding orientation may be more specific than another.

Nuclear factors associated with transcriptional regulation

Abstract: Constitutive and tissue-specific protein factors which bind to transcriptional regulatory elements of Ig genes (promoter and enhancer) are described. The factors were identified and isolated by an improved assay for protein-DNA binding. Genes encoding factors which positively regulate transcription can be isolated and employed to enhance transription of Ig genes. In particular, NF-kB, the gene encoding NF-kB, IkB and the gene encoding IkB and uses therefor.

3BP-1, an SH3 domain binding protein, has GAP activity for Rac and inhibits growth factor-induced membrane ruffling in fibroblasts.

Abstract: The SH3 binding protein, 3BP-1, was originally cloned as a partial cDNA from an expression library using the Abl SH3 domain as a probe. In addition to an SH3 binding domain, 3BP-1 displayed homology to a class of GTPase activating proteins (GAPs) active against Rac and Rho proteins. We report here a full length cDNA of 3BP-1 which extends the homology to GAP proteins previously noted. 3BP-1 functions in vitro as a GAP with a specificity for Rac-related G proteins. Microinjection of the 3BP-1 protein into serum-starved fibroblasts produces an inhibition of platelet-derived growth factor (PDGF)-induced membrane ruffling mediated by Rac. Co-injection of 3BP-1 with an activated Rac mutant that is unresponsive to GAPs, counter-acts this inhibition. 3BP-1 does not show in vitro activity towards Rho and, in agreement with this finding, microinjection of 3BP-1 into fibroblasts has no effect on lysophosphatidic acid (LPA)-induced stress fiber assembly mediated by Rho. Thus 3BP-1 is a new and specific Rac GAP that can act in cells to counter Rac-mediated membrane ruffling. How its SH3 binding site interacts with its GAP activity remains to be understood.

HIV-1 Messenger RNA in Peripheral Blood Mononuclear Cells as an Early Marker of Risk for Progression to AIDS

Abstract: Objective: To establish human immunodeficiency virus type 1 (HIV-1) messenger RNA (mRNA) expression in peripheral blood mononuclear cells as a marker of risk for progression to the acquired immunod...

Lessons from people with nonprogressive HIV infection.

Abstract: Infectious diseases can be extremely variable in their manifestations, but human immunodeficiency virus (HIV) infection is notorious for its protean manifestations. One of these, the absence of any apparent progression of disease over a decade or more, is particularly intriguing. The average time from HIV infection to death is 10 years, but clinical and immunologic decline is generally evident much earlier. About 5 percent of infected people are characterized as having nonprogressive infection because they remain healthy and do not have the declining CD4+ lymphocyte counts that are evident in people with progressive disease.1 Although we remain uncertain of their . . .

Discovery of the Reverse Transcriptase

Abstract: 1995 is the 20th anniversity of the award of the Nobel Prize in Physiology or Medicine to David Baltimore and Howard Temin for the discovery of the reverse transcriptase. The prize was shared with Renato Dulbecco.

Identification of 3BP-1 in cDNA expression library by SH3 domain screening.

Abstract: Publisher Summary This chapter describes a screening procedure to assay for protein–protein interactions using a biotinylated glutathione S-transferase (GST) fusion protein probe to screen cDNA expression libraries This method is developed to allow nonradioactive probing, which has the additional advantages of a very low background signal and a low incidence of false positives Because neither the fusion protein probe nor the protein in the expression library is denatured, this assay allows the structural components of the binding reaction to remain intact This screening procedure offers a practical and highly efficient method of identifying protein–protein interactions Using this procedure to detect proteins that bound to the Abl SH3 domain, five cDNA clones were isolated out of 7 million cDNA containing plaques that were screened Of these five, three contained the identical cDNA clone termed “3BP-1,” while the other two were identical for a different clone termed “3BP-2” On sequencing these clones, only a short stretch of approximately 40 amino acids was found where sequence similarity existed between these proteins

Modular Binding Domains in Signal Transduction Proteins Review

Abstract: The transduction of a signal is a change in form of the signal as it is passed from one carrier to another. The root "duce" means"to lead" in Latin; thus, a signal is led through a cell by steps of transduction (the same root is in the words seduce and duct as well as II Duce). The earliest transduction steps that were elucidated involved massive release of small molecule "second messengers", originally cAMP, that flooded a cell with information. With the understanding that such proteins as tyrosine kinases and Ras relatives are signal transducers, came the realization that many signaling pathways are more precise, sending controlled and probably weakly amplified signals to specific targets. These intracellular signals are often maintained in macromolecular form rather than being passed to small molecules. The proteins that carry these signals are not acting in the classic fashion of enzymes that are designed to modify large numbers of substrate molecules. These signal transducers, even if they catalyze an event such as phosphorylation, generally affect small numbers of target molecules and have often separated their catalytic function from their binding regions, which can bring substrates to the catalytic centers, link the signal transducers to upstream proteins, and localize protein complexes to particular cellular subregions. The binding domains are often modular ones constructed with a common core recognition ability coupled to a fine specificity control. They modulate the interaction of proteins with other proteins and therefore determine the paths of signal transduction systems. They are generally controlled, also, so that the aggregates they form are transient, pathways forming only when the signal is being transmitted and then disaggregating when the signal has passed. The signal transduction protein must be highly integrated, with all of the elements working together to send just the appropriate quanta of signal for the specific need. The preceding paragraphs are generalizations based on scanty and fragmentary evidence. With many transduction pathways known only in outline and more sure to be found, there is a deeply exciting richness yet to be elucidated. But many investigators are now working within the framework presented above, trying to find the relevant units and understand their integration. Protein-protein interaction has long been studied, but the realization of the importance of defined binding rood-