14–3-3 Inhibits the Dictyostelium Myosin II Heavy-Chain-specific Protein Kinase C Activity by a Direct Interaction: Identification of the 14–3-3 Binding Domain

Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14–3-3 protein (Dd14–3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14–3-3 ζ isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14–3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14–3-3. This suggests that Dd14–3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14–3-3 as well as 14–3-3ζ through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14–3-3 family and demonstrate that MHC-PKC interacts directly with Dd14–3-3 and 14–3-3ζ through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.

Mammalian Transcription Factor ATF6 Is Synthesized as a Transmembrane Protein and Activated by Proteolysis in Response to Endoplasmic Reticulum Stress

The unfolded protein response (UPR) controls the levels of molecular chaperones and enzymes involved in protein folding in the endoplasmic reticulum (ER). We recently isolated ATF6 as a candidate for mammalian UPR-specific transcription factor. We report here that ATF6 constitutively expressed as a 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) in ER-stressed cells. Furthermore, we showed that the most important consequence of this conversion was altered subcellular localization; p90ATF6 is embedded in the ER, whereas p50ATF6 is a nuclear protein. p90ATF6 is a type II transmembrane glycoprotein with a hydrophobic stretch in the middle of the molecule. Thus, the N-terminal half containing a basic leucine zipper motif is oriented facing the cytoplasm. Full-length ATF6 as well as its C-terminal deletion mutant carrying the transmembrane domain is localized in the ER when transfected. In contrast, mutant ATF6 representing the cytoplasmic region translocates into the nucleus and activates transcription of the endogenous GRP78/BiP gene. We propose that ER stress-induced proteolysis of membrane-bound p90ATF6 releases soluble p50ATF6, leading to induced transcription in the nucleus. Unlike yeast UPR, mammalian UPR appears to use a system similar to that reported for cholesterol homeostasis.

Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: Targeting with a single chain antibody variable domain isolated by phage display

EGFRvIII is a mutant epidermal growth factor receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung. The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence. Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII. A single chain antibody variable domain (scFv) phage display library of 8 × 106 members was made from the spleen of one immunized mouse. A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide. This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A. Purified immunotoxin had a Kd of 22 nM for peptide and a Kd of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse fibroblasts transfected with EGFRvIII and an IC50 of 7–10 ng/ml (110–160 pM) on transfected glioblastoma cells. There was no cytotoxic activity at 1000 ng/ml on the untransfected parent glioblastoma cell line. The immunotoxin was completely stable upon incubation at 37°C for 24 h in human serum. The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.

A repressible female-specific lethal genetic system for making transgenic insect strains suitable for a sterile-release program

We have developed a tetracycline-repressible female-specific lethal genetic system in the vinegar fly Drosophila melanogaster. One component of the system is the tetracycline-controlled transactivator gene under the control of the fat body and female-specific transcription enhancer from the yolk protein 1 gene. The other component consists of the proapoptotic gene hid under the control of a tetracycline-responsive element. Males and females of a strain carrying both components are viable on medium supplemented with tetracycline, but only males survive on normal medium. A strain with such properties would be ideal for a sterile-insect release program, which is most effective when only males are released in the field.

The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D

The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR/PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1.302 to HIgR/PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR/PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR/PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.

Mammalian Scratch: A neural-specific Snail family transcriptional repressor

Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. We report the isolation of a gene encoding a mammalian Snail family member that is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, we detect hScrt expression in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix–loop–helix (bHLH) transcription factors. We show that hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation.

Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions.

A neural system for human visual working memory

Working memory is the process of actively maintaining a representation of information for a brief period of time so that it is available for use. In monkeys, visual working memory involves the concerted activity of a distributed neural system, including posterior areas in visual cortex and anterior areas in prefrontal cortex. Within visual cortex, ventral stream areas are selectively involved in object vision, whereas dorsal stream areas are selectively involved in spatial vision. This domain specificity appears to extend forward into prefrontal cortex, with ventrolateral areas involved mainly in working memory for objects and dorsolateral areas involved mainly in working memory for spatial locations. The organization of this distributed neural system for working memory in monkeys appears to be conserved in humans, though some differences between the two species exist. In humans, as compared with monkeys, areas specialized for object vision in the ventral stream have a more inferior location in temporal cortex, whereas areas specialized for spatial vision in the dorsal stream have a more superior location in parietal cortex. Displacement of both sets of visual areas away from the posterior perisylvian cortex may be related to the emergence of language over the course of brain evolution. Whereas areas specialized for object working memory in humans and monkeys are similarly located in ventrolateral prefrontal cortex, those specialized for spatial working memory occupy a more superior and posterior location within dorsal prefrontal cortex in humans than in monkeys. As in posterior cortex, this displacement in frontal cortex also may be related to the emergence of new areas to serve distinctively human cognitive abilities.

Ligand-activated site-specific recombination in mice.

Current mouse gene targeting technology is unable to introduce somatic mutations at a chosen time and/or in a given tissue. We report here that conditional site-specific recombination can be achieved in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand-binding domain of the human estrogen receptor (ER) resulting in a tamoxifen-dependent Cre recombinase, Cre-ERT, which is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ERT under the control of a cytomegalovirus promoter. We show that excision of a chromosomally integrated gene flanked by loxP sites can be induced by administration of tamoxifen to these transgenic mice, whereas no excision could be detected in untreated animals. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.

Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system.

Many biological processes rely upon protein-protein interactions. Hence, detailed analysis of these interactions is critical for their understanding. Due to the complexities involved, genetic approaches are often needed. In yeast and phage, genetic characterizations of protein complexes are possible. However, in multicellular organisms, such characterizations are limited by the lack of powerful selection systems. Herein we describe genetic selections that allow single amino acid changes that disrupt protein-protein interactions to be selected from large libraries of randomly generated mutant alleles. The strategy, based on a yeast reverse two-hybrid system, involves a first-step negative selection for mutations that affect interaction, followed by a second-step positive selection for a subset of these mutations that maintain expression of full-length protein (two-step selection). We have selected such mutations in the transcription factor E2F1 that affect its ability to heterodimerize with DP1. The mutations obtained identified a putative helix in the marked box, a region conserved among E2F family members, as an important determinant for interaction. This two-step selection procedure can be used to characterize any interaction domain that can be tested in the two-hybrid system.