Induction of cyclin D1 by simian virus 40 small tumor antigen

Cell-cycle progression is mediated by a coordinated interaction between cyclin-dependent kinases and their target proteins including the pRB and E2F/DP-1 complexes. Immunoneutralization and antisense experiments have established that the abundance of cyclin D1, a regulatory subunit of the cyclin-dependent kinases, may be rate-limiting for G1 phase progression of the cell cycle. Simian virus 40 (SV40) small tumor (t) antigen is capable of promoting G1 phase progression and augments substantially the efficiency of SV40 transformation through several distinct domains. In these studies, small t antigen stimulated cyclin D1 promoter activity 7-fold, primarily through an AP-1 binding site at −954 with additional contributions from a CRE site at −57. The cyclin D1 AP-1 and CRE sites were sufficient for activation by small t antigen when linked to an heterologous promoter. Point mutations of small t antigen between residues 97–103 that reduced PP2A binding were partially defective in the induction of the cyclin D1 promoter. These mutations also reduced activation of MEK1 and two distinct members of the mitogen-activated protein kinase family, the ERKs (extracellular signal regulated kinases) and the SAPKs (stress-activated protein kinases), in transfected cells. Dominant negative mutants of either MEK1, ERK or SEK1, reduced small t-dependent induction of the cyclin D1 promoter. SV40 small t induction of the cyclin D1 promoter involves both the ERK and SAPK pathways that together may contribute to the proliferative and transformation enhancing activity of small t antigen.

Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and “APS reductase” activity

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector λYES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5′-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35S-labeled substrate to acid-volatile 35S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel “APS reductase” enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of ≈1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5

HIV-1 entry into CD4+ cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

The small GTPase RalA targets filamin to induce filopodia

The Ras-related small GTPases Rac, Rho, Cdc42, and RalA bind filamin, an actin filament-crosslinking protein that also links membrane and other intracellular proteins to actin. Of these GTPases only RalA binds filamin in a GTP-specific manner, and GTP-RalA elicits actin-rich filopods on surfaces of Swiss 3T3 cells and recruits filamin into the filopodial cytoskeleton. Either a dominant negative RalA construct or the RalA-binding domain of filamin 1 specifically block Cdc42-induced filopod formation, but a Cdc42 inhibitor does not impair RalA’s effects, which, unlike Cdc42, are Rac independent. RalA does not generate filopodia in filamin-deficient human melanoma cells, whereas transfection of filamin 1 restores the functional response. RalA therefore is a downstream intermediate in Cdc42-mediated filopod production and uses filamin in this pathway.

Small molecule developmental screens reveal the logic and timing of vertebrate development

Much has been learned about vertebrate development by random mutagenesis followed by phenotypic screening and by targeted gene disruption followed by phenotypic analysis in model organisms. Because the timing of many developmental events is critical, it would be useful to have temporal control over modulation of gene function, a luxury frequently not possible with genetic mutants. Here, we demonstrate that small molecules capable of conditional gene product modulation can be identified through developmental screens in zebrafish. We have identified several small molecules that specifically modulate various aspects of vertebrate ontogeny, including development of the central nervous system, the cardiovascular system, the neural crest, and the ear. Several of the small molecules identified allowed us to dissect the logic of melanocyte and otolith development and to identify critical periods for these events. Small molecules identified in this way offer potential to dissect further these and other developmental processes and to identify novel genes involved in vertebrate development.