Molecular cloning and characterization of a cellular phosphoprotein that interacts with a conserved C-terminal domain of adenovirus E1A involved in negative modulation of oncogenic transformation.

The adenovirus type 2/5 E1A proteins transform primary baby rat kidney (BRK) cells in cooperation with the activated Ras (T24 ras) oncoprotein. The N-terminal half of E1A (exon 1) is essential for this transformation activity. While the C-terminal half of E1A (exon 2) is dispensable, a region located between residues 225 and 238 of the 243R E1A protein negatively modulates in vitro T24 ras cooperative transformation as well as the tumorigenic potential of E1A/T24 ras-transformed cells. The same C-terminal domain is also required for binding of a cellular 48-kDa phosphoprotein, C-terminal binding protein (CtBP). We have cloned the cDNA for CtBP via yeast two-hybrid interaction cloning. The cDNA encodes a 439-amino acid (48 kDa) protein that specifically interacts with exon 2 in yeast two-hybrid, in vitro protein binding, and in vivo coimmunoprecipitation analyses. This protein requires residues 225-238 of the 243R E1A protein for interaction. The predicted protein sequence of the isolated cDNA is identical to amino acid sequences obtained from peptides prepared from biochemically purified CtBP. Fine mapping of the CtBP-binding domain revealed that a 6-amino acid motif highly conserved among the E1A proteins of various human and animal adenoviruses is required for this interaction. These results suggest that interaction of CtBP with the E1A proteins may play a critical role in adenovirus replication and oncogenic transformation.

Molecular cloning and characterization of a calmodulin-dependent phosphodiesterase enriched in olfactory sensory neurons.

The sensing of an odorant by an animal must be a rapid but transient process, requiring an instant response and also a speedy termination of the signal. Previous biochemical and electrophysiological studies suggest that one or more phosphodiesterases (PDEs) may play an essential role in the rapid termination of the odorant-induced cAMP signal. Here we report the molecular cloning, expression, and characterization of a cDNA from rat olfactory epithelium that encodes a member of the calmodulin-dependent PDE family designated as PDE1C. This enzyme shows high affinity for cAMP and cGMP, having a Km for cAMP much lower than that of any other neuronal Ca2+/calmodulin-dependent PDE. The mRNA encoding this enzyme is highly enriched in olfactory epithelium and is not detected in six other tissues tested. However, RNase protection analyses indicate that other alternative splice variants related to this enzyme are expressed in several other tissues. Within the olfactory epithelium, this enzyme appears to be expressed exclusively in the sensory neurons. The high affinity for cAMP of this Ca2+/calmodulin-dependent PDE and the fact that its mRNA is highly concentrated in olfactory sensory neurons suggest an important role for it in a Ca(2+)-regulated olfactory signal termination.

Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).

VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP- and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. Here, the identification of an 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrated that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data revealed that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting of VASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with our recent identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, our present results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation.

The loss of female sex pheromone after mating in the corn earworm moth Helicoverpa zea: identification of a male pheromonostatic peptide.

Female moths often become depleted of sex pheromone after mating as the various components of virgin behavior are switched off. In examining a potential male contribution to these events in the corn earworm moth Helicoverpa zea, we have characterized a basic polypeptide from the tissues producing (accessory glands) and storing (duplex) the seminal fluids. The peptide evokes the depletion of sex pheromone when injected into virgin females. This pheromonostatic peptide (PSP) is 57 amino acids long and contains a single disulfide bridge. It is blocked at the N terminus with pyroglutamate and at the C terminus by amidation. As little as 23 ng of peptide evokes the near-complete depletion of pheromone in decapitated (neck-ligated) females that had been injected with pheromone biosynthesis-activating neuropeptide. Activity is approximately 15-fold less in intact virgins, showing that the head limits the expression of activity in these injected females. Females mated to surgically impaired males, capable of producing a spermatophore but not transferring spermatozoa or seminal fluids, are depleted of pheromone by injected peptide. Females whose abdominal nerve cords have been severed are not depleted of pheromone after mating. Thus, neural signals either descending or ascending via the nerve cord are required for the depletion of pheromone after mating. PSP, from the seminal fluids, may participate in this process by direct or indirect action on the glandular tissue; if so, it represents an unusual mechanism in insects for the regulation by seminal fluids of postmating reproductive behavior.