Aminopeptidase O contains a functional nucleolar localization signal and is implicated in vascular biology

We have identified a gene trap integration into Aminopeptidase O, the gene encoding a member of the M1 family of metalloproteases. Using the betagal reporter of the gene trap vector, we have revealed that at least some ApO isoforms are expressed predominantly in embryonic and adult blood vessels leading us to propose that ApO plays a role in vascular cell biology. The protein produced from an engineered Gfp-ApO fusion cDNA localises to the nucleolus in transfected COS7 cells. We confirm that indeed the APO protein contains a functional nucleolar localisation domain by demonstrating that GFP-APO fusion proteins that lack the predicted nucleolar localisation signal are retained in the cytoplasm. We report the existence of multiple alternatively spliced Apo isoforms that differ with respect to the presence of exons encoding important functional domains. Alternative splicing predictably produces protein products with or without the catalytic domain and/or a nucleolar localisation signal and therefore likely represents an important mechanism in regulating the biological activity of APO that has been reported to cleave one of the peptides of the renin angiotensin pathway.     

A FAK/Src chimera with gain-of-function properties promotes formation of large peripheral adhesions associated with dynamic actin assembly

Formation of a complex between the tyrosine kinases FAK and Src is a key integrin-mediated signaling event implicated in cell motility, survival, and proliferation. Past studies indicate that FAK functions in the complex primarily as a "scaffold," acting to recruit and activate Src within cell/matrix adhesions. To study the cellular impact of FAK-associated Src signaling we developed a novel gain-of-function approach that involves expressing a chimeric protein with the FAK kinase domain replaced by the Src kinase domain. This FAK/Src chimera is subject to adhesion-dependent activation and promotes tyrosine phosphorylation of p130Cas and paxillin to higher steady-state levels than is achieved by wild-type FAK. When expressed in FAK -/- mouse embryo fibroblasts, the FAK/Src chimera resulted in a striking cellular phenotype characterized by unusual large peripheral adhesions, enhanced adhesive strength, and greatly reduced motility. Live cell imaging of the chimera-expressing FAK -/- cells provided evidence that the large peripheral adhesions are associated with a dynamic actin assembly process that is sensitive to a Src-selective inhibitor. These findings suggest that FAK-associated Src kinase activity has the capacity to promote adhesion integrity and actin assembly.     

Focal Adhesion Kinase Modulates Cell Adhesion Strengthening via Integrin Activation

Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces.     

Evaluation of mass trapping and mating disruption for managing Prionus californicus (Coleoptera: Cerambycidae) in hop production yards

Larvae of Prionus californicus Motschulsky (Coleoptera: Cerambycidae) feed on the roots of many types of woody perennial crops and are serious pests of hop in the northwestern United States. The adult males are strongly attracted to a volatile sex pheromone, (3R,5S)-3,5-dimethyldodecanoic acid, that is produced by females. Here, we summarize the results of field experiments that evaluated the potential for using the synthetic pheromone (in a blend of all four possible stereoisomers) to manage infestations of P. californicus in commercial hop yards by mass trapping or mating disruption. Our research provides evidence that mass trapping may be effective in reducing mating success of the females: positioning surrogate females (sentinel traps baited with a low dose of pheromone) within a square of eight pheromone-baited traps resulted in an 88% reduction in the number of wild males that reached the sentinel traps compared with sentinel traps that were surrounded by traps baited with blank lures. Similarly, surrogate females that were surrounded by pheromone lures (without traps) were reached by 84% fewer wild males than surrogate females surrounded by blank lures, suggesting that mating disruption also may be effective. A mark-recapture experiment indicated that male P. californicus were attracted to traps baited with 1 mg of pheromone from as far away as 585 m. These studies indicate that 3,5-dimethyldodecanoic acid has very good potential for managing P. californicus in hop yards, and perhaps in other crops where it is a pest.     

Focal adhesion kinase is abundant in developing blood vessels and elevation of its phosphotyrosine content in vascular smooth muscle cells is a rapid response to angiotensin II

Focal adhesion kinase (FAK) is a structurally unique nonreceptor protein-tyrosine kinase that localizes to focal adhesion plaques. Regulation of its activity has been implicated in diverse signaling pathways, including those mediated by extracellular matrix/integrin interactions, G-protein coupled receptors for mitogenic neuropeptides, and certain oncogene products. To gain evidence for specific processes in which FAK may be involved in vivo, a study was initiated to determine its expression pattern during mouse development. FAK expression was detected in early embryos and appeared to be distributed throughout all cell types at about the time of neurulation. Subsequent to neural tube closure, expression became particularly abundant in the developing vasculature. This included expression in the medial layer of arteries populated by smooth muscle cells. In vitro studies using cultured rat aortic vascular smooth muscle cells demonstrate that FAK phosphotyrosine content is dramatically elevated in response to plating cells onto the adhesive glycoprotein, fibronectin. Also, enhanced tyrosine phosphorylation of FAK is observed in these cells upon stimulation with the vasoconstrictor angiotensin II. Thus, in vascular smooth muscle cells, like fibroblasts, FAK appears to play a role in signaling mechanisms induced by extracellular matrix components as well as G-protein coupled receptor agonists. The combined results of this study suggest that signaling through FAK may play an important role in blood vessel morphogenesis and function. © 1994 Wiley-Liss, Inc.     

Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions.

We have isolated and characterized genomic DNA clones for the human and chicken homologues of the mouse En-1 and En-2 genes and determined the genomic structure and predicted protein sequences of both En genes in all three species. Comparison of these vertebrate En sequences with the Xenopus En-2 [Hemmati-Brivanlou et al., 1991) and invertebrate engrailed-like genes showed that the two previously identified highly conserved regions within the En protein ]reviewed in Joyner and Hanks, 1991] can be divided into five distinct subregions, designated EH1 to EH5. Sequences 5' and 3' to the predicted coding regions of the vertebrate En genes were also analyzed in an attempt to identify cis-acting DNA sequences important for the regulation of En gene expression. Considerable sequence similarity was found between the mouse and human homologues both within the putative 5' and 3' untranslated as well as 5' flanking regions. Between the mouse and Xenopus En-2 genes, shorter stretches of sequence similarity were found within the 3' untranslated region. The 5' untranslated regions of the mouse, chicken and Xenopus En-2 genes, however, showed no similarly conserved stretches. In a preliminary analysis of the expression pattern of the human En genes, En-2 protein and RNA were detected in the embryonic and adult cerebellum respectively and not in other tissues tested. These patterns are analogous to those seen in other vertebrates. Taken together these results further strengthen the suggestion that En gene function and regulation has been conserved throughout vertebrate evolution and, along with the five highly conserved regions within the En protein, raise an interesting question about the presence of conserved genetic pathways.     

Body size influences mating success of the Eucalyptus longhorned borer (Coleoptera: Cerambycidae)

Both sexes of adultPhoracantha semipunctata F. (Coleoptera: Cerambycidae) congregate on stressedEucalyptus that are the larval hosts. In a field study, 721 adultP. semipunctata captured on host trees varied considerably in body size with the largest individuals being about twice the length of the smallest. Females that were paired with a mate were similar in size to solitary females, suggesting that the probability of a female being mated was not affected by her size. However, large males had greater success than smaller males in obtaining mates. MaleP. semipunctata rely on antennal contact to locate and identify females on the larval host. Therefore, the rate at which males search for mates is a function of the area swept by their antennae per unit time. Because of their greater antennal spread, large males were able to search for females at double the rate of the smallest males. Large males also dominated in aggressive contests for females. The superior abilities of large maleP. semipunctata in both locating and defending mates account for the influence of body size on mating success.     

Template-based docking of a prolactin receptor proline-rich motif octapeptide to FKBP12: implications for cytokine receptor signaling.

A conserved proline-rich motif (PRM) in the cytoplasmic domain of cytokine receptors has been suggested to be a signaling switch regulated by the action of the FK506 binding protein (FKBP) family of peptidylprolyl isomerases (O'Neal KD, Yu-Lee LY, Shearer WT, 1995, Ann NY Acad Sci 766:282-284). We have docked the prolactin receptor PRM (Ile1-Phe2-Pro3-Pro4-Val5-Pro6-Gly7-Pro8) to the ligand binding site of FKBP12. The procedure involved conformational search restricted by NMR restraints (O'Neal KD et al., 1996, Biochem J 315:833-844), energy minimization of the octapeptide conformers so obtained, template-based docking of a selected conformer to FKBP12, and energy refinement of the resulting complex. The template used was the crystal structure of a cyclic FK506-peptide hybrid bound to FKBP12. Val5-Pro6 of the PRM was taken to be the biologically relevant Xaa-Pro bond. The docked conformer is stabilized by two intramolecular hydrogen bonds, N7H7-->O4 and N2H2-->O8, and two intermolecular ones, Ile56; N-H-->O = C:Pro6 and Tyr82:O-H-->O = C:Gly7. This conformer features a Type I beta-turn and has extensive hydrophobic contacts with the FKBP12 binding surface. The observed interactions support the hypothesis that FKBP12 catalyzes cis-trans isomerization in the PRM when it is part of the longer cytoplasmic domain of a cytokine receptor, and suggest a significant role for the PRM in signal transduction.