Does testosterone determine dominance in the house sparrow Passer domesticus? An experimental test

Badges of status function in many birds within a social context to establish dominance hierarchies and reduce antagonistic encounters. In order to maintain the honesty of the signalling system, such badges must be costly to produce or to maintain. The chest bib of the house sparrow functions as a badge of status and changes in size are known to be controlled by testosterone levels. We sought to test the relative importance of testosterone as opposed to bib size in determining dominance within a group of male house sparrows. We did this by manipulating testosterone levels independently during both breeding and post‐breeding seasons in experimental birds and examining the effect of testosterone titre, as well as corticosterone titre relative to bib size on dominance levels. Dominance hierarchies within the groups were tested during both the breeding and post‐breeding phases. We compared the results of these tests with dominance among intact (unmanipulated) birds. Results suggested that the breeding season dominance levels were largely determined by testosterone levels as well as bib size, whereas the post‐breeding dominance levels were determined by post‐breeding testosterone titre and previous breeding season dominance level. Within unmanipulated birds, basal corticosterone levels were significantly, negatively correlated with dominance level, but only during the breeding season. The influence of breeding season dominance on post‐breeding dominance suggests social history is important in determining dominance interactions as well as current testosterone levels and bib size.     

Continuous phosphorylation of GAP-43 and MARCKS by long-term TPA treatment in SK-N-SH human neuroblastoma cells

Long-term treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) down-regulates select protein kinase C (PKC) isozymes and may differentially affect PKC substrates. We investigated the role of PKC down-regulation on phosphorylation of two PKC substrates, the 43 kDa growth-associated protein (GAP-43) and the myristoylated alanine-rich C-kinase substrate (MARCKS) in SK-N-SH human neuroblastoma cells. Cells were treated with 70 nM TPA for 15 min, 17 or 72 h. Phosphorylation of MARCKS and GAP-43 was elevated throughout 72 h of TPA. The magnitude and peptidic sites of phosphorylation in GAP-43 and MARCKS were similar after all TPA treatments. GAP-43, but not MARCKS, content was increased after 17 and 72 h of TPA. The ratio of GAP-43 phosphorylation to content was elevated throughout 17 h but returned to control by 72 h as content increased. PKC epsilon and alpha isozyme content was greatly reduced after 72 h of TPA but membranes retained 23% of PKC activity. Only PKC epsilon translocated to membranes after 15 min TPA. GAP-43 content after 72 h of TPA was increased in subcellular fractions in which significant PKC epsilon isozyme concentration remained. These results demonstrate that continuous TPA differentially affected phosphorylation of PKC substrate proteins and regulation of PKC isozyme content in SK-N-SH cells.     

Relative dependence of different outputs of the Saccharomyces cerevisiae pheromone response pathway on the MAP kinase Fus3p

Fus3p and Kss1p act at the end of a conserved signaling cascade that mediates numerous cellular responses for mating. To determine the role of Fus3p in different outputs, we isolated and characterized a series of partial-function fus3 point mutants for their ability to phosphorylate a substrate (Ste7p), activate Ste12p, undergo G1 arrest, form shmoos, select partners, mate, and recover. All the mutations lie in residues that are conserved among MAP kinases and are predicted to affect either enzyme activity or binding to Ste7p or substrates. The data argue that Fus3p regulates the various outputs assayed through the phosphorylation of multiple substrates. Different levels of Fus3p function are required for individual outputs, with the most function required for shmoo formation, the terminal output. The ability of Fus3p to promote shmoo formation strongly correlates with its ability to promote G1 arrest, suggesting that the two events are coupled. Fus3p promotes recovery through a mechanism that is distinct from its ability to promote G1 arrest and may involve a mechanism that does not require kinase activity. Moreover, catalytically inactive Fus3p inhibits the ability of active Fus3p to activate Ste12p and hastens recovery without blocking G1 arrest or shmoo formation. These results raise the possibility that in the absence of sustained activation of Fus3p, catalytically inactive Fus3p blocks further differentiation by restoring mitotic growth. Finally, suppression analysis argues that Kss1p contributes to the overall pheromone response in a wild-type strain, but that Fus3p is the critical kinase for all of the outputs tested.     

Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.     

Developmental stress, social rank and song complexity in the European starling (Sturnus vulgaris)

Bird song is a sexually selected trait and females have been shown to prefer males that sing more complex songs. However, for repertoire size to be an honest signal of male quality it must be associated with some form of cost. This experiment investigates the effects of food restriction and social status during development on song complexity in the European starling (Sturnus vulgaris). Birds that experienced an unpredictable food supply early in life produced a significantly smaller repertoire of song phrases than those with a constant food supply. Social status during development was also significantly correlated with repertoire size, with dominant birds producing more phrase types. This study therefore provides novel evidence that social as well as nutritional history may be important in shaping the song signal in this species.     

Flt-1-dependent survival characterizes the epithelial-mesenchymal transition of colonic organoids

Aberrant cell survival and resistance to apoptosis are hallmarks of tumor invasion and progression to metastatic disease, but the mechanisms involved are poorly understood. The epithelial-mesenchymal transition (EMT), a process that facilitates progression to invasive cancer, provides a superb model for studying such survival mechanisms. Here, we used a unique spheroid culture system that recapitulates the structure of the colonic epithelium and undergoes an EMT in response to cytokine stimulation to study this problem. Our data reveal that the EMT results in the increased expression of both VEGF and Flt-1, a tyrosine kinase VEGF receptor, and that the survival of these cells depends on a VEGF/Flt-1 autocrine pathway. Perturbation of Flt-1 function by either a blocking antibody or adenoviral expression of soluble Flt-1, which acts in a dominant-negative fashion, caused massive apoptosis only in cells that underwent EMT. This pathway was critical for the survival of other invasive colon carcinoma cell lines, and we observed a correlative upregulation of Flt-1 expression linked to in vivo human cancer progression. A role for Flt-1 in cell survival is unprecedented and has significant implications for Flt-1 function in tumor progression, as well as in other biological processes, including angiogenesis and development.