The aspartate receptor of the Escherichia coli and Salmonella typhimurium chemotaxis pathway generates a transmembrane signal that regulates the activity of the cytoplasmic kinase CheA. Previous studies have identified a region of the cytoplasmic domain that is critical to receptor adaptation and kinase regulation. This region, termed the adaptation subdomain, contains a high density of acidic residues, including specific glutamate residues that serve as receptor adaptation sites. However, the mechanism of signal propagation through this region remains poorly understood. This study uses site-directed mutagenesis to neutralize each acidic residue within the subdomain to probe the hypothesis that electrostatics in this region play a significant role in the mechanism of kinase activation and modulation. Each point mutant was tested for its ability to regulate chemotaxis in vivo and kinase activity in vitro. Four point mutants (D273N, E281Q, D288N, and E477Q) were found to superactivate the kinase relative to the wild-type receptor, and all four of these kinaseactivating substitutions are located along the same intersubunit interface as the adaptation sites. These activating substitutions retained the wild-type ability of the attractant-occupied receptor to inhibit kinase activity. When combined in a quadruple mutant (D273N/E281Q/D288N/E477Q), the four charge-neutralizing substitutions locked the receptor in a kinase-superactivating state that could not be fully inactivated by the attractant. Similar lock-on character was observed for a charge reversal substitution, D273R. Together, these results implicate the electrostatic interactions at the intersubunit interface as a major player in signal transduction and kinase regulation. The negative charge in this region destabilizes the local structure in a way that enhances conformational dynamics, as detected by disulfide trapping, and this effect is reversed by charge neutralization of the adaptation sites. Finally, two substitutions (E308Q and E463Q) preserved normal kinase activation in vitro but blocked cellular chemotaxis in vivo, suggesting that these sites lie within the docking site of an adaptation enzyme, CheR or CheB. Overall, this study highlights the importance of electrostatics in signal transduction and regulation of kinase activity by the cytoplasmic domain of the aspartate receptor.The aspartate receptor of bacterial chemotaxis is one of the best-characterized members of a large family of cell surface receptors that modulate two-component signaling pathways (reviewed in refs 1-6). Through regulation of the associated histidine kinase CheA, the aspartate receptor controls cellular chemotaxis in response to changing concentrations of the chemoattractant aspartate, allowing the cell to move up an attractant concentration gradient. The receptor is an oligomer composed of three homodimers that assemble to form a trimer of dimers, as illustrated in Figure 1A. The chemotaxis signaling pathway is triggered by aspartate † Support provided by NIH Grant GM ...

Annexin A1 is a glucocorticoid-regulated, anti-inflammatory protein, which plays an important role as an endogenous regulator of the inflammatory response. Many of these anti-inflammatory properties are retained in the N-terminal annexin A1 peptide Ac1-25, which is released from the full-length protein by a neutrophil elastase. To elucidate whether the anti-inflammatory activity of the bioactive peptide is solely a result of immediate post-translational effects, which include the shedding of L-selectin or also involve transcriptional changes affecting leukocyte function, we recorded global gene expression changes in human monocytes stimulated with exogenously applied Ac1-25. Applying stringent selection criteria, we show that approximately 100 genes are up-regulated, and approximately 230 are down-regulated by a factor of at least two in the Ac1-25-treated monocytes. It is important that the profiling reveals that Ac1-25 induces an anti-inflammatory phenotype by down-regulating proinflammatory and up-regulating anti-inflammatory mediators. These effects, elicited by exogenously applied Ac1-25, depend, to different extents, on ERK1/2 and p38 signaling pathways. This identifies the annexin A1 N-terminal peptide as a stimulus, eliciting not only short-term, post-translational effects in human monocytes but also transcriptional changes, defining a more anti-inflammatory profile.