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Primer extension analysis was employed to show that the divergently transcribed regB and senC-regA-hvrA transcripts contain stable 5' ends 43 nucleotides apart within the regB-senC intergenic region in Rhodobacter capsulatus. Findings indicated that autoregulation by RegA is a factor contributing to the maintenance of an optimal low level of RegA expression that permits responsiveness to activation by phosphorylation.

Use of T7 RNA polymerase expression system helps isolate two trans-acting factors, RegA and a truncated portion of RegB (RegB'), associated with anaerobic induction of light harvesting and reaction center gene expression in Rhodobacter capsulatus. In vitro analysis of autophosphorylation and phosphotransfer activities of these segments reveals the ability of RegB' to autophosphorylate and transfer a phosphate to RegA. Autophosphorylation in RegB occurs at a conserved histidine, and the transferred phosphate is accepted by RegA at a conserved aspartate.

The high affinity binding site (Site1) of the human growth hormone (hGH) binds to its cognate receptor (hGHR) via a concave surface patch containing about 35 residues. Using 167 sequences from a shotgun alanine scanning analysis of Site1, we have determined that over half of these residues can be simultaneously changed to an alanine or a non-isosteric amino acid while still retaining a high affinity interaction. Among these hGH variants the distribution of the mutation is highly variable throughout the interface, although helix 4 is more conserved than the other binding elements. Kinetic and thermodynamic analyses were performed on 11 representative hGH Site1 variants that contained 14–20 mutations. Generally, the tightest binding variants showed similar associated rate constants (kon) as the wild-type (wt) hormone, indicating that their binding proceeds through a similar transition state intermediate. However, calorimetric analyses indicate very different thermodynamic partitioning: wt-hGH binding exhibits favorable enthalpy and entropy contributions, whereas the variants display highly favorable enthalpy and highly unfavorable entropy contributions. The heat capacities (ACp) on binding measured for wt-hGH and its variants are significantly larger than normally seen for typical protein-protein interactions, suggesting large conformational or solvation effects. The multiple Site1 mutations are shown to indirectly affect binding of the second receptor at Site2 through an allosteric mechanism. We show that the stability of the ternary hormone. receptor complex reflects the affinity of the Site2 binding and is surprisingly exempt from changes in Site1 affinity, directly demonstrating that dissociation of the active signaling complex is a stepwise process. [ABSTRACT FROM AUTHOR]

Investigates whether regulated expression of a regulatory gene cluster is necessary for controlling photosynthesis gene expression in response to anaerobiosis in Rhodobacter capsulatus. Identification of transcription start sites; Characterization of promoter mutants; Beta-galactosidase activity measurements of wild-type and mutant promoter expression.

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling for multiple receptor tyrosine kinases and several G-protein-coupled receptors. In this study, phosphopeptide affinity enrichment and mass spectrometry identified serine 523 (Ser523) in JAK2 as a site of phosphorylation. A phosphoserine 523 antibody revealed that Ser523 is rapidly but transiently phosphorylated in response to growth hormone (GH). MEK1 inhibitor UO126 suppresses GH-dependent phosphorylation of Ser523, suggesting that extracellular signal-regulated kinases (ERKs) 1 and/or 2 or another kinase downstream of MEK1 phosphorylate Ser523 in response to GH. Other ERK activators, phorbol 12-myristate 13-acetate and epidermal growth factor, also stimulate phosphorylation of Ser523. When Ser523 in JAK2 was mutated, JAK2 kinase activity as well as GH-dependent tyrosyl phosphorylation of JAK2 and Stat5 was enhanced, suggesting that phosphorylation of Ser523 inhibits JAK2 kinase activity. We hypothesize that phosphorylation of Ser523 in JAK2 by ERKs 1 and/or 2 or other as-yet-unidentified kinases acts in a negative feedback manner to dampen activation of JAK2 in response to GH and provides a mechanism by which prior exposure to environmental factors that regulate Ser523 phosphorylation might modulate the cell's response to GH. [ABSTRACT FROM AUTHOR]