Anaerobic induction of light harvesting and reaction center gene expression involves two transacting factors termed RegA and RegB. Sequence and mutational analysis has indicated that RegA and RegB constitute cognate components of a prokaryotic sensory transduction cascade with RegB comprising a membrane-spanning sensor kinase and RegA a cytosolic response regulator. In this study we have purified RegA, as well as a truncated portion of RegB (RegB') and undertaken an in vitro analysis of autophosphorylation and phosphotransfer activities. Incubation of RegB' with [gamma-32P]ATP and MgCl2 resulted in phosphorylation of RegB' (RegB' approximately P) over a 20-min incubation period. Incubation of RegB' approximately P with RegA resulted in rapid transfer of the phosphate from RegB' to RegA. In analogy to other characterized prokaryotic sensory transduction components, mutational and chemical stability studies also indicate that RegB' is autophosphorylated at a conserved histidine and that RegA accepts the phosphate from RegB at a conserved aspartate.

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-yetunidentified 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.

BackgroundMany receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how each hormone binding event triggers the appropriate response.FindingsHere, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway.ConclusionsThe findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0080-8) contains supplementary material, which is available to authorized users.

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