Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

G(s) protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical G(s)-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor

Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity

Alkylated epidermal creatine kinase as a biomarker for sulfur mustard exposure: comparison to adducts of albumin and DNA in an in vivo rat study

Sulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys(282) was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (mu LC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys(282) in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study

Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

G(s) protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical G(s)-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor