title: Human Cystathionine-β-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium.
abstract: Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP) but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP) causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity.
authors.affiliation: Department of Pharmacy, University of Naples, Federico II, Via D. Montesano, 49, Naples, Italy; Interdepartmental Centre for Sexual Medicine, University of Naples, Federico II, Via Sergio Pansini 5, Naples, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: d'Emmanuele di Villa Bianca
authors.affiliation: Department of Pharmacy, University of Naples, Federico II, Via D. Montesano, 49, Naples, Italy.
authors.forename: Emma
authors.initials: E
authors.lastname: Mitidieri
authors.affiliation: Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via Sergio Pansini 5, Naples, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Esposito
authors.affiliation: Department of Pharmacy, University of Naples, Federico II, Via D. Montesano, 49, Naples, Italy.
authors.forename: Erminia
authors.initials: E
authors.lastname: Donnarumma
authors.forename: Erminia
authors.initials: E
authors.lastname: Donnarumm
authors.affiliation: Department of Pharmacy, University of Naples, Federico II, Via D. Montesano, 49, Naples, Italy.
authors.forename: Annapina
authors.initials: A
authors.lastname: Russo
authors.affiliation: Interdepartmental Centre for Sexual Medicine, University of Naples, Federico II, Via Sergio Pansini 5, Naples, Italy; Department of Neurosciences, Human Reproduction and Odontostomatology, University of Naples, Federico II, Naples, Italy.
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Fusco
authors.affiliation: Department of Pharmacy, University of Naples, Federico II, Via D. Montesano, 49, Naples, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Ianaro
authors.affiliation: Interdepartmental Centre for Sexual Medicine, University of Naples, Federico II, Via Sergio Pansini 5, Naples, Italy; Department of Neurosciences, Human Reproduction and Odontostomatology, University of Naples, Federico II, Naples, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Mirone
authors.affiliation: Department of Pharmacy, University of Naples, Federico II, Via D. Montesano, 49, Naples, Italy; Interdepartmental Centre for Sexual Medicine, University of Naples, Federico II, Via Sergio Pansini 5, Naples, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Cirino
authors.affiliation: Department of Pharmacy, University of Naples, Federico II, Via D. Montesano, 49, Naples, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Russo
authors.affiliation: Department of Pharmacy, University of Naples, Federico II, Via D. Montesano, 49, Naples, Italy; Interdepartmental Centre for Sexual Medicine, University of Naples, Federico II, Via Sergio Pansini 5, Naples, Italy.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Sorrentino
mesh_terms: D000368:Aged; D045744:Cell Line, Tumor; D003541:Cystathionine beta-Synthase / Q000378:metabolism*; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D006862:Hydrogen Sulfide / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000378:metabolism; D019459:Urothelium / Q000166:cytology / Q000378:metabolism
chemical_list: D012694:Serine; D003541:Cystathionine beta-Synthase; D006862:Hydrogen Sulfide