title: Phosphorylation of proteins during human myometrial contractions: A phosphoproteomic approach.
abstract: Phasic myometrial contractility is a key component of human parturition and the contractions are driven by reversible phosphorylation of myosin light chains catalyzed by the calcium (Ca2+)-dependent enzyme myosin light chain kinase (MYLK). Other yet unknown phosphorylation or de-phosphorylation events may contribute to myometrial contraction and relaxation. In this study we have performed a global phosphoproteomic analysis of human myometrial tissue using tandem mass tagging to detect changes in the phosphorylation status of individual myometrial proteins during spontaneous and oxytocin-driven phasic contractions. We were able to detect 22 individual phosphopeptides whose relative ratio changed (fold > 2 or < 0.5) in response to spontaneous or oxytocin-stimulated contraction. The most significant changes in phosphorylation were to MYLK on serine 1760, a site associated with reductions in calmodulin binding and subsequent kinase activity. Phosphorylated MYLK (ser1760) increased significantly during spontaneous (9.83 ± 3.27 fold, P < 0.05) and oxytocin -induced (18.56 ± 8.18 fold, P < 0.01) contractions and we were able to validate these data using immunoblotting. Pathway analysis suggested additional proteins involved in calcium signalling, cGMP-PRKG signalling, adrenergic signalling and oxytocin signalling were also phosphorylated during contractions. This study demonstrates that a global phosphoproteomic analysis of myometrial tissue is a sensitive approach to detect changes in the phosphorylation of proteins during myometrial contractions, and provides a platform for further validation of these changes and for identification of their functional significance.
authors.affiliation: University of Bristol, School of Clinical Sciences, Dorothy Hodgkin Building, Whitson Street, Bristol, BS1 3NY, UK. Electronic address: Claire.hudson@bristol.ac.uk.
authors.forename: Claire A
authors.initials: CA
authors.lastname: Hudson
authors.affiliation: University of Bristol, School of Clinical Sciences, Dorothy Hodgkin Building, Whitson Street, Bristol, BS1 3NY, UK; Department of Obstetrics and Gynaecology, St Michael's Hospital, Southwell Street, Bristol, BS2 8EG, UK. Electronic address: A.LopezBernal@bristol.ac.uk.
authors.forename: Andrés
authors.initials: A
authors.lastname: López Bernal
mesh_terms: D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002147:Calmodulin / Q000737:chemistry; D005260:Female; D006801:Humans; D009215:Myometrium / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010121:Oxytocin / Q000502:physiology*; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D020543:Proteome; D040901:Proteomics; D015398:Signal Transduction; D014590:Uterine Contraction
chemical_list: D002147:Calmodulin; D010748:Phosphopeptides; D020543:Proteome; D010121:Oxytocin; D009219:Myosin-Light-Chain Kinase; D002118:Calcium
keywords: Contractility; Myometrium; Myosin light chain kinase; Parturition; Phosphoproteomics; Uterus