Differential Regulation of Myosin Regulatory Light Chain Phosphorylation by Protein Kinase C Isozymes in Human Uterine Myocytes. (July 2019)

• Record Type: Journal Article
• Authors: Mitchell, Bryan F.; Chi, Mei; Surgent, Elle; Sorochan, Bailey M.; Tracey, Curtis N.; Aguilar, Hector N.; Mongin, Maily; Zielnik, Barbara
• Is Part Of: Reproductive sciences. Volume 26:Number 7(2019:Jul.)
• Page Start: 988
• Page End: 996
• Publication Date: 2019-07
• Subjects: CPI-17 -- myosin regulatory light chains -- parturition -- preterm labor -- protein kinase C -- uterine contractility; Reproductive health -- Periodicals; Reproduction -- Periodicals; 612.6
• DOI: 10.1177/1933719118802062
• Languages: English
• ISSNs: 1933-7191

Abstract

Background: Preterm birth is the most common cause of neonatal morbidity and mortality and a common precedent to lifelong disability. Current treatment has minimal efficacy. Objective: We assessed the role of isozymes of the protein kinase C (PKC) family in regulating the phosphorylation of myosin regulatory light chains (RLCs), which regulate uterine contractility. We also explored the mechanisms through which these isozymes function. Study Design: We used a previously characterized and validated quantitative in-cell Western (ICW) assay to measure site-specific phosphorylations on myosin RLC and CPI-17. Cultures of human uterine myocytes (hUM) were treated with the potent contractile stimulant oxytocin to induce uterine contractility or a pharmacological mimic of diacyl-glycerol to stimulate the conventional and novel isozymes of the PKC family. Combinations of isozyme-selective inhibitors were used to determine the effects of the conventional and novel classes of isozymes. Results: Stimulation of PKC using phospho-dibutyrate caused immediate, concentration-dependent inhibition of uterine activity ex vivo . Using the ICW assay with hUM, the oxytocin-stimulated increase in the pro-contractile phosphorylations of myosin RLCs at serine 19 and threonine 18 was completely inhibited by prior treatment with phorbol-12-myristate-13-acetate, which stimulates both convention and novel classes of isozymes. Our results suggest that the conventional class of isozymes cause a reduction in phosphorylations at serine 19 and threonine 18 by reducing activity of myosin light chain kinase. The novel class of isozymes has 2 mechanisms of action: the first is activation of CPI-17 through phosphorylation at threonine 38, which results in reduced activity of myosin light chain phosphatase and increased levels of activated myosin RLC; the second is increased phosphorylation of the N-terminal region of myosin RLC. Conclusions: Specific agonists for the conventional isozymes or inhibitors of the novel isozymes of the PKC family could be useful pharmacological agents for regulation of uterine activity.