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Differential Regulation of Myosin Regulatory Light Chain Phosphorylation by Protein Kinase C Isozymes in Human
Bryan F Mitchell1,2, Mei Chi3, Elle Surgent3
1Department of Obstetrics and Gynecology, The Women and Children's Health Research Institute, University of Alberta, Edmonton, Alberta, Canada. brymitch@ualberta.ca.
Insights
Protein kinase C (PKC) isozymes regulate uterine contractility. Conventional PKC isozymes inhibit uterine activity, while novel PKC isozymes promote it, offering therapeutic targets for preterm birth.
Area of Science:
- Biochemistry
- Cell Biology
- Reproductive Biology
Background:
- Preterm birth is a leading cause of neonatal mortality and lifelong disability.
- Current treatments for preterm birth have limited effectiveness.
Purpose of the Study:
- To investigate the role of protein kinase C (PKC) isozymes in regulating uterine contractility.
- To explore the mechanisms by which PKC isozymes influence myosin regulatory light chain (RLC) phosphorylation and uterine muscle function.
Main Methods:
- Quantitative in-cell Western (ICW) assay to measure RLC phosphorylation in human uterine myocytes (hUM).
- Stimulation of hUM with oxytocin or diacyl-glycerol mimics.
- Use of isozyme-selective inhibitors to differentiate PKC conventional and novel class functions.
Main Results:
- PKC activation inhibited uterine activity ex vivo.
- Conventional PKC isozymes reduced pro-contractile RLC phosphorylation by inhibiting myosin light chain kinase.
- Novel PKC isozymes increased RLC phosphorylation via CPI-17 activation and direct RLC phosphorylation.
Conclusions:
- Conventional and novel PKC isozymes differentially regulate uterine contractility.
- Targeting specific PKC isozymes may offer novel therapeutic strategies for managing preterm labor and other uterine disorders.
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 serine19 and threonine18 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 serine19 and threonine18 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 threonine38, 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.
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