Differential Regulation of Myosin Regulatory Light Chain Phosphorylation by Protein Kinase C Isozymes in Human Uterine Myocytes

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 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.

Keywords

CPI-17 myosin regulatory light chains parturition preterm labor protein kinase C uterine contractility

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References

March of Dimes, The Partnership for Maternal, Newborn & Child Health, Save the Children, World Health Organization. Born Too Soon: The Global Action Report on Preterm Birth . Geneva: World Health Organization; 2012.

Ikebe

Hartshorne

. Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. J Biol Chem. 1985;260(18):10027–10031.

Taggart

Arthur

Zielnik

Mitchell

. Molecular pathways regulating contractility in rat uterus through late gestation and parturition. Am J Obstet Gynecol. 2012;207(1):76. e15–e24.

Savineau

Mironneau

. An analysis of the action of phorbol 12, 13-dibutyrate on mechanical activity in rat uterine smooth muscle. J Pharmacol Exp Ther. 1990;255(1):133–139.

Phillippe

. Protein kinase C, an inhibitor of oxytocin-stimulated phasic myometrial contractions. Biol Reprod. 1994;50(4):855–859.

Hayashi

Senba

Yazawa

Brautigan

Eto

. Defining the structural determinants and a potential mechanism for inhibition of myosin phosphatase by the protein kinase C-potentiated inhibitor protein of 17 kDa. J Biol Chem. 2001;276(43):39858–39863.

Arthur

Taggart

Zielnik

Wong

Mitchell

. Relationship between gene expression and function of uterotonic systems in the rat during gestation, uterine activation and both term and preterm labour. J Physiol. 2008;586(24):6063–6076.

Aguilar

Zielnik

Tracey

Mitchell

. Quantification of rapid Myosin regulatory light chain phosphorylation using high-throughput in-cell Western assays: comparison to Western immunoblots. PloS one. 2010;5(4):e9965.

Aguilar

Tracey

Tsang

McGinnis

Mitchell

. Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes. PloS one. 2011;6(6):e20903.

10.

Aguilar

Tracey

Zielnik

Mitchell

. Rho-kinase mediates diphosphorylation of myosin regulatory light chain in cultured uterine, but not vascular smooth muscle cells. J Cell Mol Med. 2012;16(12):2978–2989.

11.

Hurd

Fomin

Natarajan

Brown

Bigsby

Singh

. Expression of protein kinase C isozymes in nonpregnant and pregnant human myometrium. Am J Obstet Gynecol. 2000;183(6):1525–1531.

12.

Eude

Paris

Cabrol

Ferre

Breuiller-Fouche

. Selective protein kinase C isoforms are involved in endothelin-1-induced human uterine contraction at the end of pregnancy. Biol Reprod. 2000;63(5):1567–1573.

13.

Ozaki

Yasuda

Kim

. Possible role of the protein kinase C/CPI-17 pathway in the augmented contraction of human myometrium after gestation. Br J Pharmacol. 2003;140(7):1303–1312.

14.

Massenavette

Paul

Corriveau

Pasquier

Rousseau

. Phorbol 12,13-dibutyrate-induced protein kinase C activation triggers sustained contracture in human myometrium in vitro. Am J Obstet Gynecol. 2017;217(3):358. e1–358. e9.

15.

Nishikawa

Sellers

Adelstein

Hidaka

. Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase. J Biol Chem. 1984;259(14):8808–8814.

16.

Bengur

Robinson

Appella

Sellers

. Sequence of the sites phosphorylated by protein kinase C in the smooth muscle myosin light chain. J Biol Chem. 1987;262(16):7613–7617.

17.

Ikebe

Hartshorne

Elzinga

. Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase. Phosphorylation sites and effects of phosphorylation. J Biol Chem. 1987;262(20):9569–9573.

18.

Ikebe

Reardon

Mitsui

Ikebe

. Role of the N-terminal region of the regulatory light chain in the dephosphorylation of myosin by myosin light chain phosphatase. J Biol Chem. 1999;274(42):30122–30126.

19.

Turbedsky

Pollard

Bresnick

. A subset of protein kinase C phosphorylation sites on the myosin II regulatory light chain inhibits phosphorylation by myosin light chain kinase. Biochemistry. 1997;36(8):2063–2067.

20.

Ikebe

Inagaki

Kanamaru

Hidaka

. Phosphorylation of smooth muscle myosin light chain kinase by Ca2+-activated, phospholipid-dependent protein kinase. J Biol Chem. 1985;260(8):4547–4550.

21.

Eichholtz

de Bont

de Widt

Liskamp

Ploegh

. A myristoylated pseudosubstrate peptide, a novel protein kinase C inhibitor. J Biol Chem. 1993;268(3):1982–1986.

22.

Mizuno

Noda

Ueda

. UCN-01, an anti-tumor drug, is a selective inhibitor of the conventional PKC subfamily. FEBS Lett. 1995;359(2-3):259–261.

23.

Gschwendt

Dieterich

Rennecke

Kittstein

Mueller

Johannes

. Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett. 1996;392(2):77–80.

24.

Xue

Zhang

. Prenatal hypoxia causes a sex-dependent increase in heart susceptibility to ischemia and reperfusion injury in adult male offspring: role of protein kinase C epsilon. J Pharmacol Exp Ther. 2009;330(2):624–632.

25.

Toth

Kiss

Gergely

Walsh

Hartshorne

Erdodi

. Phosphorylation of MYPT1 by protein kinase C attenuates interaction with PP1 catalytic subunit and the 20 kDa light chain of myosin. FEBS Lett. 2000;484(2):113–117.

26.

Eto

Regulation of cellular protein phosphatase-1 (PP1) by phosphorylation of the CPI-17 family, C-kinase-activated PP1 inhibitors. J Biol Chem. 2009;284(51):35273–35277.

27.

Perjes

Skoumal

Tenhunen

. Apelin increases cardiac contractility via protein kinase cepsilon- and extracellular signal-regulated kinase-dependent mechanisms. PloS one. 2014;9(4):e93473.

28.

Zhu

Zang

. Rho kinase acts as a downstream molecule to participate in protein kinase cepsilon regulation of vascular reactivity after hemorrhagic shock in rats. Shock. 2014;42(3):239–245.

29.

Mochly-Rosen

Das

Grimes

. Protein kinase C, an elusive therapeutic target? Nat Rev Drug Discov. 2012;11(12):937–957.