DIFFERENTIAL PHOSPHORYLATION OF MYOSIN LIGHT CHAIN KINASE ISOFORMS IN HUMAN PREGNANT MYOMETRIUM IN RESPONSE TO CONTRACTILE AND RELAXING AGONISTS

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 498-514-Female Reproductive Endocrinology
Basic/Translational
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-503
Claire A Hudson*1 and Andres Lopez Bernal2
1University of Bristol, 2University of Bristol, Bristol, United Kingdom
Increased understanding of uterine contractility is necessary to prevent preterm labour and other complications of delivery in order to decrease neonatal mortality and morbidity. At present the management of preterm labour is hampered by our poor knowledge of the hormonal and biochemical regulation of uterine activation and relaxation. Myosin light chain kinase (MYLK) is a calmodulin-dependent enzyme pivotal for the process of smooth muscle contraction due to its ability to phosphorylate myosin light chain (MYL) in response to elevated intracellular calcium.  In addition to regulation by calcium, it is thought that phosphorylation of MYLK at specific residues, including serine 1760 near the calmodulin binding domain, can cause a decrease in kinase activity (1).  Phosphorylation of MYLK at specific residues in human myometrium has not been fully studied although evidence from  other types of smooth muscle has demonstrated that several agonists such as carbachol, KCl, isoproterenol and phorbol ester are involved (2).  Here we demonstrate phosphorylation of MYLK (Ser 1760) in primary myometrial cells after stimulation with the protein kinase C-activator phorbol myristate acetate (PMA) and by agents that stimulate cAMP-dependent protein kinases, including forskolin, 8-cpt-cAMP and the prostaglandin E2 receptor agonist, butaprost.  No increase in phosphorylation was seen in response to oxytocin (OXT). However, under these conditions, it is a lower molecular weight (MW) form of MYLK that is preferentially phosphorylated with little change to the main band equivalent to the smooth muscle isoform of MYLK (110kDa).  Conversely, the phosphorylation of the 110kD protein at Ser 1760 is increased in fresh tissue during spontaneous and OXT-induced contractions compared to relaxed tissue, in parallel to changes in other phosho-proteins reported by us previously (3).  RT-PCR from both fresh myometrial tissue and cultured cell extracts confirms that two additional isoforms equivalent to MYLK 3A and 4 are expressed at the mRNA level (both resulting in lower MW proteins), although to a lesser extent than the full length mRNA.  Isoform-specific siRNA design and RT-PCR in tandem with analysis of MYLK phosphorylation are now being used to confirm the identity of the lower MW isoform.   Studying differential phosphorylation of individual MYLK isoforms during episodes of contraction and relaxation will increase our understanding of the mechanism of myometrial activation and may help explain why in some women the process of spontaneous labour is triggered preterm.

1.            Conti, M. A., and Adelstein, R. S. (1981) J Biol Chem 256, 3178-3181

2.            Stull, J. T., Hsu, L. C., Tansey, M. G., and Kamm, K. E. (1990) J Biol Chem 265, 16683-16690

3.            Hudson, C. A., Heesom, K. J., and Lopez Bernal, A. (2012) Mol Hum Reprod 18, 265-279

Nothing to Disclose: CAH, AL

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: Supported Action Medical Research SP4612