Amino Acid Sequence and Phosphorylation Status of Myosin Light Chain MLC20 During Vascular Smooth Muscle Cell Maturation

Myosin light chain kinase (MLCK) is an important enzymatic regulator of vascular contractility that phosphorylates the regulatory protein, myosin light chain 20 (MLC20). We have recently demonstrated that the specific activity of MLCK is strikingly greater in fetal vs. adult sheep carotid arteries. Because cell-specific splicing isoforms of myosin-related genes have been identified in vascular smooth muscle, defining a late stage in the differentiation pathway from the synthetic to contractile phenotype, we tested the hypothesis that developmental alteration in MLCK activity may be at least partially dependent on developmental regulation of MLC20 isoforms. We also tested the hypothesis that developmental differences in baseline phosphorylation of MLC20 may influence MLCK activity. MLC20 was sequenced using a combination of biochemical purification and electron-spray ionization-mass spectrometry (ESI-MS). Phosphorylation of MLC20 was quantified by urea gel electrophoresis and immunoblotting with antibodies against both total MLC and SER19-phospho-MLC. Our results demonstrate a high level of amino acid sequence conservation in both N-terminal and C-terminal regions of sheep MLC20 in comparison to other mammalian species and are consistent with the hypothesis that developmental shifts in baseline phosphorylation influence MLCK activity.