Myosin light chain isoforms and their phosphorylation in arterial smooth muscle

Arterial smooth muscle myosin contains nonphosphorylated and phosphorylated light chains that appear as 4 spots on two-dimensional, Coomassie blue-stained gel electrophoretograms at the 20,000-molecular weight level (referred to as spots 4 through 1 in order of decreasing isoelectric points). Anti-light chain recognizes the proteins in all 4 light chain spots. Complete dephosphorylation of light chain in muscle homogenate, by inhibiting myosin light chain kinase and by adding phosphatase, leads to 2 spots on two-dimensional gel electrophoretograms; both spots are visible on immunoblots. Stimulation (K+ or stretch) of smooth muscle results in increased light chain phosphorylation. Autoradiography of the gel electrophoretograms reveals that radioactive components are contained in spots 3, 2, 1, and in an additional spot with lower isoelectric point, referred to as spot 0. Phosphoamino acid analysis shows that spots 3 and 1 contain phosphoserine, whereas spots 2 and 0 contain phosphoserine and phosphothreonine. Two-dimensional phosphopeptide mapping of the trypsin-digested proteins from spots 3 and 1 shows predominantly 2 peptides; whereas from spots 2 and 0, it shows 5 peptides. Sodium dodecyl sulfate gel electrophoresis of the phosphopeptides obtained with Staphylococcus aureus V8 digestion gives identical maps for spots 3 and 2, which are different from the identical maps of spots 1 and 0. The results suggest that arterial smooth muscle myosin contains 2 nonphosphorylated 20,000-dalton light chain isoforms with different amino acid sequences and that each isoform can be mono- and diphosphorylated.