Turnover rates at regulatory phosphorylation sites on myosin II in endothelial cells.
Assembly and motor activity of non-muscle myosin II can be regulated by phosphorylation. Because myosin II-containing structures undergo continuous assembly, disassembly, and remodeling in living cells, especially during cell migration, myosin II should undergo frequent phosphorylation and dephosphorylation. This study examines the turnover of phosphate on myosin II in stationary and migrating endothelial cells. Cultured bovine aortic endothelial cells were metabolically labeled with (32)P-phosphate, and the incorporation of phosphate into myosin II was assessed by quantitative phosphor imaging of electrophoretic gels of myosin II immunoadsorbed from cell lysates. Likewise, phosphate turnover was measured upon chasing the (32)P with unlabeled phosphate. Phosphate incorporated very slowly into heavy chains, taking >8 h to plateau, and turned over at </=12.7% per hour. Regulatory light chains became completely labeled in </=4 h, and turnover occurred at two rates: 49% turned over at 20% per hour, the remainder at 67% per hour. Peptide mapping showed light chain phosphorylation at serine 19 and threonine 18, but phosphate turnover was the same in mono- and diphosphorylated lights chains, indicating that rates are not different at the two sites. When cells were stimulated to migrate by wounding a confluent monolayer, the rate of light chain dephosphorylation increased and the rate of phosphate incorporation decreased causing a net 10% dephosphorylation of light chains. This process persisted during migration and returned to baseline when the wound was closed. There was no effect on heavy chain phosphates. Light chain dephosphorylation may facilitate migration by mobilizing myosin II during cytoskeletal remodeling.
Kolega
J
J
Department of Anatomy and Cell Biology, State University of New York at Buffalo School of Medicine and Biomedical Sciences, Buffalo, New York 14214, USA. kolega@buffalo.edu
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D009218:Myosins / Q000378:metabolism* / Q000502:physiology; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013912:Threonine / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology
D018994:Myosin Light Chains; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D013912:Threonine; D012694:Serine; D009218:Myosins