analysis of the role of ser1ser2thr9 phosphorylation on myosin ii assembly and function in live cells分析ser1ser2thr9磷酸化作用的肌球蛋白ii组装和功能在活细胞中.pdfVIP

Beach et al. BMC Cell Biology 2011, 12:52 /1471-2121/12/52 RESEARCH ARTICLE Open Access Analysis of the role of Ser1/Ser2/Thr9 phosphorylation on myosin II assembly and function in live cells 1,2 1 1 1* Jordan R Beach , Lucila S Licate , James F Crish and Thomas T Egelhoff Abstract Background: Phosphorylation of non-muscle myosin II regulatory light chain (RLC) at Thr18/Ser19 is well established as a key regulatory event that controls myosin II assembly and activation, both in vitro and in living cells. RLC can also be phosphorylated at Ser1/Ser2/Thr9 by protein kinase C (PKC). Biophysical studies show that phosphorylation at these sites leads to an increase in the Km of myosin light chain kinase (MLCK) for RLC, thereby indirectly inhibiting myosin II activity. Despite unequivocal evidence that PKC phosphorylation at Ser1/Ser2/Thr9 can regulate myosin II function in vitro, there is little evidence that this mechanism regulates myosin II function in live cells. Results: The purpose of these studies was to investigate the role of Ser1/Ser2/Thr9 phosphorylation in live cells. To do this we utilized phospho-specific antibodies and created GFP-tagged RLC reporters with phosphomimetic aspartic acid substitutions or unphosphorylatable alanine substitutions at the putative inhibitory sites or the previously characterized activation sites. Cell lines stably expressing the RLC-GFP constructs were assayed for myosin recruitment during cell division, the ability to complete cell division, and myosin assembly levels under resting or spreading conditions. Our data shows that manipulation of the activation sites (Thr18/Ser19) significantly alters myosin II function in a number of these assays while manipulation of the putative i