Analysis of the role of Ser1/Ser2/Thr9 phosphorylation on myosin II assembly and function in live cells

Abstract

BackgroundPhosphorylation 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.ResultsThe 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 inhibitory sites (Ser1/Ser2/Thr9) does not.ConclusionsThese studies suggest that inhibitory phosphorylation of RLC is not a substantial regulatory mechanism, although we cannot rule out its role in other cellular processes or perhaps other types of cells or tissues in vivo.

Mesenchymal Chemotaxis Requires Selective Inactivation of Myosin II at the Leading Edge via a Noncanonical PLCγ/PKCα Pathway

“…This inhibitory phosphorylation of this site was discovered in the 1980s, but it has received comparatively little attention relative to Thr18/Ser19 phosphorylation. Although phosphorylation of Ser1/2 is dispensable for cytokinesis in HeLa cells (Beach et al, 2011), it is important for disassembly of stress fibers in response to PDGF or PMA (Komatsu and Ikebe, 2007). Recently, phosphorylation of Ser1/2 was identified as a critical regulatory mechanism for T cell polarization (Liu et al, 2013).…”

Various Themes of Myosin Regulation

“…Though the effect is evident in in vitro studies with purified proteins, PKC phosphorylation of the RLC has not been studied extensively in vivo. It is not regarded as major regulatory mechanism for nonmuscle myosin-2 function during cell division of HeLa cells and primary human keratinocytes as well as the assembly of nonmuscle myosin-2a in spreading and resting HeLa cells [33]. However, nonmuscle myosin-2a inhibition after phosphorylation of Ser-1/Ser-2 of the RLC at the leading edge is required for mesenchymal chemotaxis in fibroblasts [34].…”

Myosin II isoform co-assembly and differential regulation in mammalian systems

“…In vitro data shows that this phosphorylation directly decreases the myosin's actin-activated ATPase activity and inhibits the interaction between MCLK and RLC, further lowering the activation state. The effect of this putative inhibitory phosphorylation on NM2 in cells remains unclear [2;8;42]. Collectively, activating and inhibiting phosphorylations of the RLC represent potential but unproven mechanisms for isoform-specific NM2 regulation.…”