Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Abstract

Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLCphosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.C lass II myosins, the first of 37 known myosin classes (1, 2) to be discovered and originally thought to be specific for muscle, are now known to occur widely in nonmuscle cells in numerous species. Human nonmuscle cells express three different myosin 2 paralogs: nonmuscle myosins (NM) NM2A, NM2B, and NM2C. Like all class II myosins (3, 4), NM2 monomers are hexapeptides of two identical heavy chains (HC), two essential light chains (ELC), and two regulatory light chains (RLC). The HCs of NM2A, NM2B, and NM2C are encoded by different genes, have 60-80% sequence similarity, and isoforms of each are derived from alternative splicing (4).Each HC has an N-terminal globular motor domain with an ATPase site and an actin-binding site, followed by a lever arm that binds one ELC and one RLC, and a long tail that homodimerizes to form a coiled-coil α-helix. The HC ends with a short C-terminal nonhelical tailpiece (5). Like most class II myosins, NM2 monomers assemble into bipolar filaments by antiparallel association of their elongated coiled-coil helical tails. The filaments of NM2s are appreciably smaller than filaments of muscle myosins, with lengths of about 300 nm, consisting of ∼30 monomers for filaments of NM2A and NM2B and ∼14 monomers for filaments of NM2C (5).As described originally for smooth muscle myosin based on light-scattering data (6-8), filaments of unphosp...

Multiple S100 protein isoforms and C-terminal phosphorylation contribute to the paralog-selective regulation of nonmuscle myosin 2 filaments

“…In the presence of Ca 2ϩ , S100A4 depolymerizes filaments, which were formed either by MLCK-induced RLC phosphorylation of NM2A in the presence of ATP or by unphosphorylated myosin in the absence of ATP. Disassembly appeared to be more effective in the case of filaments formed upon RLC phosphorylation, indicating possible differences in the overall stability of the two types of filaments, similarly to the results of Liu et al (17). Fitting of co-sedimentation data using a quadratic binding equation yielded a K dep of 26 nM for filaments formed by RLC phosphorylation and a K dep of 139 nM for unphosphorylated RLC filaments.…”

“…NM2 paralogs can assemble to form functional filaments in cells. The primary regulatory step of this self-assembly is the phosphorylation of the regulatory light chain by myosin light chain kinase (MLCK) and Rho-associated protein kinase (15)(16)(17)(18). In the absence of phosphorylation and in the presence of ATP at physiological ionic strength, NM2 molecules adopt a structure wherein the two heads make an asymmetric interaction, and the tail region makes hairpin turns in two places to fold into a compact conformation.…”

Polymerization pathway of mammalian nonmuscle myosin 2s

“…Notably, 10 of the 13 filaments in Fig. 3 have folded structures within a bare zone between the two ends of mature filaments (5,6). (More extensive images of filaments with folded structures are shown in Figs.…”

“…Phosphorylation of NM2B. Phosphorylation of the RLC was performed as described previously (6). pNM2B was aliquoted and stored in liquid N 2 .…”

“…The polymerization and depolymerization of NM2 filaments have been studied in vitro with NM2s extracted from cells (4,14) and, more recently, with purified recombinant NM2s (5,6). Based on their similarity to SM2, it has been proposed, and generally accepted, that the disassembly of filaments of RLCunphosphorylated NM2s by the addition of ATP and filament reassembly after RLC-phosphorylation, as measured by light scattering (5,21,22), results from a shift of the monomer conformation from unfolded to folded upon the addition of ATP and back to the unfolded conformation after RLCphosphorylation.…”