Results presented here demonstrate that EGF stimulation induces transient assembly and phosphorylation of NMHC-IIA and NMHC-IIB in human breast carcinoma cells. In accordance with data demonstrating that the NMHC-II isoforms have unique cellular functions, the kinetics of assembly and phosphorylation differ for the A and B isoforms. In addition, there are significant differences in the kinetics of RLC monophosphorylation (P-S19) and diphosphorylation (P-T18/P-S19). Our analyses show that RLC phosphorylation precedes or is coincident with the assembly of the two myosin-II isoforms, consistent with the idea that RLC phosphorylation promotes filament assembly.
With respect to NMHC-IIA, the peak of heavy-chain phosphorylation occurs temporally between the two peaks of myosin-IIA assembly. Localization studies indicate that the peaks of assembly correspond to different cellular distributions of myosin-IIA, with peak 1 associated with cortical structures and peak 2 associated with stress fibers. These observations suggest that NMHC-IIA phosphorylation may promote the release of myosin-IIA from cortical filaments, thus providing a mechanism for recycling of myosin-IIA monomers and the turnover of myosin-IIA filaments. The physiological relevance of NMHC-IIA phosphorylation is supported by the high levels of phosphorylation observed 3 min after EGF stimulation (0.59 mol phosphate per mol NMHC-IIA polypeptide chain). For cytosolic myosin-IIA, the RLC is phosphorylated as well, albeit at a lower stoichiometry than observed for the heavy chain (0.18 mol phosphate per mol RLC). This low stoichiometry is not entirely unexpected because our kinetic assays indicate that phosphate turnover on the RLC is rapid after EGF stimulation. At this time, it is unclear if concomitant RLC dephosphorylation and NMHC-IIA phosphorylation contribute to the release of myosin-IIA from the cytoskeleton since we cannot determine the phosphorylation status of the heavy and light chains within the same myosin-IIA molecule.
Phosphopeptide maps of the endogenous myosin-IIA from EGF-stimulated cells demonstrate that the heavy chain is phosphorylated on the CK2 site (S1943), which is located in the C-terminal tailpiece of the heavy chain (Murakami et al., 1998
). CK2 recognizes a minimal consensus sequence of S/T-X-X-D/E; however, additional acidic residues at positions −2 to + 7 are needed for efficient phosphorylation of substrates (Meggio et al., 1994
). The amino acid sequence surrounding the CK2 site on the NMHC-IIA (GDGpSDEEVD) is enriched in the acidic residues that are required for recognition by CK2. In addition, the observation that the NMHC-IIA is an in vitro substrate of CK2 (Murakami et al., 1998
; Dulyaninova et al., 2005
) and our finding that CK2 displays increased association with myosin-IIA in EGF-stimulated cells suggest that CK2 directly mediates phosphorylation of NMHC-IIA in growth factor–stimulated breast cancer cells.
We demonstrated previously that phosphorylation of myosin-IIA on S1943 reduces filament formation and regulates the binding of S100A4 (Dulyaninova et al., 2005
). In vitro assembly assays and in vivo expression studies demonstrated that the alanine, and aspartic/glutamic acid substitutions at this position mimic the assembly properties of nonphosphorylated and phosphorylated wild-type myosin-IIA, respectively. Thus with respect to filament assembly, the monoanionic side chains of aspartic and glutamic acid reproduce the function of the dianionic phosphate group at S1943. Interestingly, aspartic and glutamic acid substitutions do not inhibit S100A4 binding, demonstrating that the addition of a single charged residue does not fully mimic the spatial and charge contributions of a dianionic phosphate group at position 1943.
The studies presented here demonstrate that NMHC-IIA phosphorylation has pronounced effects on the motility of MDA-MB-231 cells. The expression of constitutively phosphorylated or nonphosphorylatable analogs of the NMHC-IIA has opposing effects on cell migration and growth factor–stimulated cell protrusion. Cells expressing NMHC-IIA S1943D or E displayed enhanced directional migration into a wound and cell protrusion, whereas cells expressing NMHC-IIA S1943A exhibited reduced migration and protrusion. Our studies indicate that NMHC-IIA S1943A and S1943D coassemble with wild-type NMHC-IIA. If heavy-chain phosphorylation regulates myosin-IIA filament turnover, then incorporation of NMHC-IIA S1943A monomers into filaments would reduce filament turnover, resulting in the formation stable filaments. Stable myosin-IIA filaments could allow for increased contractile force, augmented retrograde flow and reduced cell protrusion. Conversely, incorporation of NMHC-IIA S1943D or E monomers into filaments would be expected to reduce filament stability, and as a consequence, decreased contractility. This would inhibit retrograde flow and allow for enhanced cell protrusion.
Our findings are consistent with published studies demonstrating a role for myosin-II motor function in retrograde flow and the regulation of protrusion. For example, microinjection of NEM-inactivated myosin S1 fragments reduces F-actin flow and enhances the extension of growth cones in neurons (Lin et al., 1996
). Inhibition of myosin-II activity with protein kinase inhibitors attenuates F-actin flow in the cell center of sea urchin coelomocytes (Henson et al., 1999
) and in the lamella of epithelial cells (Gupton et al., 2002
), and treatment of neurons with blebbistatin significantly reduces F-actin retrograde flow and increases cell protrusion (Medeiros et al., 2006
). More recently, studies of spreading mouse embryonic fibroblasts have indicated that myosin-IIA, and not myosin-IIB, primarily regulates retrograde flow (Cai et al., 2006
). The studies presented here indicate that heavy-chain phosphorylation provides another regulatory mechanism for modulating contractile force during cell motility and protrusion.
In addition, our studies suggest that the phosphorylation status of the NMHC-IIA also impacts the turnover of focal adhesions. We detected more stable focal adhesions in cells expressing NMHC-IIA S1943A after EGF stimulation as evidenced by paxillin pY118 staining. This observation is consistent with previous studies showing that a reduction in cytoskeletal contractility induces the disassembly of focal adhesions (Chrzanowska-Wodnicka and Burridge, 1996
). Although our analyses did not reveal any gross alterations in focal adhesions in cells expressing NMHC-IIA S1943D or E, we detected reduced Y118 phosphorylation of paxillin in these cells after EGF stimulation. These findings suggest that reduced myosin-IIA filament assembly or filament instability also affects focal adhesions either at the level of adhesion assembly or in their rate of turnover. Taken together, these studies support a direct role for NMHC-IIA phosphorylation in modulating myosin-IIA assembly during cell migration.