Initial stages of tumor cell metastasis involve an epithelialmesenchyme transition that involves activation of amoeboid migration and loss of cell-cell adhesion. The actomyosin cytoskeleton has fundamental but poorly understood roles in these events. Myosin II, an abundant force-producing protein, has roles in cell body translocation and retraction of the posterior of the cell during migration. Recent studies have suggested that this protein may also have roles in leading edge protrusive events. The metastasis-promoting protein metastasin-1, a regulator of myosin II assembly, colocalizes with myosin IIA at the leading edge of cancer cells, suggesting direct roles for myosin II in metastatic behavior. We have assessed the roles of specific myosin II isoforms during lamellar spreading of MDA-MB-231 breast cancer cells on extracellular matrix. We find that the two major myosin II isoforms IIA and IIB are both expressed in these cells, and both are recruited dramatically to the lamellar margin during active spreading on fibronectin. There is also a transient increase in regulatory light chain phosphorylation that correlates the recruitment of myosin IIA and myosin IIB into this spreading margin. Pharmacologic inhibition of myosin II or myosin light chain kinase dramatically reduced spreading. Depletion of myosin IIA via small interfering RNA impaired migration but enhanced lamellar spreading, whereas depletion of myosin IIB impaired not only migration but also impaired initial rates of lamellar spreading. These results indicate that both isoforms are critical for the mechanics of cell migration, with myosin IIB seeming to have a preferential role in the mechanics of lamellar protrusion. (Cancer Res 2006; 66(9): 4725-33)

Myosin II filament assembly in Dictyostelium discoideum is regulated via phosphorylation of residues located in the carboxyl-terminal portion of the myosin II heavy chain (MHC) tail. A series of novel protein kinases in this system are capable of phosphorylating these residues in vitro, driving filament disassembly. Previous studies have demonstrated that at least three of these kinases (MHCK A, MHCK B, and MHCK C) display differential localization patterns in living cells. We have created a collection of single, double, and triple gene knockout cell lines for this family of kinases. Analysis of these lines reveals that three MHC kinases appear to represent the majority of cellular activity capable of driving myosin II filament disassembly, and reveals that cytokinesis defects increase with the number of kinases disrupted. Using biochemical fractionation of cytoskeletons and in vivo measurements via fluorescence recovery after photobleaching (FRAP), we find that myosin II overassembly increases incrementally in the mutants, with the MHCK A ؊ /B ؊ /C ؊ triple mutant showing severe myosin II overassembly. These studies suggest that the full complement of MHC kinases that significantly contribute to growth phase and cytokinesis myosin II disassembly in this organism has now been identified. INTRODUCTIONMyosin II plays fundamental roles in a variety of cellular contractile processes, ranging from cytokinesis to cell migration and developmental morphogenesis (Ridley et al., 2003;Baumann, 2004;Van Haastert and Devreotes, 2004). A critical feature of nonmuscle myosin II isoforms is that cellular function requires the assembly of this motor protein into bipolar filament structures that can interact with cortical actin arrays. Filament assembly in nonmuscle cells is dynamic and subject to both spatial and temporal regulation. In mammalian nonmuscle cells, phosphorylation of the myosin II regulatory light chain (RLC) is a widely cited model for assembly regulation, with RLC phosphorylation favoring filament assembly (Scholey et al., 1980). However, there is also strong experimental support for other models of mammalian nonmuscle myosin II assembly control, including evidence for monomer sequestration by the S100 protein metastasin 1 (Li et al., 2003), and biochemical evidence that MHC phosphorylation may modulate filament assembly (Murakami et al., 1998;Murakami et al., 2000). Definitive in vivo studies to distinguish relative contributions of each mechanism have as yet not been performed.The simple amoeba Dictyostelium discoideum contains a single myosin II heavy chain gene, which serves cellular roles that are conserved with those of myosin II in other organisms. Biochemical and cellular studies in this system have established that myosin II filament assembly in vivo involves a dynamic equilibrium between a large pool of disassembled molecules and a pool of assembled filaments that associate with the cortical cytoskeleton. This equilibrium appears regulated by a variety of events ranging from chemoattractant receptor st...

Myosin heavy chain kinase (MHCK) A phosphorylates mapped sites at the C-terminal tail of Dictyostelium myosin II heavy chain, driving disassembly of myosin filaments both in vitro and in vivo. MHCK A is organized into three functional domains that include an N-terminal coiled-coil region, a central kinase catalytic domain unrelated to conventional protein kinases, and a WD repeat domain at the C terminus. MHCK B is a homologue of MHCK A that possesses structurally related catalytic and WD repeat domains. In the current study, we explored the role of the WD repeat domains in defining the activities of both MHCK A and MHCK B using recombinant bacterially expressed truncations of these kinases either with or without their WD repeat domains. We demonstrate that substrate targeting is a conserved function of the WD repeat domains of both MHCK A and MHCK B and that this targeting is specific for Dictyostelium myosin II filaments. We also show that the mechanism of targeting involves direct binding of the WD repeat domains to the myosin substrate. To our knowledge, this is the first report of WD repeat domains physically targeting attached kinase domains to their substrates. The examples presented here may serve as a paradigm for enzyme targeting in other systems.

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.