Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells (original) (raw)
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A Proteomic Study of Myosin II Motor Proteins during Tumor Cell Migration
Journal of Molecular Biology, 2011
Myosin II motor proteins play important roles in cell migration. Although myosin II filament assembly plays a key role in stabilization of focal contacts at the leading edge of migrating cells, the mechanisms and signaling pathways regulating localized assembly of lamellipodial myosin II filaments are poorly understood. We performed proteomic analysis of myosin IIA heavy chain (MHC) phosphorylation sites in MDA-MB 231 breast cancer cells to identify MHC phosphorylation sites activated during integrin engagement and lamellar extension on fibronectin. Fibronectin-activated MHC phosphorylation was identified on novel and previously recognized consensus sites for phosphorylation by Protein Kinase C (PKC) and Casein Kinase II (CK II). S1943, a CK-II consensus site, was highly phosphorylated in response to matrix engagement, and phosphoantibody staining revealed phosphorylation on myosin II assembled into leading edge lamellae. Surprisingly, neither pharmacological nor siRNA reduction in CKII activity reduced this stimulated S1943 phosphorylation. Our data demonstrate that S1943 phosphorylation is upregulated during lamellar protrusion, and that CKII does not appear to be the kinase responsible for this matrix-induced phosphorylation event.
Cancer Research, 2006
Initial stages of tumor cell metastasis involve an epithelial-mesenchyme 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 lamella...
Journal of Biological Chemistry, 2003
Epidermal growth factor (EGF) stimulation of prostate metastatic tumor cells results in transient phosphorylation and cellular localization of non-muscle myosin heavy chain II-B (NMHC II-B) with kinetics similar to those seen in chemotaxis. We demonstrate that expression of 18-and 72-kDa fragments derived from the NMHC II-B C terminus that contain EGF-dependent NMHC II-B phosphorylation sites serve as dominantnegative mutations for EGF-dependent NMHC II-B phosphorylation and localization. Both fragments inhibited the EGF-dependent phosphorylation by competing with NMHC II-B on the myosin heavy chain kinase. However, only expression of the 72-kDa fragment resulted in cells with abnormalities in cell shape, focal adhesions, and chemotaxis. We found that the 72-kDa (but not 18-kDa) fragment is capable of self-assembly. To our knowledge, these results provide the first strong evidence that EGF-dependent NMHC II-B phosphorylation is required for the cellular localization of NMHC II-B and that NMHC II-B is required for normal cell attachment and for chemotactic response.
Nonmuscle myosin IIb is involved in the guidance of fibroblast migration
2004
Although myosin II is known to play an important role in cell migration, little is known about its specific functions. We have addressed the function of one of the isoforms of myosin II, myosin IIB, by analyzing the movement and mechanical characteristics of fibroblasts where this protein has been ablated by gene disruption. Myosin IIB null cells displayed multiple unstable and disorganized protrusions, although they were still able to generate a large fraction of traction forces when cultured on flexible polyacrylamide substrates. However, the traction forces were highly disorganized relative to the direction of cell migration. Analysis of cell migration patterns indicated an increase in speed and decrease in persistence, which were likely responsible for the defects in directional movements as demonstrated with Boyden chambers. In addition, unlike control cells, mutant cells failed to respond to mechanical signals such as compressing forces and changes in substrate rigidity. Immunofluorescence staining indicated that myosin IIB was localized preferentially along stress fibers in the interior region of the cell. Our results suggest that myosin IIB is involved not in propelling but in directing the cell movement, by coordinating protrusive activities and stabilizing the cell polarity.
Myosin II heavy chain isoforms are phosphorylated in an EGF-dependent manner
Journal of Cell Science, 2001
To explore the involvement and regulation of the nonmuscle myosin II heavy chains isoforms, MHC-A and MHC-B in the chemotaxis of metastatic tumor cells,we analyzed the changes in phosphorylation and cellular localization of these isoforms upon stimulation of prostate tumor cells with epidermal growth factor(EGF). EGF stimulation of prostate tumor cells resulted in transient increases in MHC-A and MHC-B phosphorylation and subcellular localization with quite different kinetics. Furthermore, the kinetics of subcellular localization correlated with the in vivo kinetics of MHC-B phosphorylation but not of MHC-A phosphorylation, suggesting different modes of regulation for these myosin II isoforms. We further showed that protein kinase C (PKC) is involved in the EGF-dependent phosphorylation of MHC-A and MHC-B. To our knowledge, this is the first report demonstrating that MHC phosphorylation might regulate its subcellular localization and that the EGF signal is transmitted to MHC-A and M...
Molecular Biology of the Cell, 2019
Though many cancers are known to show up-regulation of nonmuscle myosin (NM) IIA and IIB, the mechanism by which NMIIs aid in cancer development remains unexplored. Here we demonstrate that tumor-generating, fibroblast-like cells isolated from 3-methylcholanthrene (3MC)-induced murine tumor exhibit distinct phospho-dependent localization of NMIIA and NMIIB at the perinuclear area and tip of the filopodia and affect cell migration differentially. While NMIIA-KD affects protrusion dynamics and increases cell directionality, NMIIB-KD lowers migration speed and increases filopodial branching. Strategically located NMIIs at the perinuclear area colocalize with the linker of nucleoskeleton and cytoskeleton (LINC) protein Nesprin2 and maintain the integrity of the nuclear-actin cap. Interestingly, knockdown of NMIIs results in altered expression of genes involved in epithelial-to-mesenchymal transition, angiogenesis, and cellular senescence. NMIIB-KD cells display down-regulation of Gsc an...
Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells
PLoS ONE, 2013
Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rhodependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors.
Proceedings of the National Academy of Sciences, 2011
Despite functional significance of nonmuscle myosin II in cell migration and invasion, its role in epithelial-mesenchymal transition (EMT) or TGF-β signaling is unknown. Analysis of normal mammary gland expression revealed that myosin IIC is expressed in luminal cells, whereas myosin IIB expression is up-regulated in myoepithelial cells that have more mesenchymal characteristics. Furthermore, TGFβ induction of EMT in nontransformed murine mammary gland epithelial cells results in an isoform switch from myosin IIC to myosin IIB and increased phosphorylation of myosin heavy chain (MHC) IIA on target sites known to regulate filament dynamics (S1916, S1943). These expression and phosphorylation changes are downstream of heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of TGF-β signaling. E1 knockdown drives cells into a migratory, invasive mesenchymal state and concomitantly up-regulates MHC IIB expression and MHC IIA phosphorylation. Abrogation of myosin IIB expression in the E1 knockdown cells has no effect on 2D migration but significantly reduced transmigration and macrophage-stimulated collagen invasion. These studies indicate that transition between myosin IIC/myosin IIB expression is a critical feature of EMT that contributes to increases in invasive behavior.
Class I Myosins, molecular motors involved in cell migration and cancer
Cell Adhesion & Migration, 2022
Class I Myosins are a subfamily of motor proteins with ATPase activity and a characteristic structure conserved in all myosins: A N-Terminal Motor Domain, a central Neck and a C terminal Tail domain. Humans have eight genes for these myosins. Class I Myosins have different functions: regulate membrane tension, participate in endocytosis, exocytosis, intracellular trafficking and cell migration. Cell migration is influenced by many cellular components including motor proteins, like myosins. Recently has been reported that changes in myosin expression have an impact on the migration of cancer cells, the formation of infiltrates and metastasis. We propose that class I myosins might be potential markers for future diagnostic, prognostic or even as therapeutic targets in leukemia and other cancers.
Molecular biology of the cell, 2006
Nonmuscle myosin II is an important component of the cytoskeleton, playing a major role in cell motility and chemotaxis. We have previously demonstrated that, on stimulation with epidermal growth factor (EGF), nonmuscle myosin heavy chain II-B (NMHC-IIB) undergoes a transient phosphorylation correlating with its cellular localization. We also showed that members of the PKC family are involved in this phosphorylation. Here we demonstrate that of the two conventional PKC isoforms expressed by prostate cancer cells, PKCbetaII and PKCgamma, PKCgamma directly phosphorylates NMHC-IIB. Overexpression of wild-type and kinase dead dominant negative PKCgamma result in both altered NMHC-IIB phosphorylation and subcellular localization. We have also mapped the phosphorylation sites of PKCgamma on NMHC-IIB. Conversion of the PKCgamma phosphorylation sites to alanine residues, reduces the EGF-dependent NMHC-IIB phosphorylation. Aspartate substitution of these sites reduces NMHC-IIB localization i...