Publications by authors named "Thomas Egelhoff"

37 Publications

Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner.

Proc Natl Acad Sci U S A 2019 07 24;116(31):15550-15559. Epub 2019 Jun 24.

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Jacksonville, FL 32224;

The ability of glioblastoma to disperse through the brain contributes to its lethality, and blocking this behavior has been an appealing therapeutic approach. Although a number of proinvasive signaling pathways are active in glioblastoma, many are redundant, so targeting one can be overcome by activating another. However, these pathways converge on nonredundant components of the cytoskeleton, and we have shown that inhibiting one of these-the myosin II family of cytoskeletal motors-blocks glioblastoma invasion even with simultaneous activation of multiple upstream promigratory pathways. Myosin IIA and IIB are the most prevalent isoforms of myosin II in glioblastoma, and we now show that codeleting these myosins markedly impairs tumorigenesis and significantly prolongs survival in a rodent model of this disease. However, while targeting just myosin IIA also impairs tumor invasion, it surprisingly increases tumor proliferation in a manner that depends on environmental mechanics. On soft surfaces myosin IIA deletion enhances ERK1/2 activity, while on stiff surfaces it enhances the activity of NFκB, not only in glioblastoma but in triple-negative breast carcinoma and normal keratinocytes as well. We conclude myosin IIA suppresses tumorigenesis in at least two ways that are modulated by the mechanics of the tumor and its stroma. Our results also suggest that inhibiting tumor invasion can enhance tumor proliferation and that effective therapy requires targeting cellular components that drive both proliferation and invasion simultaneously.
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http://dx.doi.org/10.1073/pnas.1902847116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681735PMC
July 2019

ROCK Inhibition Promotes the Development of Chondrogenic Tissue by Improved Mass Transport.

Tissue Eng Part A 2018 08 23;24(15-16):1218-1227. Epub 2018 Apr 23.

2 Case Center for Multimodal Evaluation of Engineered Cartilage, Case Western Reserve University , Cleveland, Ohio.

Human mesenchymal stem cell (hMSC)-based chondrogenesis is a key process used to develop tissue engineered cartilage constructs from stem cells, but the resulting constructs have inferior biochemical and biomechanical properties compared to native articular cartilage. Transforming growth factor β containing medium is commonly applied to cell layers of hMSCs, which aggregate upon centrifugation to form 3-D constructs. The aggregation process leads to a high cell density condition, which can cause nutrient limitations during long-term culture and, subsequently, inferior quality of tissue engineered constructs. Our objective is to modulate the aggregation process by targeting RhoA/ROCK signaling pathway, the chief modulator of actomyosin contractility, to enhance the end quality of the engineered constructs. Through ROCK inhibition, repression of cytoskeletal tension in chondrogenic hMSCs was achieved along with less dense aggregates with enhanced transport properties. ROCK inhibition also led to significantly increased cartilaginous extracellular matrix accumulation. These findings can be used to create an improved microenvironment for hMSC-derived tissue engineered cartilage culture. We expect that these findings will ultimately lead to improved cartilaginous tissue development from hMSCs.
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http://dx.doi.org/10.1089/ten.TEA.2017.0438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080111PMC
August 2018

Myosin IIA Heavy Chain Phosphorylation Mediates Adhesion Maturation and Protrusion in Three Dimensions.

J Biol Chem 2017 02 4;292(8):3099-3111. Epub 2017 Jan 4.

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195. Electronic address:

Non-muscle myosin II (NMII) is a conserved force-producing cytoskeletal enzyme with important but poorly understood roles in cell migration. To investigate myosin heavy chain (MHC) phosphorylation roles in 3D migration, we expressed GFP-tagged NMIIA wild-type or mutant constructs in cells depleted of endogenous NMIIA protein. We find that individual mutation or double mutation of Ser-1916 or Ser-1943 to alanine potently blocks recruitment of GFP-NM-IIA filaments to leading edge protrusions in 2D, and this in turn blocks maturation of anterior focal adhesions. When placed in 3D collagen gels, cells expressing wild-type GFP MHC-IIA behave like parental cells, displaying robust and active formation and retraction of protrusions. However, cells depleted of NMIIA or cells expressing the mutant GFP MHC-IIA display severe defects in invasion and in stabilizing protrusions in 3D. These studies reveal an NMIIA-specific role in 3D invasion that requires competence for NMIIA phosphorylation at Ser-1916 and Ser-1943. In sum, these results demonstrate a critical and previously unrecognized role for NMIIA phosphorylation in 3D invasion.
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http://dx.doi.org/10.1074/jbc.M116.733402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336147PMC
February 2017

Increased cancer stem cell invasion is mediated by myosin IIB and nuclear translocation.

Oncotarget 2016 Jul;7(30):47586-47592

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.

Despite many advances in the treatment of breast cancer, it remains one of the leading causes of death among women. One hurdle for effective therapy is the treatment of the highly invasive and tumorigenic subpopulation of tumors called cancer stem cells (CSCs). CSCs, when stimulated with EGF, migrate through a physiological 3D collagen matrix at a higher velocity than non-stem cancer cells (non-SCCs). This increased invasion is due, in part, by an enhanced nuclear translocation ability of CSCs. We observed no difference between CSC and non-SCC in cellular migration rates on a 2D surface. Furthermore, during transwell migration using large diameter transwell pores, both CSC and non-SCC populations migrated with similar efficiency. However, when challenged with more restrictive transwells, CSCs were dramatically more capable of transwell migration. These results implicate nuclear translocation as a major rate limiting factor for CSC dissemination. We further show that non-muscle myosin IIB is critical for this enhanced nuclear translocation and the ability for cancer stem cells to efficiently migrate through restrictive 3D environments. These studies suggest that cytoskeletal elements upregulated in CSCs, such as myosin IIB, may be valuable targets for intervention in cancer stem cell dispersal from tumors.
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http://dx.doi.org/10.18632/oncotarget.9896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5216963PMC
July 2016

Transforming Growth Factor-β Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2-Dependent Bladder Cancer Cell Migration and Invasion.

Am J Pathol 2016 05 14;186(5):1351-60. Epub 2016 Mar 14.

Department of Pathology, University of California San Diego, La Jolla, California. Electronic address:

Our prior work identified the mammalian target of rapamycin complex 2 (mTORC2) as a key regulator of bladder cancer cell migration and invasion, although upstream growth factor mediators of this pathway in bladder cancer have not been well delineated. We tested whether transforming growth factor (TGF)-β, which can function as a promotility factor in bladder cancer cells, could regulate mTORC2-dependent bladder cancer cell motility and invasion. In human bladder cancers, the highest levels of phosphorylated SMAD2, a TGF-β signaling intermediate, were present in high-grade invasive bladder cancers and associated with more frequent recurrence and decreased disease-specific survival. Increased expression of TGF-β isoforms, receptors, and signaling components was detected in invasive high-grade bladder cancer cells that expressed Vimentin and lacked E-cadherin. Application of TGF-β induced phosphorylation of the Ser473 residue of AKT, a selective target of mTORC2, in a SMAD2- and SMAD4-independent manner and increased bladder cancer cell migration in a modified scratch wound assay and invasion through Matrigel. Inhibition of TGF-β receptor I using SB431542 ablated TGF-β-induced migration and invasion. A similar effect was seen when Rictor, a key mTORC2 component, was selectively silenced. Our results suggest that TGF-β can induce bladder cancer cell invasion via mTORC2 signaling, which may be applicable in most bladder cancers.
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http://dx.doi.org/10.1016/j.ajpath.2016.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861762PMC
May 2016

Matrix-driven Myosin II Mediates the Pro-fibrotic Fibroblast Phenotype.

J Biol Chem 2016 Mar 13;291(12):6083-95. Epub 2016 Jan 13.

From the Department of Pathobiology and Respiratory Institute,

Pro-fibrotic mesenchymal cells are known to be the key effector cells of fibroproliferative disease, but the specific matrix signals and the induced cellular responses that drive the fibrogenic phenotype remain to be elucidated. The key mediators of the fibroblast fibrogenic phenotype were characterized using a novel assay system that measures fibroblast behavior in response to actual normal and fibrotic lung tissue. Using this system, we demonstrate that normal lung promotes fibroblast motility and polarization, while fibrotic lung immobilizes the fibroblast and promotes myofibroblast differentiation. These context-specific phenotypes are surprisingly both mediated by myosin II. The role of myosin II is supported by the observation of an increase in myosin phosphorylation and a change in intracellular distribution in fibroblasts on fibrotic lung, as compared with normal lung. Moreover, loss of myosin II activity has opposing effects on protrusive activity in fibroblasts on normal and fibrotic lung. Loss of myosin II also selectively inhibits myofibroblast differentiation in fibroblasts on fibrotic lung. Importantly, these findings are recapitulated by varying the matrix stiffness of polyacrylamide gels in the range of normal and fibrotic lung tissue. Comparison of the effects of myosin inhibition on lung tissue with that of polyacrylamide gels suggests that matrix fiber organization drives the fibroblast phenotype under conditions of normal/soft lung, while matrix stiffness drives the phenotype under conditions of fibrotic/stiff lung. This work defines novel roles for myosin II as a key regulatory effector molecule of the pro-fibrotic phenotype, in response to biophysical properties of the matrix.
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http://dx.doi.org/10.1074/jbc.M115.712380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813589PMC
March 2016

Non-muscle myosin IIB is critical for nuclear translocation during 3D invasion.

J Cell Biol 2015 Aug 10;210(4):583-94. Epub 2015 Aug 10.

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 441195 Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195

Non-muscle myosin II (NMII) is reported to play multiple roles during cell migration and invasion. However, the exact biophysical roles of different NMII isoforms during these processes remain poorly understood. We analyzed the contributions of NMIIA and NMIIB in three-dimensional (3D) migration and in generating the forces required for efficient invasion by mammary gland carcinoma cells. Using traction force microscopy and microfluidic invasion devices, we demonstrated that NMIIA is critical for generating force during active protrusion, and NMIIB plays a major role in applying force on the nucleus to facilitate nuclear translocation through tight spaces. We further demonstrate that the nuclear membrane protein nesprin-2 is a possible linker coupling NMIIB-based force generation to nuclear translocation. Together, these data reveal a central biophysical role for NMIIB in nuclear translocation during 3D invasive migration, a result with relevance not only to cancer metastasis but for 3D migration in other settings such as embryonic cell migration and wound healing.
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http://dx.doi.org/10.1083/jcb.201502039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539979PMC
August 2015

Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer.

Stem Cells 2015 Jul 15;33(7):2114-2125. Epub 2015 May 15.

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.

Advanced cancers display cellular heterogeneity driven by self-renewing, tumorigenic cancer stem cells (CSCs). The use of cell lines to model CSCs is challenging due to the difficulty of identifying and isolating cell populations that possess differences in self-renewal and tumor initiation. To overcome these barriers in triple-negative breast cancer (TNBC), we developed a CSC system using a green fluorescent protein (GFP) reporter for the promoter of the well-established pluripotency gene NANOG. NANOG-GFP+ cells gave rise to both GFP+ and GFP(-) cells, and GFP+ cells possessed increased levels of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 and elevated self-renewal and tumor initiation capacities. GFP+ cells also expressed mesenchymal markers and demonstrated increased invasion. Compared with the well-established CSC markers CD24(-) /CD44(+) , CD49f, and aldehyde dehydrogenase (ALDH) activity, our NANOG-GFP reporter system demonstrated increased enrichment for CSCs. To explore the utility of this system as a screening platform, we performed a flow cytometry screen that confirmed increased CSC marker expression in the GFP+ population and identified new cell surface markers elevated in TNBC CSCs, including junctional adhesion molecule-A (JAM-A). JAM-A was highly expressed in GFP+ cells and patient-derived xenograft ALDH+ CSCs compared with the GFP(-) and ALDH(-) cells, respectively. Depletion of JAM-A compromised self-renewal, whereas JAM-A overexpression induced self-renewal in GFP(-) cells. Our data indicate that we have defined and developed a robust system to monitor differences between CSCs and non-CSCs in TNBC that can be used to identify CSC-specific targets for the development of future therapeutic strategies.
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http://dx.doi.org/10.1002/stem.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494654PMC
July 2015

Rac1-dependent phosphorylation and focal adhesion recruitment of myosin IIA regulates migration and mechanosensing.

Curr Biol 2015 Jan 24;25(2):175-186. Epub 2014 Dec 24.

Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Background: Cell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Nonmuscle myosin II (MII) is a critical mediator of contractility and FA dynamics in cell migration. Signaling downstream of the small GTPase Rac1 also regulates FA and actin dynamics, but its role in regulation of MII during migration is less clear.

Results: We found that Rac1 promotes association of MIIA with FA. Live-cell imaging showed that, whereas most MIIA at the leading edge assembled into dorsal contractile arcs, a substantial subset assembled in or was captured within maturing FA, and this behavior was promoted by active Rac1. Protein kinase C (PKC) activation was necessary and sufficient for integrin- and Rac1-dependent phosphorylation of MIIA heavy chain (HC) on serine1916 (S1916) and recruitment to FA. S1916 phosphorylation of MIIA HC and localization in FA was enhanced during cell spreading and ECM stiffness mechanosensing, suggesting upregulation of this pathway during physiological Rac1 activation. Phosphomimic and nonphosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA minifilaments in FA. S1916 phosphorylation was also sufficient to promote the rapid assembly of FAs to enhance cell migration and for the modulation of traction force, spreading, and migration by ECM stiffness.

Conclusions: Our study reveals for the first time that Rac1 and integrin activation regulates MIIA HC phosphorylation through a PKC-dependent mechanism that promotes MIIA association with FAs and acts as a critical modulator of cell migration and mechanosensing.
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http://dx.doi.org/10.1016/j.cub.2014.11.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302036PMC
January 2015

Mammalian target of rapamycin complex 2 (mTORC2) is a critical determinant of bladder cancer invasion.

PLoS One 2013 27;8(11):e81081. Epub 2013 Nov 27.

Department of Pathology, University of California San Diego, La Jolla, California, United States of America.

Bladder cancer is the fourth most common cause of cancer in males in the United States. Invasive behavior is a major determinant of prognosis. In this study, we identified mammalian target of rapamycin complex 2 (mTORC2) as a central regulator of bladder cancer cell migration and invasion. mTORC2 activity was assessed by the extent of phosphorylation of Ser473 in AKT and determined to be approximately 5-fold higher in specimens of invasive human bladder cancer as opposed to non-invasive human bladder cancer. The immortalized malignant bladder cell lines, UMUC-3, J82 and T24 demonstrated higher baseline mTORC2 activity relative to the benign bladder papilloma-derived cell line RT4 and the normal urothelial cell line HU1. The malignant bladder cancer cells also demonstrated increased migration in transwell and denudation assays, increased invasion of matrigel, and increased capacity to invade human bladder specimens. Gene silencing of rictor, a critical component of mTORC2, substantially inhibited bladder cancer cell migration and invasion. This was accompanied by a significant decrease in Rac1 activation and paxillin phosphorylation. These studies identify mTORC2 as a major target for neutralizing bladder cancer invasion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081081PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842329PMC
December 2014

Keratin 5-Cre-driven excision of nonmuscle myosin IIA in early embryo trophectoderm leads to placenta defects and embryonic lethality.

Dev Biol 2013 Oct 30;382(1):136-48. Epub 2013 Jul 30.

Department of Cellular and Molecular Medicine NC10, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

In studies initially focused on roles of nonmuscle myosin IIA (NMIIA) in the developing mouse epidermis, we have discovered that a previously described cytokeratin 5 (K5)-Cre gene construct is expressed in early embryo development. Mice carrying floxed alleles of the nonmuscle myosin II heavy chain gene (NMHC IIA(flox/flox)) were crossed with the K5-Cre line. The progeny of newborn pups did not show a Mendelian genotype distribution, suggesting embryonic lethality. Analysis of post-implantation conceptuses from embryonic day (E)9.5 to E13.5 revealed poorly developed embryos and defective placentas, with significantly reduced labyrinth surface area and blood vessel vascularization. These results suggested the novel possibility that the bovine K5 promoter-driven Cre-recombinase was active early in trophoblast-lineage cells that give rise to the placenta. To test this possibility, K5-Cre transgenic mice were crossed with the mT/mG reporter mouse in which activation of GFP expression indicates Cre transgene expression. We observed activation of K5-Cre-driven GFP expression in the ectoplacental cone, in the extraembryonic ectoderm, and in trophoblast giant cells in the E6.5 embryo. In addition, we observed GFP expression at E11.5 to E13.5 in both the labyrinth of the placenta and the yolk sac. NMIIA expression was detected in these same cell types in normal embryos, as well as in E13.5 yolk sac and labyrinth. These findings taken together suggest that NMHC IIA may play critical roles in the early trophoblast-derived ectoplacental cone and extraembryonic ectoderm, as well as in the yolk sac and labyrinth tissues that form later. Our findings are consistent with phenotypes of constitutive NMIIA knockout mice made earlier, that displayed labyrinth and yolk sac-specific defects, but our findings extend those observations by suggesting possible NMIIA roles in trophoblast lineages as well. These results furthermore demonstrate that K5-Cre gene constructs, previously reported to be activated starting at approximately E12.5 in the forming epidermis, may be widely useful as drivers for activation of cre/lox based gene excision in early embryo extraembronic trophoblast tissues as well.
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http://dx.doi.org/10.1016/j.ydbio.2013.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4186751PMC
October 2013

Release of nonmuscle myosin II from the cytosolic domain of tumor necrosis factor receptor 2 is required for target gene expression.

Sci Signal 2013 Jul 16;6(284):ra60. Epub 2013 Jul 16.

Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

Tumor necrosis factor-α (TNF-α) elicits its biological activities through activation of TNF receptor 1 (TNFR1, also known as p55) and TNFR2 (also known as p75). The activities of both receptors are required for the TNF-α-induced proinflammatory response. The adaptor protein TNFR-associated factor 2 (TRAF2) is critical for either p55- or p75-mediated activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, as well as for target gene expression. We identified nonmuscle myosin II (myosin) as a binding partner of p75. TNF-α-dependent signaling by p75 and induction of target gene expression persisted substantially longer in cells deficient in myosin regulatory light chain (MRLC; a component of myosin) than in cells replete in myosin. In resting endothelial cells, myosin was bound constitutively to the intracellular region of p75, a region that overlaps with the TRAF2-binding domain, and TNF-α caused the rapid dissociation of myosin from p75. At early time points after exposure to TNF-α, p75 activated Rho-associated kinase 1 (ROCK1). Inhibition of ROCK1 activity blocked TNF-α-dependent phosphorylation of MRLC and the dissociation of myosin from p75. ROCK1-dependent release of myosin was necessary for the TNF-α-dependent recruitment of TRAF2 to p75 and for p75-specific activation of NF-κB and MAPK signaling. Thus, our findings have revealed a previously uncharacterized, noncanonical regulatory function of myosin in cytokine signaling.
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http://dx.doi.org/10.1126/scisignal.2003743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837481PMC
July 2013

Myosin heavy chain kinases play essential roles in Ca2+, but not cAMP, chemotaxis and the natural aggregation of Dictyostelium discoideum.

J Cell Sci 2012 Oct 16;125(Pt 20):4934-44. Epub 2012 Aug 16.

Developmental Studies Hybridoma Bank, Department of Biology, University of Iowa, Iowa City, 52242, USA.

Behavioral analyses of the deletion mutants of the four known myosin II heavy chain (Mhc) kinases of Dictyostelium discoideum revealed that all play a minor role in the efficiency of basic cell motility, but none play a role in chemotaxis in a spatial gradient of cAMP generated in vitro. However, the two kinases MhckA and MhckC were essential for chemotaxis in a spatial gradient of Ca(2+), shear-induced directed movement, and reorientation in the front of waves of cAMP during natural aggregation. The phenotypes of the mutants mhckA(-) and mhckC(-) were highly similar to that of the Ca(2+) channel/receptor mutant iplA(-) and the myosin II phosphorylation mutant 3XALA, which produces constitutively unphosphorylated myosin II. These results demonstrate that IplA, MhckA and MhckC play a selective role in chemotaxis in a spatial gradient of Ca(2+), but not cAMP, and suggest that Ca(2+) chemotaxis plays a role in the orientation of cells in the front of cAMP waves during natural aggregation.
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http://dx.doi.org/10.1242/jcs.112474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517097PMC
October 2012

Oxidized LDL/CD36 interaction induces loss of cell polarity and inhibits macrophage locomotion.

Mol Biol Cell 2012 Aug 20;23(16):3057-68. Epub 2012 Jun 20.

Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.

Cell polarization is essential for migration and the exploratory function of leukocytes. However, the mechanism by which cells maintain polarity or how cells revert to the immobilized state by gaining cellular symmetry is not clear. Previously we showed that interaction between oxidized low-density lipoprotein (oxLDL) and CD36 inhibits macrophage migration; in the current study we tested the hypothesis that oxLDL/CD36-induced inhibition of migration is the result of intracellular signals that regulate cell polarity. Live cell imaging of macrophages showed that oxLDL actuated retraction of macrophage front end lamellipodia and induced loss of cell polarity. Cd36 null and macrophages null for Vav, a guanine nucleotide exchange factor (GEF), did not show this effect. These findings were caused by Rac-mediated inhibition of nonmuscle myosin II, a cell polarity determinant. OxLDL induced dephosphorylation of myosin regulatory light chain (MRLC) by increasing the activity of Rac. Six-thioguanine triphosphate (6-thio-GTP), which inhibits Vav-mediated activation of Rac, abrogated the effect of oxLDL. Activation of the Vav-Rac-myosin II pathway by oxidant stress may induce trapping of macrophages at sites of chronic inflammation such as atherosclerotic plaque.
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http://dx.doi.org/10.1091/mbc.E11-12-1051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418302PMC
August 2012

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

BMC Cell Biol 2011 Dec 2;12:52. Epub 2011 Dec 2.

Department of Cell Biology, Lerner Research Institute NC-10, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

Background: Phosphorylation 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.

Results: The 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.

Conclusions: These 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.
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http://dx.doi.org/10.1186/1471-2121-12-52DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257205PMC
December 2011

Myosin II isoform switching mediates invasiveness after TGF-β-induced epithelial-mesenchymal transition.

Proc Natl Acad Sci U S A 2011 Nov 24;108(44):17991-6. Epub 2011 Oct 24.

Department of Cell Biology, Cleveland Clinic, Cleveland, OH 44195, USA.

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, TGF-β 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.
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http://dx.doi.org/10.1073/pnas.1106499108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207690PMC
November 2011

Dictyostelium huntingtin controls chemotaxis and cytokinesis through the regulation of myosin II phosphorylation.

Mol Biol Cell 2011 Jul 11;22(13):2270-81. Epub 2011 May 11.

Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Abnormalities in the huntingtin protein (Htt) are associated with Huntington's disease. Despite its importance, the function of Htt is largely unknown. We show that Htt is required for normal chemotaxis and cytokinesis in Dictyostelium discoideum. Cells lacking Htt showed slower migration toward the chemoattractant cAMP and contained lower levels of cortical myosin II, which is likely due to defects in dephosphorylation of myosin II mediated by protein phosphatase 2A (PP2A). htt(-) cells also failed to maintain myosin II in the cortex of the cleavage furrow, generating unseparated daughter cells connected through a thin cytoplasmic bridge. Furthermore, similar to Dictyostelium htt(-) cells, siRNA-mediated knockdown of human HTT also decreased the PP2A activity in HeLa cells. Our data indicate that Htt regulates the phosphorylation status of myosin II during chemotaxis and cytokinesis through PP2A.
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http://dx.doi.org/10.1091/mbc.E10-11-0926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128529PMC
July 2011

Role of B regulatory subunits of protein phosphatase type 2A in myosin II assembly control in Dictyostelium discoideum.

Eukaryot Cell 2011 Apr 25;10(4):604-10. Epub 2011 Feb 25.

Department of Cell Biology, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA.

In Dictyostelium discoideum, myosin II resides predominantly in a soluble pool as the result of phosphorylation of the myosin heavy chain (MHC), and dephosphorylation of the MHC is required for myosin II filament assembly, recruitment to the cytoskeleton, and force production. Protein phosphatase type 2A (PP2A) was identified in earlier studies in Dictyostelium as a key biochemical activity that can drive MHC dephosphorylation. We report here gene targeting and cell biological studies addressing the roles of candidate PP2A B regulatory subunits (phr2aBα and phr2aBβ) in myosin II assembly control in vivo. Dictyostelium phr2aBα- and phr2aBβ-null cells show delayed development, reduction in the assembly of myosin II in cytoskeletal ghost assays, and defects in cytokinesis when grown in suspension compared to parental cell lines. These results demonstrate that the PP2A B subunits phr2aBα and phr2aBβ contribute to myosin II assembly control in vivo, with phr2aBα having the predominant role facilitating MHC dephosphorylation to facilitate filament assembly.
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http://dx.doi.org/10.1128/EC.00296-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3127641PMC
April 2011

A proteomic study of myosin II motor proteins during tumor cell migration.

J Mol Biol 2011 Apr 18;407(5):673-86. Epub 2011 Feb 18.

Department of Cell Biology, Lerner College of Medicine, Case Western Reserve University, The Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

Myosin II motor proteins play important roles in cell migration. Although myosin II filament assembly plays a key role in the stabilization of focal contacts at the leading edge of migrating cells, the mechanisms and signaling pathways regulating the localized assembly of lamellipodial myosin II filaments are poorly understood. We performed a proteomic analysis of myosin heavy chain (MHC) phosphorylation sites in MDA-MB 231 breast cancer cells to identify MHC phosphorylation sites that are 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 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 reduction nor small inhibitory RNA reduction in CK-II activity reduced this stimulated S1943 phosphorylation. Our data demonstrate that S1943 phosphorylation is upregulated during lamellar protrusion, and that CK-II does not appear to be the kinase responsible for this matrix-induced phosphorylation event.
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http://dx.doi.org/10.1016/j.jmb.2011.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3072708PMC
April 2011

INSIGHTS INTO THE ROLES OF NON-MUSCLE MYOSIN IIA IN HUMAN KERATINOCYTE MIGRATION.

Cell Mol Bioeng 2009 Nov;2(4):486-494

Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106.

Epidermal cell migration is a key factor in wound healing responses, regulated by the F-actin-myosin II systems. Previous reports have established the importance of non-muscle myosin II (NMII) in regulating cell migration. However, the role of NMII in primary human keratinocytes has not been investigated. In this study we used a microfabrication-based two-dimensional migration assay to examine the role of NMII in keratinocyte migration. We developed confluent cell islands of various sizes (0.025 - 0.25 mm(2)) and quantified migration as Fold Increase in island area over time. We report here that NMII was expressed and activated in migrating keratinocytes. Inhibition of NMIIA motor activity with blebbistatin increased migration significantly in all cell island sizes in six hours compared to control. Inhibition of Rho-kinase by Y-27632 did not alter migration while inhibition of myosin light chain kinase by ML-7 suppressed migration significantly in six hours. Both blebbistatin and Y-27632 induced formation of large membrane ruffles and elongated tails. In contrast, ML-7 blocked cell spreading, resulting in a rounded morphology. Taken together, these data suggest that NMIIA decreases migration in keratinocytes, but the mechanism may be differentially regulated by upstream kinases.
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http://dx.doi.org/10.1007/s12195-009-0094-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2883784PMC
November 2009

A microfluidic imaging chamber for the direct observation of chemotactic transmigration.

Biomed Microdevices 2010 Jun;12(3):543-53

Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106, USA.

To study the roles of nonmuscle myosin II (NM-II) during invasive cell migration, microfluidic migration chambers have been designed and fabricated using photo- and soft-lithography microfabrication techniques. The chamber consists of two channels separated by a vertical barrier with multiple bays of pores with widths varying from 6 microm to 16 microm, and lengths varying from 25 microm to 50 microm. The cells are plated in the channel on one side of the barrier while a chemoattractant is flowed through the channel on the other side of the barrier. In these chambers, cells can be observed with transmitted light or fluorescence optics while they chemotax through various sized pores that impose differential mechanical resistance to transmigration. As an initial test of this device, we compared breast-cancer cell chemotactic transmigration through different pore sizes with and without inhibition of NM-II. Two distinct rates were observed as cells attempted to pull their nucleus through the smaller pores, and the faster nuclear transit mode was critically dependent on NM-II motor activity. The ability to monitor cells as they chemotax through pores of different dimensions within a single experimental system provides novel information on how pore size affects cell morphology and migration rate, providing a dramatic improvement of imaging potential relative to other in vitro transmigration systems such as Boyden chambers.
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http://dx.doi.org/10.1007/s10544-010-9411-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2886973PMC
June 2010

Novel regulation and dynamics of myosin II activation during epidermal wound responses.

Exp Cell Res 2010 Apr 2;316(6):980-91. Epub 2010 Feb 2.

Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

Wound healing in the skin is an important and complex process that involves 3-dimensional tissue reorganization, including matrix and chemokine-triggered cell migration, paracrine signaling, and matrix remodeling. The molecular signals and underlying mechanisms that stimulate myosin II activity during skin wound healing have not been elucidated. To begin understanding the signaling pathways involved in the activation of myosin II in this process, we have evaluated myosin II activation in migrating primary human keratinocytes in response to scratch wounding in vitro. We report here that myosin II activation and recruitment to the cytoskeleton in wounded keratinocytes are biphasic. Post-wounding, a rapid phosphorylation of myosin II regulatory light chain (RLC) occurs with resultant translocation of myosin IIA to the cell cortex, far in advance of the later polarization and cell migration. During this acute-phase of myosin II activation, pharmacological approaches reveal p38-MAP kinase and cytosolic calcium as having critical roles in the phosphorylation driving cytoskeletal assembly. Although p38-MAPK has known roles in keratinocyte migration, and known roles in leading-edge focal complex dynamics, to our knowledge this is the first report of p38-MAPK acting as an upstream activator of myosin II phosphorylation and assembly during any type of wound response.
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http://dx.doi.org/10.1016/j.yexcr.2010.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835573PMC
April 2010

Roles of an unconventional protein kinase and myosin II in amoeba osmotic shock responses.

Traffic 2009 Dec 22;10(12):1773-84. Epub 2009 Sep 22.

Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

The contractile vacuole (CV) is a dynamic organelle that enables Dictyostelium amoeba and other protist to maintain osmotic homeostasis by expelling excess water. In the present study, we have uncovered a mechanism that coordinates the mechanics of the CV with myosin II, regulated by VwkA, an unconventional protein kinase that is conserved in an array of protozoa. Green fluorescent protein (GFP)-VwkA fusion proteins localize persistently to the CV during both filling and expulsion phases of water. In vwkA null cells, the established CV marker dajumin still localizes to the CV, but these structures are large, spherical and severely impaired for discharge. Furthermore, myosin II cortical localization and assembly are abnormal in vwkA null cells. Parallel analysis of wild-type cells treated with myosin II inhibitors or of myosin II null cells also results in enlarged CVs with impaired dynamics. We suggest that the myosin II cortical cytoskeleton, regulated by VwkA, serves a critical conserved role in the periodic contractions of the CV, as part of the osmotic protective mechanism of protozoa.
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http://dx.doi.org/10.1111/j.1600-0854.2009.00992.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783991PMC
December 2009

Myosin II recruitment during cytokinesis independent of centralspindlin-mediated phosphorylation.

J Biol Chem 2009 Oct 6;284(40):27377-83. Epub 2009 Aug 6.

Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.

During cell division, the mechanisms by which myosin II is recruited to the contractile ring are not fully understood. Much recent work has focused on a model in which spatially restricted de novo filament assembly occurs at the cell equator via localized myosin II regulatory light chain (RLC) phosphorylation, stimulated by the RhoA-activating centralspindlin complex. Here, we show that a recombinant myosin IIA protein that assembles constitutively and is incapable of binding RLC still displays strong localization to the furrow in mammalian cells. Furthermore, this RLC-deficient myosin II efficiently drives cytokinesis, demonstrating that centralspindlin-based RLC phosphorylation is not necessary for myosin II localization during furrowing. Myosin II truncation analysis further reveals two distinct myosin II tail properties that contribute to furrow localization: a central tail domain mediating cortical furrow binding to heterologous binding partners and a carboxyl-terminal region mediating co-assembly with existing furrow myosin IIA or IIB filaments.
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http://dx.doi.org/10.1074/jbc.M109.028316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785666PMC
October 2009

Multiple regulatory steps control mammalian nonmuscle myosin II assembly in live cells.

Mol Biol Cell 2009 Jan 29;20(1):338-47. Epub 2008 Oct 29.

Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.

To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (DeltaIQ2) destabilizes the 10S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Deltatailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Deltatailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6S/10S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.
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http://dx.doi.org/10.1091/mbc.e08-04-0372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613126PMC
January 2009

Cardiac myocyte cytokinesis: The contractile ring is the thing.

J Mol Cell Cardiol 2006 Oct 24;41(4):592-4. Epub 2006 Aug 24.

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http://dx.doi.org/10.1016/j.yjmcc.2006.07.013DOI Listing
October 2006

Distinct roles of nonmuscle myosin II isoforms in the regulation of MDA-MB-231 breast cancer cell spreading and migration.

Cancer Res 2006 May;66(9):4725-33

Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA.

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 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.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-4236DOI Listing
May 2006

Multiple myosin II heavy chain kinases: roles in filament assembly control and proper cytokinesis in Dictyostelium.

Mol Biol Cell 2005 Sep 29;16(9):4256-66. Epub 2005 Jun 29.

Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan.

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.
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http://dx.doi.org/10.1091/mbc.e05-03-0219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196335PMC
September 2005

Identification and characterization of a novel alpha-kinase with a von Willebrand factor A-like motif localized to the contractile vacuole and Golgi complex in Dictyostelium discoideum.

Mol Biol Cell 2005 May 23;16(5):2248-62. Epub 2005 Feb 23.

Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106-4970, USA.

We have identified a new protein kinase in Dictyostelium discoideum that carries the same conserved class of "alpha-kinase" catalytic domain as reported previously in myosin heavy chain kinases (MHCKs) in this amoeba but that has a completely novel domain organization. The protein contains an N-terminal von Willebrand factor A (vWFA)-like motif and is therefore named VwkA. Manipulation of VwkA expression level via high copy number plasmids (VwkA++ cells) or gene disruption (vwkA null cells) results in an array of cellular defects, including impaired growth and multinucleation in suspension culture, impaired development, and alterations in myosin II abundance and assembly. Despite sequence similarity to MHCKs, the purified protein failed to phosphorylate myosin II in vitro. Autophosphorylation activity, however, was enhanced by calcium/calmodulin, and the enzyme can be precipitated from cellular lysates with calmodulin-agarose, suggesting that VwkA may directly bind calmodulin. VwkA is cytosolic in distribution but enriched on the membranes of the contractile vacuole and Golgi-like structures in the cell. We propose that VwkA likely acts indirectly to influence myosin II abundance and assembly behavior and possibly has broader roles than previously characterized alpha kinases in this organism, which all seem to be MHCKs.
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http://dx.doi.org/10.1091/mbc.e04-07-0639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087232PMC
May 2005

Actin activation of myosin heavy chain kinase A in Dictyostelium: a biochemical mechanism for the spatial regulation of myosin II filament disassembly.

J Biol Chem 2005 Jan 14;280(4):2879-87. Epub 2004 Nov 14.

Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.

Studies in Dictyostelium discoideum have established that the cycle of myosin II bipolar filament assembly and disassembly controls the temporal and spatial localization of myosin II during critical cellular processes, such as cytokinesis and cell locomotion. Myosin heavy chain kinase A (MHCK A) is a key enzyme regulating myosin II filament disassembly through myosin heavy chain phosphorylation in Dictyostelium. Under various cellular conditions, MHCK A is recruited to actin-rich cortical sites and is preferentially enriched at sites of pseudopod formation, and thus MHCK A is proposed to play a role in regulating localized disassembly of myosin II filaments in the cell. MHCK A possesses an aminoterminal coiled-coil domain that participates in the oligomerization, cellular localization, and actin binding activities of the kinase. In the current study, we show that the interaction between the coiled-coil domain of MHCK A and filamentous actin leads to an approximately 40-fold increase in the initial rate of kinase catalytic activity. Actin-mediated activation of MHCK A involves increased rates of kinase autophosphorylation and requires the presence of the coiled-coil domain. Structure-function analyses revealed that the coiled-coil domain alone binds to actin filaments (apparent K(D) = 0.9 microm) and thus mediates the direct interaction with F-actin required for MHCK A activation. Collectively, these results indicate that MHCK A recruitment to actin-rich sites could lead to localized activation of the kinase via direct interaction with actin filaments, and thus this mode of kinase regulation may represent an important mechanism by which the cell achieves localized disassembly of myosin II filaments required for specific changes in cell shape.
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http://dx.doi.org/10.1074/jbc.M410803200DOI Listing
January 2005
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