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1.
Article in English | MEDLINE | ID: mdl-38700016

ABSTRACT

Myosin phosphatase targeting subunit1 (MYPT1) is a critical subunit of myosin phosphatase (MP), which brings PP1Cδ phosphatase and its substrate together. We previously showed that MYPT1 depletion resulted in oblique chromatid segregation. Therefore, we hypothesized that MYPT1 may control microtubule-dependent motor activity. Dynein, a minus-end microtubule motor, is known to be involved in mitotic spindle assembly. We thus examined whether MYPT1 and dynein may interact. Proximity ligation assay and co-immunoprecipitation revealed that MYPT1 and dynein intermediate chain (DIC) were associated. We found that DIC phosphorylation is increased in MYPT1-depleted cells in vivo, and that MP was able to dephosphorylate DIC in vitro. MYPT1 depletion also altered the localization and motility of Rab7-containing vesicles. MYPT1-depletion dispersed the perinuclear Rab7 localization to the peripheral in interphase cells. The dispersed Rab7 localization was rescued by microinjection of a constitutively active, truncated MYPT1 mutant, supporting that MP is responsible for the altered Rab7 localization. Analyses of Rab7 vesicle trafficking also revealed that minus-end transport was reduced in MYPT1-depleted cells. These results suggest an unexpected role of MP: MP controls dynein activity in both mitotic and interphase cells, possibly by dephosphorylating dynein subunits including DIC.

2.
J Immunol ; 207(3): 938-949, 2021 08 01.
Article in English | MEDLINE | ID: mdl-34301846

ABSTRACT

Migration of mature dendritic cells (DCs) to lymph nodes is critical for the initiation of adaptive immunity. CCR7, a G-protein-coupled receptor for CCL19/21 chemokines, is known to be essential for chemotaxis of mature DCs, but the molecular mechanism linking inflammation to chemotaxis remains unclear. We previously demonstrated that fascin1, an actin-bundling protein, increases chemotaxis of mature mouse DCs. In this article, we demonstrated that fascin1 enhanced IL-6 secretion and signaling of mature mouse DCs. Furthermore, we demonstrated that IL-6 signaling is required for chemotaxis. Blockage of IL-6 signaling in wild-type DCs with an anti-IL-6 receptor α (IL-6Rα) Ab inhibited chemotaxis toward CCL19. Likewise, knockout of IL-6Rα inhibited chemotaxis of bone marrow-derived DCs. The addition of soluble IL-6Rα and IL-6 rescued chemotaxis of IL-6Rα knockout bone marrow-derived DCs, underscoring the role of IL-6 signaling in chemotaxis. We found that IL-6 signaling is required for internalization of CCR7, the initial step of CCR7 recycling. CCR7 recycling is essential for CCR7-mediated chemotaxis, explaining why IL-6 signaling is required for chemotaxis of mature DCs. Our results have identified IL-6 signaling as a new regulatory pathway for CCR7/CCL19-mediated chemotaxis and suggest that rapid migration of mature DCs to lymph nodes depends on inflammation-associated IL-6 signaling.


Subject(s)
Antigens, Differentiation/metabolism , Dendritic Cells/immunology , Interleukin-6/metabolism , Microfilament Proteins/metabolism , Receptors, CCR7/metabolism , Receptors, Odorant/metabolism , Animals , Antibodies, Blocking/pharmacology , Antigens, Differentiation/genetics , Cell Differentiation , Cells, Cultured , Chemotaxis , Gene Expression Regulation , Mice , Mice, Knockout , Microfilament Proteins/genetics , Receptors, Interleukin-6/genetics , Receptors, Interleukin-6/immunology , Receptors, Interleukin-6/metabolism , Receptors, Odorant/genetics , Signal Transduction
3.
Gastroenterology ; 146(5): 1386-96.e1-17, 2014 May.
Article in English | MEDLINE | ID: mdl-24462734

ABSTRACT

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is often lethal because it is highly invasive and metastasizes rapidly. The actin-bundling protein fascin has been identified as a biomarker of invasive and advanced PDAC and regulates cell migration and invasion in vitro. We investigated fascin expression and its role in PDAC progression in mice. METHODS: We used KRas(G12D) p53(R172H) Pdx1-Cre (KPC) mice to investigate the effects of fascin deficiency on development of pancreatic intraepithelial neoplasia (PanIn), PDAC, and metastasis. We measured levels of fascin in PDAC cell lines and 122 human resected PDAC samples, along with normal ductal and acinar tissues; we associated levels with patient outcomes. RESULTS: Pancreatic ducts and acini from control mice and early-stage PanINs from KPC mice were negative for fascin, but approximately 6% of PanIN3 and 100% of PDAC expressed fascin. Fascin-deficient KRas(G12D) p53(R172H) Pdx1-Cre mice had longer survival times, delayed onset of PDAC, and a lower PDAC tumor burdens than KPC mice; loss of fascin did not affect invasion of PDAC into bowel or peritoneum in mice. Levels of slug and fascin correlated in PDAC cells; slug was found to regulate transcription of Fascin along with the epithelial-mesenchymal transition. In PDAC cell lines and cells from mice, fascin concentrated in filopodia and was required for their assembly and turnover. Fascin promoted intercalation of filopodia into mesothelial cell layers and cell invasion. Nearly all human PDAC samples expressed fascin, and higher fascin histoscores correlated with poor outcomes, vascular invasion, and time to recurrence. CONCLUSIONS: The actin-bundling protein fascin is regulated by slug and involved in late-stage PanIN and PDAC formation in mice. Fascin appears to promote formation of filopodia and invasive activities of PDAC cells. Its levels in human PDAC correlate with outcomes and time to recurrence, indicating it might be a marker or therapeutic target for pancreatic cancer.


Subject(s)
Carcinoma in Situ/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Carrier Proteins/metabolism , Microfilament Proteins/metabolism , Pancreatic Neoplasms/metabolism , Transcription Factors/metabolism , Animals , Carcinoma in Situ/genetics , Carcinoma in Situ/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/mortality , Carcinoma, Pancreatic Ductal/secondary , Carrier Proteins/genetics , Cell Line, Tumor , Disease Models, Animal , Disease Progression , Epithelial-Mesenchymal Transition , Humans , Mice , Mice, Knockout , Microfilament Proteins/deficiency , Microfilament Proteins/genetics , Neoplasm Invasiveness , Neoplasm Recurrence, Local , Neoplasm Staging , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/mortality , Pancreatic Neoplasms/pathology , Prognosis , Pseudopodia/metabolism , RNA Interference , Snail Family Transcription Factors , Survival Analysis , Time Factors , Transcription Factors/genetics , Transfection
4.
J Immunol ; 191(12): 6156-64, 2013 Dec 15.
Article in English | MEDLINE | ID: mdl-24244012

ABSTRACT

Ag-presenting dendritic cells (DCs) must survive bacterial infection to present Ag information to naive T cells. The greater ability of DCs' host defense is evident from the report that DCs are more resistant to Listeria monocytogenes than macrophages. However, the molecular mechanism of this resistance is unclear. We found that Listeria replicate more slowly in wild-type DCs compared with fascin1 knockout DCs. This finding is significant because fascin1, an actin-bundling protein, is specifically and greatly induced upon maturation of dendritic cells, but not other blood cells, including macrophages and neutrophils. Infection by Listeria makes phagosomes more acidic in wild-type DCs than in fascin1 knockout DCs, suggesting that fascin1 facilitates phagolysosomal fusion for killing of phagocytosed Listeria. We further found that fascin1 binds to LC3, an autophagosome marker, both in vivo and in vitro. Listeria are associated with LC3 to a greater extent in wild-type DCs than in fascin1 knockout DCs, suggesting that fascin1 facilitates autophagy for eradication of cytoplasmic Listeria. Taken together, our results suggest that fascin1 plays critical roles in the survival of DCs during Listeria infection, allowing DCs to function in innate and adaptive immunity.


Subject(s)
Dendritic Cells/microbiology , Listeria monocytogenes/immunology , Listeriosis/immunology , Microfilament Proteins/physiology , Animals , Autophagy/physiology , Dendritic Cells/immunology , Dendritic Cells/ultrastructure , Immunity, Innate , Listeria monocytogenes/growth & development , Mice , Mice, Knockout , Microfilament Proteins/deficiency , Microscopy, Electron , Microtubule-Associated Proteins/metabolism , Phagosomes/chemistry , Phagosomes/microbiology , Phagosomes/physiology , Protein Binding , Receptors, Odorant , Vacuoles/chemistry , Vacuoles/physiology
5.
Biol Open ; 2(11): 1187-91, 2013.
Article in English | MEDLINE | ID: mdl-24244855

ABSTRACT

The actin bundling protein fascin 1 is not expressed in adult epithelial tissues, but during development it is transiently expressed in many different cell types, and later in adults it is expressed in a subset of immune cells, nervous tissues, endothelial cells, smooth muscle cells and pericytes. In contrast to the wealth of knowledge about the role of fascin 1 in cancer cell migration and invasion, little is known about the involvement of fascin 1 in angiogenesis. We speculated that as angiogenesis involves migration and invasion of tissues by endothelial cells, fascin 1 might have a role in both normal and tumour angiogenesis. Here, we provide evidence that loss of fascin 1 causes relatively minor reductions to angiogenesis during embryonic, postnatal and cancerous development by examining E12.5 hindbrains, postnatal retinas and B16F0 tumour cell allografts in fascin 1-null mice. We also find that in fascin 1 null tissues, endothelial cells display reduced filopodia formation during sprouting. We thus propose that fascin 1 expression promotes angiogenesis via filopodia formation, but is largely dispensable for both normal and tumour angiogenesis.

6.
Development ; 140(10): 2203-11, 2013 May.
Article in English | MEDLINE | ID: mdl-23633513

ABSTRACT

Fascins, a family of actin-bundling proteins, are expressed in a spatially and temporally restricted manner during development and often in cancer. Fascin 1 has a clear role in cell migration in vitro, but its role in vivo in mammals is not well understood. Here, we investigate the role of fascin 1 in the melanocyte lineage and in melanoma cells. Fascin 1 knockout causes hypopigmentation in adult mice owing to migration and cell cycle progression defects in melanoblasts, the melanocyte precursor cell. Study of live embryo skin explants reveals that E14.5 fascin 1-null melanoblasts migrate slower, and generate fewer and thinner pseudopods. By contrast, fascin 1 expression drives faster migration and lamellipodia protrusion in melanocytes in vitro. In addition, fascin 1 depletion retards melanoblast proliferation in vivo and melanoma cell growth in vitro. These data indicate that fascin 1 not only promotes cell migration in mouse melanocytes but it also has a role in growth and cell cycle progression.


Subject(s)
Carrier Proteins/physiology , Gene Expression Regulation, Developmental , Melanocytes/cytology , Microfilament Proteins/physiology , Animals , Carrier Proteins/genetics , Cell Cycle , Cell Line, Tumor , Cell Movement , Cell Proliferation , Humans , Melanoma/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microfilament Proteins/genetics , Pigmentation , Skin/pathology , Time Factors
7.
Crit Rev Immunol ; 32(1): 11-21, 2012.
Article in English | MEDLINE | ID: mdl-22428853

ABSTRACT

Fascin-1 is an actin-bundling protein that shares no homology with other actin-bundling proteins. It is greatly induced upon maturation of dendritic cells (DCs). However, fascin-1 is not expressed in other primary blood cells, including macrophages and neutrophils, indicating a unique role of fascin-1 in the function of DCs upon maturation. An increasing body of evidence has shown that fascin-1 plays critical roles in maturation-associated DC functions, including dynamic assembly of veil-like membrane protrusions, disassembly of podosomes, migration to lymph nodes, and the assembly of the immunological synapse. Pathological analyses of fascin-1 expression revealed that fascin-1 is a useful marker of diseases of immune cells, including Langerhans cell histiocytosis and Hodgkin diseases. Furthermore, attempts have been made to explore the use of a fascin-1 promoter for DNA vaccination because it is strong and specific to DCs.


Subject(s)
Carrier Proteins/immunology , Dendritic Cells/immunology , Microfilament Proteins/immunology , Animals , Biomarkers/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Differentiation , Dendritic Cells/cytology , Humans , Microfilament Proteins/chemistry , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Promoter Regions, Genetic , Vaccines, DNA/immunology
8.
Arch Biochem Biophys ; 510(2): 76-82, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21396909

ABSTRACT

At mitosis, cells undergo drastic alterations in morphology and cytoskeletal organization including cell rounding during prophase, mitotic spindle assembly during prometaphase and metaphase, chromatid segregation in anaphase, and cytokinesis during telophase. It is well established that myosin II is a motor responsible for cytokinesis. Recent reports have indicated that myosin II is also involved in spindle assembly and karyokinesis. In this review, we summarize current understanding of the functions of myosin II in mitosis and cytokinesis of higher eukaryotes, and discuss the roles of possible upstream molecules that control myosin II in these mitotic events.


Subject(s)
Cytokinesis , Mitosis , Myosin-Light-Chain Kinase/metabolism , Myosin-Light-Chain Phosphatase/metabolism , Animals , Humans , Myosin Type II/metabolism
9.
J Biol Chem ; 286(12): 10825-33, 2011 Mar 25.
Article in English | MEDLINE | ID: mdl-21252232

ABSTRACT

Myosin phosphatase is a heterotrimeric holoenzyme consisting of myosin phosphatase-targeting subunit 1 (MYPT1), a catalytic subunit of PP1Cß, and a 20-kDa subunit of an unknown function. We have previously reported that myosin phosphatase also controls mitosis, apparently by antagonizing polo-like kinase 1 (PLK1). Here we found that depletion of MYPT1 by siRNA led to precocious chromatid segregation when HeLa cells were arrested at metaphase by a proteasome inhibitor, MG132, or by Cdc20 depletion. Consistently, cyclin B1 and securin were not degraded, indicating that the chromatid segregation is independent of the anaphase-promoting complex/cyclosome. Precocious segregation induced by MYPT1 depletion requires PLK1 activity because a PLK1 inhibitor, BI-2536, blocked precocious segregation. Furthermore, the expression of an unphosphorylatable mutant of SA2 (SCC3 homologue 2), a subunit of the cohesin complex, prevented precocious chromatid segregation induced by MYPT1 depletion. It has been shown that SA2 at centromeres is protected from phosphorylation by PP2A phosphatase recruited by Shugoshin (Sgo1), whereas SA2 along chromosome arms is phosphorylated by PLK1, leading to SA2 dissociation at chromosome arms. Taken together, our results suggest that hyperactivation of PLK1 caused by MYPT1 reduction could override the counteracting PP2A phosphatase, resulting in precocious chromatid segregation. We propose that SA2 at the centromeres is protected by two phosphatases. One is PP2A directly dephosphorylating SA2, and the other is myosin phosphatase counteracting PLK1.


Subject(s)
Centromere/metabolism , Chromosome Segregation/physiology , Chromosomes, Human/metabolism , Myosin-Light-Chain Phosphatase/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Centromere/genetics , Chromosome Segregation/drug effects , Chromosomes, Human/genetics , HeLa Cells , Humans , Mutation , Myosin-Light-Chain Phosphatase/genetics , Phosphorylation/drug effects , Phosphorylation/physiology , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Pteridines/pharmacology , Polo-Like Kinase 1
10.
J Immunol ; 186(5): 2850-9, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-21263068

ABSTRACT

Dendritic cells (DCs) play central roles in innate and adaptive immunity. Upon maturation, DCs assemble numerous veil-like membrane protrusions, disassemble podosomes, and travel from the peripheral tissues to lymph nodes to present Ags to T cells. These alterations in morphology and motility are closely linked to the primary function of DCs, Ag presentation. However, it is unclear how and what cytoskeletal proteins control maturation-associated alterations, in particular, the change in cell migration. Fascin1, an actin-bundling protein, is specifically and greatly induced upon maturation, suggesting a unique role for fascin1 in mature DCs. To determine the physiological roles of fascin1, we characterized bone marrow-derived, mature DCs from fascin1 knockout mice. We found that fascin1 is critical for cell migration: fascin1-null DCs exhibit severely decreased membrane protrusive activity. Importantly, fascin1-null DCs have lower chemotactic activity toward CCL19 (a chemokine for mature DCs) in vitro, and in vivo, Langerhans cells show reduced emigration into draining lymph nodes. Morphologically, fascin1-null mature DCs are flatter and fail to disassemble podosomes, a specialized structure for cell-matrix adhesion. Expression of exogenous fascin1 in fascin1-null DCs rescues the defects in membrane protrusive activity, as well as in podosome disassembly. These results indicate that fascin1 positively regulates migration of mature DCs into lymph nodes, most likely by increasing dynamics of membrane protrusions, as well as by disassembling podosomes.


Subject(s)
Cell Differentiation/immunology , Cell Movement/immunology , Dendritic Cells/immunology , Microfilament Proteins/physiology , Animals , Biomarkers/metabolism , Cell Differentiation/genetics , Cell Line, Tumor , Cell Membrane/immunology , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Movement/genetics , Cell Surface Extensions/immunology , Cell Surface Extensions/pathology , Cell Surface Extensions/ultrastructure , Dendritic Cells/metabolism , Dendritic Cells/ultrastructure , Female , Lymph Nodes/immunology , Lymph Nodes/metabolism , Lymph Nodes/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins/deficiency , Microfilament Proteins/genetics , Receptors, Odorant
11.
Cell Motil Cytoskeleton ; 66(8): 524-34, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19343791

ABSTRACT

Fascin1, an actin-bundling protein, has been demonstrated to be critical for filopodia formation in cultured cells, and thus is believed to be vital in motile activities including neurite extension and cell migration. To test whether fascin1 plays such essential roles within a whole animal, we have generated and characterized fascin1-deficient mice. Unexpectedly, fascin1-deficient mice are viable and fertile with no major developmental defect. Nissl staining of serial coronal brain sections reveals that fascin1-deficient brain is grossly normal except that knockout mouse brain lacks the posterior region of the anterior commissure neuron and has larger lateral ventricle. Fascin1-deficient, dorsal root ganglion neurons are able to extend neurites in vitro as well as those from wild-type mice, although fascin1-deficient growth cones are smaller and exhibit fewer and shorter filopodia than wild-type counterparts. Likewise, fascin1-deficient, embryonic fibroblasts are able to assemble filopodia, though filopodia are fewer, shorter and short-lived. These results indicate that fascin1-mediated filopodia assembly is dispensable for mouse development. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.


Subject(s)
Embryonic Development/physiology , Microfilament Proteins/metabolism , Animals , Animals, Newborn , Brain/cytology , Brain/metabolism , Cells, Cultured , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , Male , Mice , Mice, Knockout , Microfilament Proteins/genetics , Microscopy, Fluorescence , Models, Genetic , Neurons/cytology , Neurons/metabolism , Pseudopodia/metabolism , Receptors, Odorant , Reverse Transcriptase Polymerase Chain Reaction
12.
Dev Cell ; 14(5): 787-97, 2008 May.
Article in English | MEDLINE | ID: mdl-18477460

ABSTRACT

Myosin phosphatase-targeting subunit 1 (MYPT1) binds to the catalytic subunit of protein phosphatase 1 (PP1C). This binding is believed to target PP1C to specific substrates including myosin II, thus controlling cellular contractility. Surprisingly, we found that during mitosis, mammalian MYPT1 binds to polo-like kinase 1 (PLK1). MYPT1 is phosphorylated during mitosis by proline-directed kinases including cdc2, which generates the binding motif for the polo box domain of PLK1. Depletion of PLK1 by small interfering RNAs is known to result in loss of gamma-tubulin recruitment to the centrosomes, blocking centrosome maturation and leading to mitotic arrest. We found that codepletion of MYPT1 and PLK1 reinstates gamma-tubulin at the centrosomes, rescuing the mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at its activating site (Thr210) in vivo, explaining, at least in part, the rescue phenotype by codepletion. Taken together, our results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Mitosis , Myosin-Light-Chain Phosphatase/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Amino Acid Sequence , Animals , CDC2 Protein Kinase/metabolism , COS Cells , Cell Cycle Proteins/metabolism , Cell Survival , Centrosome/enzymology , Chlorocebus aethiops , Enzyme Activation , HeLa Cells , Humans , Kinetochores/enzymology , Molecular Sequence Data , Myosin-Light-Chain Phosphatase/chemistry , Phosphorylation , Phosphothreonine/metabolism , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Protein Transport , Proto-Oncogene Proteins/metabolism , Rats , Spindle Apparatus/enzymology , Tubulin/metabolism , Polo-Like Kinase 1
14.
J Cell Biol ; 164(3): 427-39, 2004 Feb 02.
Article in English | MEDLINE | ID: mdl-14757754

ABSTRACT

We examined the role of regulatory myosin light chain (MLC) phosphorylation of myosin II in cell migration of fibroblasts. Myosin light chain kinase (MLCK) inhibition blocked MLC phosphorylation at the cell periphery, but not in the center. MLCK-inhibited cells did not assemble zyxin-containing adhesions at the periphery, but maintained focal adhesions in the center. They generated membrane protrusions all around the cell, turned more frequently, and migrated less effectively. In contrast, Rho-associated kinase (ROCK) inhibition blocked MLC phosphorylation in the center, but not at the periphery. ROCK-inhibited cells assembled zyxin-containing adhesions at the periphery, but not focal adhesions in the center. They moved faster and more straight. On the other hand, inhibition of myosin phosphatase increased MLC phosphorylation and blocked peripheral membrane ruffling, as well as turnover of focal adhesions and cell migration. Our results suggest that myosin II activated by MLCK at the cell periphery controls membrane ruffling, and that the spatial regulation of MLC phosphorylation plays critical roles in controlling cell migration of fibroblasts.


Subject(s)
Cell Movement/physiology , Cell Surface Extensions/metabolism , Fibroblasts/metabolism , Focal Adhesions/metabolism , Myosin-Light-Chain Kinase/metabolism , Protein Serine-Threonine Kinases/metabolism , Actins/metabolism , Animals , Cell Adhesion/physiology , Cell Size , Cells, Cultured , Enzyme Inhibitors/metabolism , Fibroblasts/cytology , Fibroma , Gerbillinae , Humans , Intracellular Signaling Peptides and Proteins , Microscopy, Video , Myosin-Light-Chain Phosphatase/antagonists & inhibitors , Myosin-Light-Chain Phosphatase/metabolism , Phosphorylation , rho-Associated Kinases
15.
Mol Biol Cell ; 14(5): 1745-56, 2003 May.
Article in English | MEDLINE | ID: mdl-12802051

ABSTRACT

Citron kinase is a Rho-effector protein kinase that is related to Rho-associated kinases of ROCK/ROK/Rho-kinase family. Both ROCK and citron kinase are suggested to play a role in cytokinesis. However, no substrates are known for citron kinase. We found that citron kinase phosphorylated regulatory light chain (MLC) of myosin II at both Ser-19 and Thr-18 in vitro. Unlike ROCK, however, citron kinase did not phosphorylate the myosin binding subunit of myosin phosphatase, indicating that it does not inhibit myosin phosphatase. We found that the expression of the kinase domain of citron kinase resulted in an increase in MLC di-phosphorylation. Furthermore, the kinase domain was able to increase di-phosphorylation and restore stress fiber assembly even when ROCK was inhibited with a specific inhibitor, Y-27632. The expression of full-length citron kinase also increased di-phosphorylation during cytokinesis. These observations suggest that citron kinase phosphorylates MLC to generate di-phosphorylated MLC in vivo. Although both mono- and di-phosphorylated MLC were found in cleavage furrows, di-phosphorylated MLC showed more constrained localization than did mono-phosphorylated MLC. Because citron kinase is localized in cleavage furrows, citron kinase may be involved in regulating di-phosphorylation of MLC during cytokinesis.


Subject(s)
Myosin Light Chains/metabolism , Myosin Type II/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , COS Cells , Intracellular Signaling Peptides and Proteins , Kinetics , Phosphorylation
16.
J Biol Chem ; 278(20): 17937-44, 2003 May 16.
Article in English | MEDLINE | ID: mdl-12637566

ABSTRACT

The Arp2/3 complex greatly accelerates actin polymerization, which is thought to play a major role in cell motility by inducing membrane protrusions including ruffling movements. Membrane ruffles contain a variety of actin-binding proteins, which would modulate Arp2/3-dependent actin polymerization. However, their exact roles in actin polymerization remain to be established. Because caldesmon is present in membrane ruffles, as well as in stress fibers, it may alter Arp2/3-mediated actin polymerization. We have found that caldesmon greatly retards Arp2/3-induced actin polymerization. Kinetic analyses have revealed that caldesmon inhibits the nucleation process, whereas it does not largely reduce elongation. Caldesmon is found to inhibit binding of Arp2/3 to F-actin, which apparently reduces the ability of F-actin as a secondary activator of Arp2/3-mediated nucleation. We also have found that the inhibition of the binding between actin and caldesmon either by Ca(2+)/calmodulin or by phosphorylation with cdc2 kinase reverses the inhibitory effect of caldesmon on Arp2/3-induced actin polymerization. Our results suggest that caldesmon may be a key protein that modulates membrane ruffling and that this may involve changes in caldesmon phosphorylation and/or intracellular calcium concentrations during signal transduction.


Subject(s)
Actins/metabolism , Calmodulin-Binding Proteins/pharmacology , Cell Nucleus/metabolism , Cytoskeletal Proteins/metabolism , Actin-Related Protein 2 , Actin-Related Protein 3 , Animals , Blotting, Western , Calcium/metabolism , Calmodulin/metabolism , Calmodulin-Binding Proteins/metabolism , Chickens , Dose-Response Relationship, Drug , HeLa Cells , Humans , Kinetics , Muscle, Skeletal/metabolism , Phosphorylation , Protein Binding , Rats , Signal Transduction , Time Factors
17.
Am J Clin Pathol ; 118(3): 335-43, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12219775

ABSTRACT

Langerhans cell histiocytosis (LCH) is a clonal disorder believed to be derivedfrom cells of the dendritic system. Fascin, a 55-kd actin-bundling protein, represents a highly selective marker for dendritic cells of lymphoid tissues and peripheral blood and is involved in the formation of dendritic processes in maturing epidermal Langerhans cells. Since lesional cells of LCH may represent Langerhans cells arrested at an early stage of activation, immunohistochemical expression offascin in epidermal Langerhans cells and in the lesional cells of 34 cases of LCH was evaluated in paraffin sections using an immunoalkaline phosphatase technique. Though epidermal Langerhans cells were nonreactive for fascin, lesional cells in all LCH cases exhibited immunoreactivityforfascin, CD1a, and S-100 protein. Variation in staining intensity was observed in some cases, possibly reflecting differences in cell maturation or activation. Involved tissues included bone, soft tissue, lymph node, thyroid, orbit, and extradural cranial tissue. Immunoreactivity of lesional cells of LCH for fascin supports their derivation from cells of the dendritic system and represents another alteration in the phenotype of Langerhans cells that is associated with maturation, migration, culture, or clonal expansion.


Subject(s)
Actins/metabolism , Dendritic Cells/metabolism , Histiocytosis, Langerhans-Cell/metabolism , Indoles/metabolism , Adolescent , Adult , Biomarkers , Child , Child, Preschool , Dendritic Cells/pathology , Female , Fluorescent Antibody Technique, Indirect , Histiocytosis, Langerhans-Cell/pathology , Humans , Immunoenzyme Techniques , Infant , Male , Middle Aged
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