scholarly journals h3/Acidic Calponin: An Actin-binding Protein That Controls Extracellular Signal-regulated Kinase 1/2 Activity in Nonmuscle Cells

2010 ◽  
Vol 21 (8) ◽  
pp. 1409-1422 ◽  
Author(s):  
Sarah Appel ◽  
Philip G. Allen ◽  
Susanne Vetterkind ◽  
Jian-Ping Jin ◽  
Kathleen G. Morgan
Keyword(s):  
Wound Healing ◽  
Binding Protein ◽  
Actin Binding ◽  
Cell Cortex ◽  
Extracellular Signal Regulated Kinase ◽  
Actin Binding Protein ◽  
Fibroblast Migration ◽  
Extracellular Signal ◽  
Nonmuscle Cells

Migration of fibroblasts is important in wound healing. Here, we demonstrate a role and a mechanism for h3/acidic calponin (aCaP, CNN3) in REF52.2 cell motility, a fibroblast line rich in actin filaments. We show that the actin-binding protein h3/acidic calponin associates with stress fibers in the absence of stimulation but is targeted to the cell cortex and podosome-like structures after stimulation with a phorbol ester, phorbol-12,13-dibutyrate (PDBu). By coimmunoprecipitation and colocalization, we show that extracellular signal-regulated kinase (ERK)1/2 and protein kinase C (PKC)α constitutively associate with h3/acidic calponin and are cotargeted with h3/acidic calponin in the presence of PDBu. This targeting can be blocked by a PKC inhibitor but does not require phosphorylation of h3/acidic calponin at the PKC sites S175 or T184. Knockdown of h3/acidic calponin results in a loss of PDBu-mediated ERK1/2 targeting, whereas PKCα targeting is unaffected. Caldesmon is an actin-binding protein that regulates actomyosin interactions and is a known substrate of ERK1/2. Both ERK1/2 activity and nonmuscle l-caldesmon phosphorylation are blocked by h3/acidic calponin knockdown. Furthermore, h3/acidic calponin knockdown inhibits REF52.2 migration in an in vitro wound healing assay. Our findings are consistent with a model whereby h3/acidic calponin controls fibroblast migration by regulation of ERK1/2-mediated l-caldesmon phosphorylation.

scholarly journals The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro

2001 ◽  
Vol 154 (6) ◽  
pp. 1209-1224 ◽  
Author(s):  
Åsa E.Y. Engqvist-Goldstein ◽  
Robin A. Warren ◽  
Michael M. Kessels ◽  
James H. Keen ◽  
John Heuser ◽  
...  
Keyword(s):  
Binding Protein ◽  
Coiled Coil ◽  
Actin Binding ◽  
Live Cells ◽  
Cell Cortex ◽  
Coated Pits ◽  
Actin Binding Protein ◽  
Real Time Analysis

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.

scholarly journals Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

2002 ◽  
Vol 159 (6) ◽  
pp. 993-1004 ◽  
Author(s):  
Christine L. Humphries ◽  
Heath I. Balcer ◽  
Jessica L. D'Agostino ◽  
Barbara Winsor ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Binding Protein ◽  
Coiled Coil ◽  
Actin Binding ◽  
Complex Activity ◽  
Actin Binding Protein ◽  
Coiled Coil Domain ◽  
Allele Specific ◽  
And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

scholarly journals Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Regulates Actin Organization and Cell Motility by Phosphorylating the Actin Cross-Linking Protein EPLIN

2007 ◽  
Vol 27 (23) ◽  
pp. 8190-8204 ◽  
Author(s):  
Mei-Ying Han ◽  
Hidetaka Kosako ◽  
Toshiki Watanabe ◽  
Seisuke Hattori
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Cell Motility ◽  
Stress Fiber ◽  
Mitogen Activated Protein Kinase ◽  
Cross Linking ◽  
Intact Cells ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

ABSTRACT Extracellular signal-regulated kinase (ERK) is important for various cellular processes, including cell migration. However, the detailed molecular mechanism by which ERK promotes cell motility remains elusive. Here we characterize epithelial protein lost in neoplasm (EPLIN), an F-actin cross-linking protein, as a novel substrate for ERK. ERK phosphorylates Ser360, Ser602, and Ser692 on EPLIN in vitro and in intact cells. Phosphorylation of the C-terminal region of EPLIN reduces its affinity for actin filaments. EPLIN colocalizes with actin stress fibers in quiescent cells, and stimulation with platelet-derived growth factor (PDGF) induces stress fiber disassembly and relocalization of EPLIN to peripheral and dorsal ruffles, wherein phosphorylation of Ser360 and Ser602 is observed. Phosphorylation of these two residues is also evident during wound healing at the leading edge of migrating cells. Moreover, expression of a non-ERK-phosphorylatable mutant, but not wild-type EPLIN, prevents PDGF-induced stress fiber disassembly and membrane ruffling and also inhibits wound healing and PDGF-induced cell migration. We propose that ERK-mediated phosphorylation of EPLIN contributes to actin filament reorganization and enhanced cell motility.

scholarly journals nPIST: A Novel Actin Binding Protein of trans-Golgi Network

2018 ◽  
Author(s):  
Swagata Das ◽  
Priyanka Dutta ◽  
Mohit Mazumder ◽  
Soma Seal ◽  
Kheerthana Duraivelan ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Purkinje Cells ◽  
Binding Protein ◽  
Vesicular Trafficking ◽  
Actin Binding ◽  
Perinuclear Region ◽  
Actin Binding Protein ◽  
Golgi Network

Abstractnpist is the neuronal isoform of PIST, a trans-golgi associated protein involved in major modulation of vesicular trafficking. nPIST interacts with glutamate delta2 receptor (GluRδ2) in Purkinje cells. Our study shows nPIST as a novel actin binding protein. Our structure based sequence analysis shows nPIST contains one WH2-like domain. Further our experimental analysis illustrates that fragment of nPIST consisting of WH2-like domain binds to actin. Moreover it was found that nPIST contains several regions involved in interaction with actin. The binding of nPIST to actin through multiple actin binding regions facilitated actin filament stabilization in vitro. In vivo, nPIST localized actin in perinuclear region as a blotch when ectopically expressed.

scholarly journals The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1663 ◽  
Author(s):  
Arne Velthaus ◽  
Kerstin Cornils ◽  
Jan K. Hennigs ◽  
Saskia Grüb ◽  
Hauke Stamm ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Binding Protein ◽  
Actin Binding ◽  
Bone Marrow Niche ◽  
Actin Binding Protein ◽  
Acute Myeloid

Leukemia-initiating cells reside within the bone marrow in specialized niches where they undergo complex interactions with their surrounding stromal cells. We have identified the actin-binding protein Plastin-3 (PLS3) as potential player within the leukemic bone marrow niche and investigated its functional role in acute myeloid leukemia. High expression of PLS3 was associated with a poor overall and event-free survival for AML patients. These findings were supported by functional in vitro and in vivo experiments. AML cells with a PLS3 knockdown showed significantly reduced colony numbers in vitro while the PLS3 overexpression variants resulted in significantly enhanced colony numbers compared to their respective controls. Furthermore, the survival of NSG mice transplanted with the PLS3 knockdown cells showed a significantly prolonged survival in comparison to mice transplanted with the control AML cells. Further studies should focus on the underlying leukemia-promoting mechanisms and investigate PLS3 as therapeutic target.

scholarly journals αII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts

2005 ◽  
Vol 388 (2) ◽  
pp. 631-638 ◽  
Author(s):  
Björn ROTTER ◽  
Odile BOURNIER ◽  
Gael NICOLAS ◽  
Didier DHERMY ◽  
Marie-Christine LECOMTE
Keyword(s):  
Binding Protein ◽  
Focal Adhesions ◽  
Membrane Skeleton ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Src Homology 3 ◽  
Src Homology 3 Domain ◽  
Src Homology

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of α- and β-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent α-spectrin subunit in non-erythroid cells, αII-spectrin, contains two particular spectrin repeats in its central region, α9 and α10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the α9-α10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the α10 repeat of αII-spectrin whereas EVL interacts with the Src homology 3 domain located within the α9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between αII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

scholarly journals Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein-280–deficient Melanoma Cell Line

1997 ◽  
Vol 136 (4) ◽  
pp. 845-857 ◽  
Author(s):  
C. Casey Cunningham ◽  
Nicole Leclerc ◽  
Lisa A. Flanagan ◽  
Mei Lu ◽  
Paul A. Janmey ◽  
...  
Keyword(s):  
Melanoma Cell ◽  
Binding Protein ◽  
Growth Cones ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Low Concentrations ◽  
Rheologic Property ◽  
Neuronal Growth Cones

The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.

scholarly journals A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor.

1990 ◽  
Vol 111 (4) ◽  
pp. 1477-1489 ◽  
Author(s):  
M Brink ◽  
G Gerisch ◽  
G Isenberg ◽  
A A Noegel ◽  
J E Segall ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Mutant Cell ◽  
Actin Binding ◽  
In Vitro Assays ◽  
Cell Cortex ◽  
Cross Linking ◽  
Actin Binding Proteins ◽  
Cell Extracts ◽  
Nonmuscle Cells

Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.

scholarly journals Cooperative Regulation of Extracellular Signal-Regulated Kinase Activation and Cell Shape Change by Filamin A and β-Arrestins

2006 ◽  
Vol 26 (9) ◽  
pp. 3432-3445 ◽  
Author(s):  
Mark G. H. Scott ◽  
Vincenzo Pierotti ◽  
Hélène Storez ◽  
Erika Lindberg ◽  
Alain Thuret ◽  
...  
Keyword(s):  
Shape Change ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Filamin A ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

ABSTRACT β-Arrestins (βarr) are multifunctional adaptor proteins that can act as scaffolds for G protein-coupled receptor activation of mitogen-activated protein kinases (MAPK). Here, we identify the actin-binding and scaffolding protein filamin A (FLNA) as a βarr-binding partner using Son of sevenless recruitment system screening, a classical yeast two-hybrid system, coimmunoprecipitation analyses, and direct binding in vitro. In FLNA, the βarr-binding site involves tandem repeat 22 in the carboxyl terminus. βarr binds FLNA through both its N- and C-terminal domains, indicating the presence of multiple binding sites. We demonstrate that βarr and FLNA act cooperatively to activate the MAPK extracellular signal-regulated kinase (ERK) downstream of activated muscarinic M1 (M1MR) and angiotensin II type 1a (AT1AR) receptors and provide experimental evidence indicating that this phenomenon is due to the facilitation of βarr-ERK2 complex formation by FLNA. In Hep2 cells, stimulation of M1MR or AT1AR results in the colocalization of receptor, βarr, FLNA, and active ERK in membrane ruffles. Reduction of endogenous levels of βarr or FLNA and a catalytically inactive dominant negative MEK1, which prevents ERK activation, inhibit membrane ruffle formation, indicating the functional requirement for βarr, FLNA, and active ERK in this process. Our results indicate that βarr and FLNA cooperate to regulate ERK activation and actin cytoskeleton reorganization.

scholarly journals Distribution of actin-binding protein and myosin in macrophages during spreading and phagocytosis.

1980 ◽  
Vol 84 (2) ◽  
pp. 215-224 ◽  
Author(s):  
O I Stendahl ◽  
J H Hartwig ◽  
E A Brotschi ◽  
T P Stossel
Keyword(s):  
Actin Filaments ◽  
Structural Unit ◽  
Binding Protein ◽  
Actin Binding ◽  
Rabbit Lung ◽  
Actin Binding Protein ◽  
Lung Macrophages ◽  
In Cells

Actin-binding protein (ABP) and myosin are proteins that influence the rigidity and movement, respectively, of actin filaments in vitro. We examined the distribution of ABP and myosin molecules in acetone-fixed rabbit lung macrophages by means of immunofluorescence. The staining for both of these proteins in unspread cells was quite uniform, but was reduced in the nucleus and concentrated slightly in the periphery. The peripheral accumulation of staining attenuated in uniformly spread cells, although filopodia and hyaline veils definitely stained. In cells fixed during ingestion of yeast particles, the brightest staining correlated with the disposition of organelle-excluding pseudopodia initially surrounding the yeast. After phagocytosis was complete and the yeasts resided in intracellular vacuoles, no concentration of staining around the ingested yeasts was detectable. We conclude that ABP and myosin molecules are components of the structural unit of the cell responsible for spreading and phagocytosis, the hyaline cortex, a region known to be rich in actin filaments. The findings are consistent with the theory that these molecules control the rigidity and movement of filaments in the periphery of the living macrophage.

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