NUANCE, a giant protein connecting the nucleus and actin cytoskeleton

NUANCE (NUcleus and ActiN Connecting Element) was identified as a novel protein with an α-actinin-like actin-binding domain. A human 21.8 kb cDNA of NUANCE spreads over 373 kb on chromosome 14q22.1-q22.3. The cDNA sequence predicts a 796 kDa protein with an N-terminal actin-binding domain, a central coiled-coil rod domain and a predicted C-terminal transmembrane domain. High levels of NUANCE mRNA were detected in the kidney, liver,stomach, placenta, spleen, lymphatic nodes and peripheral blood lymphocytes. At the subcellular level NUANCE is present predominantly at the outer nuclear membrane and in the nucleoplasm. Domain analysis shows that the actin-binding domain binds to Factin in vitro and colocalizes with the actin cytoskeleton in vivo as a GFP-fusion protein. The C-terminal transmembrane domain is responsible for the targeting the nuclear envelope. Thus, NUANCE is the firstα-actinin-related protein that has the potential to link the microfilament system with the nucleus.

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Microtubule Actin Cross-Linking Factor (Macf)

The Journal of Cell Biology ◽

10.1083/jcb.147.6.1275 ◽

1999 ◽

Vol 147 (6) ◽

pp. 1275-1286 ◽

Cited By ~ 150

Author(s):

Conrad L. Leung ◽

Dongming Sun ◽

Min Zheng ◽

David R. Knowles ◽

Ronald K.H. Liem

Keyword(s):

Calcium Binding ◽

Coiled Coil ◽

Specific Protein ◽

Binding Domain ◽

Full Length ◽

Actin Binding ◽

Cross Linking ◽

Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

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A Coiled-Coil Domain of Melanophilin Is Essential for Myosin Va Recruitment and Melanosome Transport in Melanocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0457 ◽

2006 ◽

Vol 17 (11) ◽

pp. 4720-4735 ◽

Cited By ~ 61

Author(s):

Alistair N. Hume ◽

Abul K. Tarafder ◽

José S. Ramalho ◽

Elena V. Sviderskaya ◽

Miguel C. Seabra

Keyword(s):

Coiled Coil ◽

Binding Domain ◽

Actin Binding ◽

Binding Studies ◽

Null Cell ◽

Coiled Coil Region ◽

Actin Binding Domain ◽

Transport Assay ◽

Myosin Va

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F–binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

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Tissue Expression and Actin Binding of a Novel N-Terminal Utrophin Isoform

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/904547 ◽

2011 ◽

Vol 2011 ◽

pp. 1-18

Author(s):

Richard A. Zuellig ◽

Beat C. Bornhauser ◽

Ralf Amstutz ◽

Bruno Constantin ◽

Marcus C. Schaub

Keyword(s):

Actin Cytoskeleton ◽

Tissue Expression ◽

Protein Product ◽

Binding Domain ◽

Actin Binding ◽

Rat Tissues ◽

Cortical Actin ◽

Actin Binding Domain ◽

Spectrin Repeats

Utrophin and dystrophin present two large proteins that link the intracellular actin cytoskeleton to the extracellular matrix via the C-terminal-associated protein complex. Here we describe a novel short N-terminal isoform of utrophin and its protein product in various rat tissues (N-utro, 62 kDa, amino acids 1–539, comprising the actin-binding domain plus the first two spectrin repeats). Using different N-terminal recombinant utrophin fragments, we show that actin binding exhibits pronounced negative cooperativity (affinity constantsK1=∼5×106andK2=∼1×105 M-1) and is Ca2+-insensitive. Expression of the different fragments in COS7 cells and in myotubes indicates that the actin-binding domain alone binds exlusively to actin filaments. The recombinant N-utro analogue binds in vitro to actin and in the cells associates to the membranes. The results indicate that N-utro may be responsible for the anchoring of the cortical actin cytoskeleton to the membranes in muscle and other tissues.

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The C-Terminus of c-Abl Is Required for Proliferation and Viability Signaling in a c-Abl/Erythropoietin Receptor Fusion Protein

Blood ◽

10.1182/blood.v92.10.3848.422k44_3848_3856 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3848-3856 ◽

Cited By ~ 3

Author(s):

K. Okuda ◽

A. D’Andrea ◽

R.A. Van Etten ◽

J.D. Griffin

Keyword(s):

Transmembrane Domain ◽

Biological Effects ◽

Sh2 Domain ◽

Binding Domain ◽

Erythropoietin Receptor ◽

Myelogenous Leukemia ◽

Actin Binding ◽

Time To Death ◽

Actin Binding Domain

Activated ABL oncogenes cause B-cell leukemias in mice and chronic myelogenous leukemia in humans. However, the mechanism of transformation is complex and not well understood. A method to rapidly and reversibly activate c-ABL was created by fusing the extra-cytoplasmic and transmembrane domain of the erythropoietin (EPO) receptor with c-ABL (EPO R/ABL). When this chimeric receptor was expressed in Ba/F3 cells, the addition of EPO resulted in a dose-dependent activation of c-ABL tyrosine kinase and was strongly antiapoptotic and weakly mitogenic. To evaluate the contributions of various ABL domains to biochemical signaling and biological effects, chimeric receptors were constructed in which the ABL SH3 domain was deleted (▵SH3), the SH2 domain was deleted (▵SH2), the C-terminal actin-binding domain was deleted (▵ABD), or kinase activity was eliminated by a point mutation, K290M (KD). The mutant receptors were stably expressed in Ba/F3 cells and analyzed for signaling defects, proliferation, viability, and EPO-induced leukemia in nude mice. When compared with the ability of the full-length EPO R/ABL receptor to induce proliferation and support viability in vitro, the ▵SH3 mutant was equivalent, the ▵SH2 mutant was moderately impaired, and the ▵ABD and KD mutants were profoundly impaired. None of these cell lines caused leukemia in mice in the absence of pharmacological doses of EPO. However, in mice treated with EPO (10 U/d), death from leukemia occurred rapidly with wild-type and ▵SH3. However, time to death was prolonged by at least twofold for ▵SH2 and greater than threefold for ▵ABD. This inducible model of ABL transformation provides a method to link specific signaling defects with specific biological defects and has shown an important role for the C-terminal actin-binding domain in proliferation and transformation in the context of this receptor/oncogene.

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The C-Terminus of c-Abl Is Required for Proliferation and Viability Signaling in a c-Abl/Erythropoietin Receptor Fusion Protein

Blood ◽

10.1182/blood.v92.10.3848 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3848-3856 ◽

Cited By ~ 3

Author(s):

K. Okuda ◽

A. D’Andrea ◽

R.A. Van Etten ◽

J.D. Griffin

Keyword(s):

Transmembrane Domain ◽

Biological Effects ◽

Sh2 Domain ◽

Binding Domain ◽

Erythropoietin Receptor ◽

Myelogenous Leukemia ◽

Actin Binding ◽

Time To Death ◽

Actin Binding Domain

Abstract Activated ABL oncogenes cause B-cell leukemias in mice and chronic myelogenous leukemia in humans. However, the mechanism of transformation is complex and not well understood. A method to rapidly and reversibly activate c-ABL was created by fusing the extra-cytoplasmic and transmembrane domain of the erythropoietin (EPO) receptor with c-ABL (EPO R/ABL). When this chimeric receptor was expressed in Ba/F3 cells, the addition of EPO resulted in a dose-dependent activation of c-ABL tyrosine kinase and was strongly antiapoptotic and weakly mitogenic. To evaluate the contributions of various ABL domains to biochemical signaling and biological effects, chimeric receptors were constructed in which the ABL SH3 domain was deleted (▵SH3), the SH2 domain was deleted (▵SH2), the C-terminal actin-binding domain was deleted (▵ABD), or kinase activity was eliminated by a point mutation, K290M (KD). The mutant receptors were stably expressed in Ba/F3 cells and analyzed for signaling defects, proliferation, viability, and EPO-induced leukemia in nude mice. When compared with the ability of the full-length EPO R/ABL receptor to induce proliferation and support viability in vitro, the ▵SH3 mutant was equivalent, the ▵SH2 mutant was moderately impaired, and the ▵ABD and KD mutants were profoundly impaired. None of these cell lines caused leukemia in mice in the absence of pharmacological doses of EPO. However, in mice treated with EPO (10 U/d), death from leukemia occurred rapidly with wild-type and ▵SH3. However, time to death was prolonged by at least twofold for ▵SH2 and greater than threefold for ▵ABD. This inducible model of ABL transformation provides a method to link specific signaling defects with specific biological defects and has shown an important role for the C-terminal actin-binding domain in proliferation and transformation in the context of this receptor/oncogene.

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The preprophase band-associated kinesin-14 OsKCH2 is a processive minus-end-directed microtubule motor

10.1101/232652 ◽

2017 ◽

Author(s):

Kuo-Fu Tseng ◽

Pan Wang ◽

Yuh-Ru Julie Lee ◽

Joel Bowen ◽

Allison M. Gicking ◽

...

Keyword(s):

Coiled Coil ◽

Preprophase Band ◽

Cytoplasmic Dynein ◽

Coiled Coils ◽

Land Plants ◽

Actin Binding ◽

Actin Binding Domain ◽

Microtubule Motor

AbstractIn animals and fungi, cytoplasmic dynein is a processive motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual homodimers. We find that CC1 but not CC2 forms the coiled-coil for OsKCH2 dimerization. Instead, CC2 functions to enable OsKCH2 processivity by enhancing its binding to microtubules. Collectively, these results show that land plants have evolved unconventional kinesin-14 homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.

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The C Terminus of Rotavirus VP4 Protein Contains an Actin Binding Domain Which Requires Cooperation with the Coiled-Coil Domain for Actin Remodeling

Journal of Virology ◽

10.1128/jvi.01598-18 ◽

2018 ◽

Vol 93 (1) ◽

Cited By ~ 2

Author(s):

Wilfried Condemine ◽

Thibaut Eguether ◽

Nathalie Couroussé ◽

Catherine Etchebest ◽

Agnes Gardet ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Coiled Coil ◽

Binding Domain ◽

Spike Protein ◽

Actin Binding ◽

Intestinal Cells ◽

Actin Remodeling ◽

Coiled Coil Domain ◽

C Terminus ◽

Actin Binding Domain

ABSTRACTThe interactions between viruses and actin cytoskeleton have been widely studied. We showed that rotaviruses remodel microfilaments in intestinal cells and demonstrated that this was due to the VP4 spike protein. Microfilaments mainly occur in the apical domain of infected polarized enterocytes and favor the polarized apical exit of viral progeny. The present work aims at the identification of molecular determinants of actin-VP4 interactions. We used various deletion mutants of VP4 that were transfected into Cos-7 cells and analyzed interactions by immunofluorescence confocal microscopy. It has been established that the C-terminal part of VP4 is embedded within viral particles when rotavirus assembles. The use of specific monoclonal antibodies demonstrated that VP4 is expressed in different forms in infected cells: classically as spike on the outer layer of virus particles, but also as free soluble protein in the cytosol. The C terminus of free VP4 was identified as interacting with actin microfilaments. The VP4 actin binding domain is unable to promote microfilament remodeling by itself; the coiled-coil domain is also required in this process. This actin-binding domain was shown to dominate a previously identified peroxisomal targeting signal, located in the three last amino acids of VP4. The newly identified actin-binding domain is highly conserved in rotavirus strains from species A, B, and C, suggesting that actin binding and remodeling is a general strategy for rotavirus exit. This provides a novel mechanism of protein-protein interactions, not involving cell signaling pathways, to facilitate rotavirus exit.IMPORTANCERotaviruses are causal agents of acute infantile viral diarrhea. In intestinal cells,in vitroas well asin vivo, virus assembly and exit do not imply cell lysis but rely on an active process in which the cytoskeleton plays a major role. We describe here a novel molecular mechanism by which the rotavirus spike protein VP4 drives actin remodeling. This relies on the fact that VP4 occurs in different forms. Besides its structural function within the virion, a large proportion of VP4 is expressed as free protein. Here, we show that free VP4 possesses a functional actin-binding domain. This domain, in coordination with a coiled-coil domain, promotes actin cytoskeleton remodeling, thereby providing the capacity to destabilize the cell membrane and allow efficient rotavirus exit.

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Specific Interaction of the Actin-binding Domain of Dystrophin with Intermediate Filaments Containing Keratin 19

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0112 ◽

2005 ◽

Vol 16 (9) ◽

pp. 4280-4293 ◽

Cited By ~ 47

Author(s):

Michele R. Stone ◽

Andrea O'Neill ◽

Dawn Catino ◽

Robert J. Bloch

Keyword(s):

Striated Muscle ◽

Specific Interaction ◽

Binding Domain ◽

Actin Binding ◽

Glycoprotein Complex ◽

Actin Binding Domain ◽

Dystrophin Glycoprotein Complex ◽

Related Proteins

Cytokeratins 8 and 19 concentrate at costameres of striated muscle and copurify with the dystrophin-glycoprotein complex, perhaps through the interaction of the cytokeratins with the actin-binding domain of dystrophin. We overexpressed dystrophin's actin-binding domain (Dys-ABD), K8 and K19, as well as closely related proteins, in COS-7 cells to assess the basis and specificity of their interaction. Dys-ABD alone associated with actin microfilaments. Expressed with K8 and K19, which form filaments, Dys-ABD associated preferentially with the cytokeratins. This interaction was specific, as the homologous ABD of βI-spectrin failed to interact with K8/K19 filaments, and Dys-ABD did not associate with desmin or K8/K18 filaments. Studies in COS-7 cells and in vitro showed that Dys-ABD binds directly and specifically to K19. Expressed in muscle fibers in vivo, K19 accumulated in the myoplasm in structures that contained dystrophin and spectrin and disrupted the organization of the sarcolemma. K8 incorporated into sarcomeres, with no effect on the sarcolemma. Our results show that dystrophin interacts through its ABD with K19 specifically and are consistent with the idea that cytokeratins associate with dystrophin at the sarcolemma of striated muscle.

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

The Journal of Cell Biology ◽

10.1083/jcb.200206113 ◽

2002 ◽

Vol 159 (6) ◽

pp. 993-1004 ◽

Cited By ~ 126

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.

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