scholarly journals Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin

2004 ◽  
Vol 164 (5) ◽  
pp. 653-659 ◽  
Author(s):  
Bruno T. Fievet ◽  
Alexis Gautreau ◽  
Christian Roy ◽  
Laurence Del Maestro ◽  
Paul Mangeat ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Binding Sites ◽  
Intramolecular Interaction ◽  
Activation Mechanism ◽  
Actin Binding ◽  
Activation Process ◽  
Cell Morphogenesis ◽  
Cellular Functions ◽  
Apical Localization

Ezrin, a membrane–actin cytoskeleton linker, which participates in epithelial cell morphogenesis, is held inactive in the cytoplasm through an intramolecular interaction. Phosphatidylinositol 4,5-bisphosphate (PIP2) binding and the phosphorylation of threonine 567 (T567) are involved in the activation process that unmasks both membrane and actin binding sites. Here, we demonstrate that ezrin binding to PIP2, through its NH2-terminal domain, is required for T567 phosphorylation and thus for the conformational activation of ezrin in vivo. Furthermore, we found that the T567D mutation mimicking T567 phosphorylation bypasses the need for PIP2 binding for unmasking both membrane and actin binding sites. However, PIP2 binding and T567 phosphorylation are both necessary for the correct apical localization of ezrin and for its role in epithelial cell morphogenesis. These results establish that PIP2 binding and T567 phosphorylation act sequentially to allow ezrin to exert its cellular functions.

scholarly journals Tropomyosin distinguishes between the two actin-binding sites of villin and affects actin-binding properties of other brush border proteins.

1987 ◽  
Vol 104 (1) ◽  
pp. 29-40 ◽  
Author(s):  
D R Burgess ◽  
K O Broschat ◽  
J M Hayden
Keyword(s):  
Epithelial Cell ◽  
Protein Interactions ◽  
Brush Border ◽  
Binding Sites ◽  
Intestinal Epithelial Cell ◽  
Actin Binding ◽  
Intestinal Epithelial ◽  
Binding Properties ◽  
Order Of Magnitude ◽  
Protein Components

The intestinal epithelial cell brush border exhibits distinct localizations of the actin-binding protein components of its cytoskeleton. The protein interactions that dictate this subcellular organization are as yet unknown. We report here that tropomyosin, which is found in the rootlet but not in the microvillus core, can bind to and saturate the actin of isolated cores, and can cause the dissociation of up to 30% of the villin and fimbrin from the cores but does not affect actin binding by 110-kD calmodulin. Low speed sedimentation assays and ultrastructural analysis show that the tropomyosin-containing cores remain bundled, and that 110-kD calmodulin remains attached to the core filaments. The effects of tropomyosin on the binding and bundling activities of villin were subsequently determined by sedimentation assays. Villin binds to F-actin with an apparent Ka of 7 X 10(5) M-1 at approximate physiological ionic strength, which is an order of magnitude lower than that of intestinal epithelial cell tropomyosin. Binding of villin to F-actin presaturated with tropomyosin is inhibited relative to that to pure F-actin, although full saturation can be obtained by increasing the villin concentration. Villin also inhibits the binding of tropomyosin to F-actin, although not to the same extent. However, tropomyosin strongly inhibits bundling of F-actin by villin, and bundling is not recovered even at a saturating villin concentration. Since villin has two actin-binding sites, both of which are required for bundling, the fact that tropomyosin inhibits bundling of F-actin under conditions where actin is fully saturated with villin strongly suggests that tropomyosin's and one of villin's F-actin-binding sites overlap. These results indicate that villin and tropomyosin could compete for actin filaments in the intestinal epithelial cell, and that tropomyosin may play a major role in the regulation of microfilament structure in these and other cells.

scholarly journals Rap1, AF6 and Pak4 cooperate with Par3 to promote ZA assembly and remodeling in the fly photoreceptor

2017 ◽  
Author(s):  
Rhian F. Walther ◽  
Mubarik Burki ◽  
Noelia Pinal ◽  
Clare Rogerson ◽  
Franck Pichaud
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Binding Protein ◽  
Small Gtpase ◽  
Actin Binding ◽  
Cell Morphogenesis ◽  
Recruitment And Retention ◽  
Actin Binding Protein ◽  
P21 Activated Kinase ◽  
Fly Photoreceptor

AbstractThe epithelial Zonula adherens (ZA) is a main adhesion compartment that enables organogenesis by allowing epithelial cells to assemble into sheets. How ZA assembly is regulated during epithelial cell morphogenesis is not fully understood. We show that during ZA morphogenesis, the function of the small GTPase Rap1 and the F-actin binding protein AF6/Cno are both linked to that of the P21-activated kinase Pak4/Mbt. We find that Rap1 and Mbt regulate each other’s localization at the ZA and cooperate in promoting ECadherin stabilization. During this process Cno regulates the recruitment of Baz at the ZA, a process that is also regulated by Arm phosphorylation by Mbt. Altogether, we propose that Rap1, Cno and Mbt regulate ZA morphogenesis by coordinating ECadherin stabilization and Baz recruitment and retention. In addition, our work uncovers a new link between two main oncogenes, Rap1 and Pak4/Mbt, in a model developing epithelial cell.

scholarly journals lncRNA MALAT1 Mediates Osteogenic Differentiation and Apoptosis in Osteoporosis by Regulating the miR-485-5p/WNT7B Axis

2021 ◽  
Author(s):  
Yuan Zhou ◽  
Zhuo Xu ◽  
Yuanyi Wang ◽  
Qiang Song ◽  
Ruofeng Yin
Keyword(s):  
Osteogenic Differentiation ◽  
Cell Apoptosis ◽  
Binding Sites ◽  
Hindlimb Unloading ◽  
Cellular Functions ◽  
Alizarin Red ◽  
Lncrna Malat1 ◽  
Non Coding Rnas

Abstract Background: Accumulating evidence demonstrates that long non-coding RNAs (lncRNAs) are associated with the development of osteoporosis. This study aimed to investigate the effects of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on osteogenic differentiation and cell apoptosis in osteoporosis. Methods: hindlimb unloading (HU) was performed to establish osteoporosis model in vivo. MicroCT was applied for pathological analysis. Microgravity (MG) was used to construct osteoporosis in vitro. The mRNA and miRNA expression was determined using RT-qPCR. Protein expression was determined using western blot. The binding sites between miR-485-5p and MALAT1/Wnt family member 7B (WNT7B) was predicted by bioinformatics analysis and verified by luciferase and RNA pull-down assays. Cellular functions were determined by ALP staining, Alizarin red staining, and flow cytometry assays. Results: MALAT1 expression was downregulated in HU mice and MG treated MC3T3-E1 cells. However, overexpression of MALAT1 upregulated the expression of Bmp4, Col1a1, Spp1, and enhanced ALP activity. Additionally, overexpression of MALAT1 inhibited apoptosis, decreased Bax and caspase-3 levels, and increased Bcl-2 level. Moreover, MALAT1 overexpression improved bone phenotype in vivo. MALAT1 functioned as a ceRNA to upregulate WNT7B. Overexpression of miR-485-5p rescued the promotion of osteogenic differentiation and the inhibition of apoptosis induced by MALAT1. Knockdown of WNT7B abolished the facilitation of osteogenic differentiation and the suppression of apoptosis induced by downregulation of miR-485-5p. Conclusion: In conclusion, MALAT1 promoted osteogenic differentiation and inhibited cell apoptosis through miR-485-5p/WNT7B axis, which suggested that MALAT1 is a potential target to alleviate osteoporosis.

The regulation of Rubisco activity

1986 ◽  
Vol 313 (1162) ◽  
pp. 337-346 ◽  
Keyword(s):  
Binding Sites ◽  
Large Subunit ◽  
Rubisco Activase ◽  
Activation Process ◽  
Ribulose Bisphosphate ◽  
Rubisco Activity ◽  
Dual Roles ◽  
Naturally Occurring

The processes of photosynthesis and photorespiration are initiated by Rubisco, but the enzyme must be activated before it will catalyse either the carboxylation or oxygenation of ribulose bisphosphate. Rubisco is activated in vitro by CO 2 and Mg 2+ . The dual roles of CO 2 as both an activator and a substrate led to anomalously high K m (CO 2 ) values until the activation requirement was recognized. During activation, CO 2 forms a carbamate at the ε-amino of a lysine residue on the large subunit of Rubisco. Under conditions thought to exist in the chloroplast during photosynthesis (10 μm CO 2 , 5 mM Mg 2+ and pH 8.0), Rubisco is only partially active since the K act (CO 2 ) is in the range 25-30 μm CO 2 . Thus the mechanism of activation as deduced from in vitro studies is incomplete. Higher activation levels can be obtained by preincubating Rubisco with phosphorylated metabolites, but these occupy ribulose bisphosphate binding sites and thus inhibit catalysis. Recently, a naturally occurring effector, which binds tightly to Rubisco and inhibits activity, has been found. This compound is synthesized in the dark and metabolized upon illumination, but its identity and physiological function are not yet known. In leaves, Rubisco is nearly fully activated at high light intensities. By analysing an Arabidopsis thaliana mutant deficient in the ability to activate Rubisco, we have determined that a soluble protein is required for the in vivo activation process. This enzyme, designated Rubisco activase, reduces the high K act (CO 2 ), observed with the isolated enzyme, to physiological levels in an illuminated reconstituted assay system containing washed thylakoid membranes, Rubisco and ribulose bisphosphate.

scholarly journals A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

2006 ◽  
Vol 17 (4) ◽  
pp. 1971-1984 ◽  
Author(s):  
Michael G. Clark ◽  
Joseph Teply ◽  
Brian K. Haarer ◽  
Susan C. Viggiano ◽  
David Sept ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Actin Filaments ◽  
Random Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Actin Binding ◽  
Interacting Protein ◽  
Actin Binding Site

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal β-propeller and a secondary actin binding site lies in a comparable location on its C-terminal β-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding- and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N- and C-terminal propeller domains.

scholarly journals Drebrin contains a cryptic F-actin–bundling activity regulated by Cdk5 phosphorylation

2013 ◽  
Vol 202 (5) ◽  
pp. 793-806 ◽  
Author(s):  
Daniel C. Worth ◽  
Catherine N. Daly ◽  
Sara Geraldo ◽  
Fazal Oozeer ◽  
Phillip R. Gordon-Weeks
Keyword(s):  
Growth Cone ◽  
Dendritic Spines ◽  
Binding Sites ◽  
Intramolecular Interaction ◽  
Mechanistic Model ◽  
Coiled Coil ◽  
Actin Binding ◽  
In Vitro Assays ◽  
Coiled Coil Domain

Drebrin is an actin filament (F-actin)–binding protein with crucial roles in neuritogenesis and synaptic plasticity. Drebrin couples dynamic microtubules to F-actin in growth cone filopodia via binding to the microtubule-binding +TIP protein EB3 and organizes F-actin in dendritic spines. Precisely how drebrin interacts with F-actin and how this is regulated is unknown. We used cellular and in vitro assays with a library of drebrin deletion constructs to map F-actin binding sites. We discovered two domains in the N-terminal half of drebrin—a coiled-coil domain and a helical domain—that independently bound to F-actin and cooperatively bundled F-actin. However, this activity was repressed by an intramolecular interaction relieved by Cdk5 phosphorylation of serine 142 located in the coiled-coil domain. Phospho-mimetic and phospho-dead mutants of serine 142 interfered with neuritogenesis and coupling of microtubules to F-actin in growth cone filopodia. These findings show that drebrin contains a cryptic F-actin–bundling activity regulated by phosphorylation and provide a mechanistic model for microtubule–F-actin coupling.

scholarly journals Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter Functions

2005 ◽  
Vol 16 (7) ◽  
pp. 3128-3139 ◽  
Author(s):  
Omar Quintero-Monzon ◽  
Avital A. Rodal ◽  
Boris Strokopytov ◽  
Steven C. Almo ◽  
Bruce L. Goode
Keyword(s):  
Crystal Structure ◽  
Point Mutations ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Cellular Functions ◽  
Multidomain Protein ◽  
Actin Binding Domain ◽  
And Function ◽  
Actin Binding Site

Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1–actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.

Cytoskeletal Bundle Mechanics

2007 ◽  
Author(s):  
Mark Bathe ◽  
Claus Heussinger ◽  
Mireille Claessens ◽  
Andreas Bausch ◽  
Erwin Frey
Keyword(s):  
Acoustic Waves ◽  
Actin Binding ◽  
Mechanical Resistance ◽  
Mechanical Forces ◽  
Mechanical Stimuli ◽  
Filamentous Actin ◽  
Cellular Functions ◽  
Mechanochemical Transduction ◽  
Cellular Locomotion

Filamentous actin (F-actin) is a stiff biopolymer that is tightly crosslinked in vivo by actin-binding proteins (ABPs) to form stiff bundles that form major constituents of a multitude of slender cytoskeletal processes including stereocilia, filopodia, microvilli, neurosensory bristles, cytoskeletal stress fibers, and the acrosomal process of sperm cells (Fig. 1). The mechanical properties of these cytoskeletal processes play key roles in a broad range of cellular functions — the bending stiffness of stereocilia mediates the mechanochemical transduction of mechanical stimuli such as acoustic waves to detect sound, the critical buckling load of filopodia and acrosomal processes determines their ability to withstand compressive mechanical forces generated during cellular locomotion and fertilization, and the entropic stretching stiffness of cytoskeletal bundles mediates cytoskeletal mechanical resistance to cellular deformation. Thus, a detailed understanding of F-actin bundle mechanics is fundamental to gaining a mechanistic understanding of cytoskeletal function.

scholarly journals Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism

2020 ◽  
Vol 6 (23) ◽  
pp. eaaz7651 ◽  
Author(s):  
Austin Zimmet ◽  
Trevor Van Eeuwen ◽  
Malgorzata Boczkowska ◽  
Grzegorz Rebowski ◽  
Kenji Murakami ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Motility ◽  
Binding Sites ◽  
Activation Mechanism ◽  
Actin Binding ◽  
Cross Linking ◽  
Specific Interactions ◽  
Related Protein ◽  
Actin Networks ◽  
Cryo Electron Microscopy

Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions, and contribution of each NPF to activation are unresolved. Here, we report the cryo–electron microscopy structure of recombinantly expressed human Arp2/3 complex with two WASP family NPFs bound and address the mechanism of activation. A cross-linking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex.

New actin mutants allow further characterization of the nucleotide binding cleft and drug binding sites

1999 ◽  
Vol 112 (9) ◽  
pp. 1325-1336
Author(s):  
L.D. Belmont ◽  
G.M. Patterson ◽  
D.G. Drubin
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Fluid Phase ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Drug Binding ◽  
Actin Binding ◽  
Nucleotide Exchange ◽  
Latrunculin A

We have generated 9 site-specific mutations in Saccharomyces cerevisiae actin. These mutants display a variety of phenotypes when expressed in vivo, including slow actin filament turnover, slow fluid-phase endocytosis, and defects in actin organization. Actin mutation D157E confers resistance to the actin-sequestering drug, latrunculin A. Latrunculin A inhibits nucleotide exchange on wild-type yeast actin but not on D157E actin, suggesting that this residue is part of the latrunculin A binding site. We have refined our earlier map of the phalloidin binding site on actin, demonstrating a requirement for residue G158 in addition to D179 and R177. The nine new actin mutants as well as a large collection of existing actin mutants were also used to identify the putative binding site of another actin binding drug, tolytoxin, on actin. The actin alleles that result in decreased sensitivity to this drug cluster at a site near the nucleotide-binding pocket. Actin purified from one of these mutants has a reduced affinity for tolytoxin. In addition, tolytoxin causes a 2.4-fold increase in the t1/2 of ATP exchange, further suggesting that this drug binds near the nucleotide-binding pocket of actin. We note that the binding sites for latrunculin A, phalloidin, and tolytoxin all map close to the actin nucleotide binding pocket.

Sign in / Sign up

Export Citation Format

Share Document