scholarly journals Are the conserved sequences in segment 1 of gelsolin important for binding actin?

1992 ◽  
Vol 116 (5) ◽  
pp. 1135-1143 ◽  
Author(s):  
M Way ◽  
B Pope ◽  
A G Weeds
Keyword(s):  
Direct Interaction ◽  
Actin Binding ◽  
Amino Acid Substitutions ◽  
Interaction Site ◽  
Conserved Sequences ◽  
Functional Sites ◽  
Deletion Mutagenesis ◽  
Polar Groups ◽  
Charged Residues ◽  
Minimal Region

The minimal region required for actin binding in the smallest of the three domains of gelsolin (termed Segment 1 or S1) was previously defined by deletion mutagenesis as residues 37-126. Further analysis of NH2-terminal deletions here redefines the minimal functional core as residues 41-126. Amino acid substitutions within this core further elucidate the nature of the interaction of segment 1 with actin. Of 26 point mutants analyzed, 14 reduced the affinity for actin. The charged residues His 119, Arg 120, Glu 121, and Gln 123 appear to be involved in direct interaction with actin. Substitutions of Leu 108, Leu 112, and Val 117 by polar groups all affect the structural stability of segment 1 and thereby reduce binding affinity. In addition replacement of Glu 126 by aspartic acid modifies the physical properties of segment 1 and weakens binding. We have further shown that changing charged residues within the highly conserved pentapeptide sequence LDDYL (residues 108-112) has no effect on actin binding. This sequence, found in a number of different actin binding proteins, does not therefore constitute part of the interaction site. Similarly, substitution of the two acidic residues by basic ones within the DESG motif of segment 1 (residues 96-99, but also found near the COOH terminus of actin) does not impair binding. These results show the dangers of predicting functional sites on the basis of conserved sequences.

Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2413-2419 ◽  
Author(s):  
José L. Alonso-Lebrero ◽  
Juan M. Serrador ◽  
Carmen Domı́nguez-Jiménez ◽  
Olga Barreiro ◽  
Alfonso Luque ◽  
...  
Keyword(s):  
Adhesion Molecules ◽  
Adhesion Molecule ◽  
Direct Interaction ◽  
Intercellular Adhesion ◽  
Actin Binding ◽  
Intercellular Adhesion Molecule ◽  
Human Neutrophils ◽  
Binding Assays ◽  
Glutathione S Transferase ◽  
Amino Terminal

Abstract In response to the chemoattractants interleukin 8, C5a,N-formyl-methionyl-leucyl-phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1), intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are redistributed to a newly formed uropod in human neutrophils. The adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the cytoskeletal protein moesin in the uropod of stimulated neutrophils. Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by isolating a complex with glutathione S-transferase fusions of the cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as moesin and ezrin by Western blot analysis. ICAM-3 and moesin also coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity assay. Direct interaction of the cytoplasmic domains of ICAM-3 and PSGL-1 with the amino-terminal domain of recombinant moesin was demonstrated by protein-protein binding assays. These results suggest that the redistribution of PSGL-1 and its association with intracellular molecules, including the ezrin-radixin-moesin actin-binding proteins, regulate functions mediated by PSGL-1 in leukocytes stimulated by chemoattractants.

scholarly journals Concerted Effects of Amino Acid Substitutions in Conserved Charged Residues and Other Residues in the Cytoplasmic Domain of PomA, a Stator Component of Na+-Driven Flagella

2004 ◽  
Vol 186 (20) ◽  
pp. 6749-6758 ◽  
Author(s):  
Hajime Fukuoka ◽  
Toshiharu Yakushi ◽  
Michio Homma
Keyword(s):  
Amino Acid ◽  
Temperature Shift ◽  
Terminal Region ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Flagellar Motor ◽  
Transmembrane Segments ◽  
Suppressor Mutations ◽  
Essential Components ◽  
Charged Residues

ABSTRACT PomA is a membrane protein that is one of the essential components of the sodium-driven flagellar motor in Vibrio species. The cytoplasmic charged residues of Escherichia coli MotA, which is a PomA homolog, are believed to be required for the interaction of MotA with the C-terminal region of FliG. It was previously shown that a PomA variant with neutral substitutions in the conserved charged residues (R88A, K89A, E96Q, E97Q, and E99Q; AAQQQ) was functional. In the present study, five other conserved charged residues were replaced with neutral amino acids in the AAQQQ PomA protein. These additional substitutions did not affect the function of PomA. However, strains expressing the AAQQQ PomA variant with either an L131F or a T132M substitution, neither of which affected motor function alone, exhibited a temperature-sensitive (TS) motility phenotype. The double substitutions R88A or E96Q together with L131F were sufficient for the TS phenotype. The motility of the PomA TS mutants immediately ceased upon a temperature shift from 20 to 42°C and was restored to the original level approximately 10 min after the temperature was returned to 20°C. It is believed that PomA forms a channel complex with PomB. The complex formation of TS PomA and PomB did not seem to be affected by temperature. Suppressor mutations of the TS phenotype were mapped in the cytoplasmic boundaries of the transmembrane segments of PomA. We suggest that the cytoplasmic surface of PomA is changed by the amino acid substitutions and that the interaction of this surface with the FliG C-terminal region is temperature sensitive.

scholarly journals Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

2008 ◽  
Vol 19 (5) ◽  
pp. 1837-1847 ◽  
Author(s):  
Christopher T. Pappas ◽  
Nandini Bhattacharya ◽  
John A. Cooper ◽  
Carol C. Gregorio
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Actin Polymerization ◽  
Striated Muscle ◽  
Solid Phase ◽  
Molecular Model ◽  
Direct Interaction ◽  
Tryptophan Fluorescence ◽  
Actin Binding

The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant “molecular ruler” nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160–164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160–164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.

scholarly journals Characterization of an F-actin-binding domain in the cytoskeletal protein vinculin.

1994 ◽  
Vol 126 (5) ◽  
pp. 1231-1240 ◽  
Author(s):  
A R Menkel ◽  
M Kroemker ◽  
P Bubeck ◽  
M Ronsiek ◽  
G Nikolai ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Direct Interaction ◽  
Binding Domain ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Permeabilized Cells ◽  
Cell Matrix ◽  
Actin Binding Domain ◽  
Major Structural Component

Vinculin, a major structural component of vertebrate cell-cell and cell-matrix adherens junctions, has been found to interact with several other junctional components. In this report, we have identified and characterized a binding site for filamentous actin. These results included studies with gizzard vinculin, its proteolytic head and tail fragments, and recombinant proteins containing various gizzard vinculin sequences fused to the maltose binding protein (MBP) of Escherichia coli. In cosedimentation assays, only the vinculin tail sequence mediated a direct interaction with actin filaments. The binding was saturable, with a dissociation constant value in the micromolar range. Experiments with deletion clones localized the actin-binding domain to a region confined by residues 893-1016 in the 170-residue-long carboxyterminal segment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction. In fixed and permeabilized cells (cell models), as well as after microinjection, proteins containing the actin-binding domain specifically decorated stress fibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli. These results corroborated the sedimentation experiments. Our data support and extend previous work showing that vinculin binds directly to actin filaments. They are consistent with a model suggesting that in adhesive cells, the NH2-terminal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament ends into the characteristic spear tip-shaped structures.

scholarly journals Identification of membrane proteins mediating the interaction of human platelets

1980 ◽  
Vol 86 (1) ◽  
pp. 77-86 ◽  
Author(s):  
D Phillips ◽  
L Jennings ◽  
H Edwards
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Membrane Surface ◽  
Direct Interaction ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Insoluble Residue ◽  
Membrane Glycoproteins ◽  
Actin Binding Protein ◽  
Activated Platelets

Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.

Kindlin-2 Maintains Vascular Barrier Integrity By Stabilizing Endothelial Adherens Junctions

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3452-3452
Author(s):  
Elzbieta Pluskota ◽  
Dorota Szpak ◽  
Katarzyna Bialkowska ◽  
Kamila Bledzka ◽  
Edward F. Plow
Keyword(s):  
Actin Filaments ◽  
Conflicts Of Interest ◽  
Umbilical Vein ◽  
Adherens Junctions ◽  
Direct Interaction ◽  
Fold Increase ◽  
Actin Binding ◽  
Binding Studies ◽  
Vascular Integrity

Abstract Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, angiogenesis, thrombosis and hemostasis. Its absence is embryonically lethal in mice, emphasizing its biological importance. In the present study, we examined the role of Kindlin-2 in regulation of vascular integrity. In vivo, Kindlin-2+/- mice showed enhanced (70%) permeability of tracheal vasculature to fluorescent beads as compared to wild-type (WT) littermates under both basal conditions or when stimulated with Platelet Activating Factor (PAF). Consistent with these in vivo observations, confluent monolayers of aortic endothelial cells (ECs) isolated from Kindlin-2+/- mice had increased (2-3-fold) baseline, PAF- and thrombin-induced permeability for Alexa-488-labeled bovine serum albumin (BSA) or FITC-dextran-10000 as compared to WT cells. Also, reduction of Kindlin-2 expression by 60-70% in human umbilical vein endothelial cell (HUVEC) monolayers with Kindlin-2-specific siRNA resulted in 3-fold increase in their baseline or thrombin/(PAF)-induced permeability of these markers. Mechanistically, Kindlin-2 co-localized with VE-cadherin and actin within adherens junctions in resting, confluent HUVEC monolayers as well as co-immunoprecipitated with these proteins and other components of adherens junctions, including α-, β- and γ-catenin. In contrast, Kindlin-2 did not co-localize or co-immunoprecipitate with any components of GAP or tight junctions. VE-cadherin-based complexes had less associated Kindlin-2 and actin in Kindlin-2+/- ECs and in HUVECs treated with Kindlin-2-specifc siRNA. Also, upon stimulation of HUVECs with PAF or thrombin, Kindlin-2 along with actin dissociated from VE-cadherin-based complexes, suggesting that Kindlin-2 stabilizes adherens junctions. In direct binding studies with recombinant proteins the SPR sensograms revealed direct interaction of Kindlin-2 with β- and γ-catenin, but not with α-catenin nor the VE-cadherin cytoplasmic tail. In addition, using actin spin-down assays we demonstrated that Kindlin-2 directly interacted with actin filaments and linked them to β- or γ-catenin. Using WT and deletion mutants of Kindlin-2 we mapped the β- and γ-catenin binding site to the F1 and F3 domains in Kindlin-2, while actin binding is very dependent on the F0 domain. Taken together, these data identify a previously unappreciated function of Kindlin-2. It plays a crucial role in maintaining vascular integrity by linking adherens junctions to actin filaments. Disclosures No relevant conflicts of interest to declare.

scholarly journals Phosphorylation of Profilin by ROCK1 Regulates Polyglutamine Aggregation

2008 ◽  
Vol 28 (17) ◽  
pp. 5196-5208 ◽  
Author(s):  
Jieya Shao ◽  
William J. Welch ◽  
Nicholas A. DiProspero ◽  
Marc I. Diamond
Keyword(s):  
Direct Interaction ◽  
Inhibitory Effect ◽  
Binding Activity ◽  
Hek293 Cells ◽  
Actin Binding ◽  
Inhibitory Effects ◽  
Binding Factor ◽  
Direct Target ◽  
Polyglutamine Aggregation ◽  
Polyglutamine Protein

ABSTRACT Y-27632, an inhibitor of the Rho-associated kinase ROCK, is a therapeutic lead for Huntington disease (HD). The downstream targets that mediate its inhibitory effects on huntingtin (Htt) aggregation and toxicity are unknown. We have identified profilin, a small actin-binding factor that also interacts with Htt, as being a direct target of the ROCK1 isoform. The overexpression of profilin reduces the aggregation of polyglutamine-expanded Htt and androgen receptor (AR) peptides. This requires profilin's G-actin binding activity and its direct interaction with Htt, which are both inhibited by the ROCK1-mediated phosphorylation of profilin at Ser-137. Y-27632 blocks the phosphorylation of profilin in HEK293 cells and primary neurons, which maintains profilin in an active state. The knockdown of profilin blocks the inhibitory effect of Y-27632 on both AR and Htt aggregation. A signaling pathway from ROCK1 to profilin thus controls polyglutamine protein aggregation and is targeted by a promising therapeutic lead for HD.

scholarly journals Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.

1996 ◽  
Vol 7 (2) ◽  
pp. 261-272 ◽  
Author(s):  
U Gottwald ◽  
R Brokamp ◽  
I Karakesisoglou ◽  
M Schleicher ◽  
A A Noegel
Keyword(s):  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
Dictyostelium Discoideum ◽  
Direct Interaction ◽  
Actin Binding ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal

In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner.

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