scholarly journals Kindlin-2 directly binds actin and regulates integrin outside-in signaling

2016 ◽  
Vol 213 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Kamila Bledzka ◽  
Katarzyna Bialkowska ◽  
Khalid Sossey-Alaoui ◽  
Julia Vaynberg ◽  
Elzbieta Pluskota ◽  
...  
Keyword(s):  
Cell Spreading ◽  
Cellular Responses ◽  
Cell System ◽  
Actin Binding ◽  
Wild Type ◽  
Cho Cell ◽  
Actin Organization ◽  
Support Cell ◽  
Direct Binding ◽  
Actin Binding Site

Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2+/− mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK47/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK47/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK47/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses.

scholarly journals Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation

2002 ◽  
Vol 156 (4) ◽  
pp. 703-713 ◽  
Author(s):  
Reed J. Kelso ◽  
Andrew M. Hudson ◽  
Lynn Cooley
Keyword(s):  
Site Directed Mutagenesis ◽  
Actin Binding ◽  
Cross Linking ◽  
Ring Canal ◽  
Major Function ◽  
Actin Organization ◽  
Ring Canals ◽  
Dependent Pathway ◽  
Dimensional Electrophoresis ◽  
Actin Binding Site

The Drosophila kelch gene encodes a member of a protein superfamily defined by the presence of kelch repeats. In Drosophila, Kelch is required to maintain actin organization in ovarian ring canals. We set out to study the actin cross-linking activity of Kelch and how Kelch function is regulated. Biochemical studies using purified, recombinant Kelch protein showed that full-length Kelch bundles actin filaments, and kelch repeat 5 contains the actin binding site. Two-dimensional electrophoresis demonstrated that Kelch is tyrosine phosphorylated in a src64-dependent pathway. Site-directed mutagenesis determined that tyrosine residue 627 is phosphorylated. A Kelch mutant with tyrosine 627 changed to alanine (KelY627A) rescued the actin disorganization phenotype of kelch mutant ring canals, but failed to produce wild-type ring canals. Electron microscopy demonstrated that phosphorylation of Kelch is critical for the proper morphogenesis of actin during ring canal growth, and presence of the nonphosphorylatable KelY627A protein phenocopied src64 ring canals. KelY627A protein in ring canals also dramatically reduced the rate of actin monomer exchange. The phenotypes caused by src64 mutants and KelY627A expression suggest that a major function of Src64 signaling in the ring canal is the negative regulation of actin cross-linking by Kelch.

scholarly journals A Role for Tissue Factor in Cell Adhesion and Migration Mediated by Interaction with Actin-binding Protein 280

1998 ◽  
Vol 140 (5) ◽  
pp. 1241-1253 ◽  
Author(s):  
Ilka Ott ◽  
Edgar G. Fischer ◽  
Yohei Miyagi ◽  
Barbara M. Mueller ◽  
Wolfram Ruf
Keyword(s):  
Cell Adhesion ◽  
Tumor Cell ◽  
Tissue Factor ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Actin Binding ◽  
Support Cell ◽  
Cell Metastasis ◽  
Tumor Cell Metastasis ◽  
And Migration

Tissue factor (TF), the protease receptor initiating the coagulation system, functions in vascular development, angiogenesis, and tumor cell metastasis by poorly defined molecular mechanisms. We demonstrate that immobilized ligands for TF specifically support cell adhesion, migration, spreading, and intracellular signaling, which are not inhibited by RGD peptides. Two-hybrid screening identified actin-binding protein 280 (ABP-280) as ligand for the TF cytoplasmic domain. Extracellular ligation of TF is necessary for ABP-280 binding. ABP-280 recruitment to TF adhesion contacts is associated with reorganization of actin filaments, but cytoskeletal adaptor molecules typically found in integrin-mediated focal contacts are not associated with TF. Chimeric molecules of the TF cytoplasmic domain and an unrelated extracellular domain support cell spreading and migration, demonstrating that the extracellular domain of TF is not involved in the recruitment of accessory molecules that influence adhesive functions. Replacement of TF's cytoplasmic Ser residues with Asp to mimic phosphorylation enhances the interaction with ABP-280, whereas Ala mutations abolish coprecipitation of ABP-280 with immobilized TF cytoplasmic domain, and severely reduce cell spreading. The specific interaction of the TF cytoplasmic domain with ABP-280 provides a molecular pathway by which TF supports tumor cell metastasis and vascular remodeling.

Integrin signaling: roles for the cytoplasmic tails of alpha IIb beta 3 in the tyrosine phosphorylation of pp125FAK

1995 ◽  
Vol 108 (12) ◽  
pp. 3817-3825 ◽  
Author(s):  
L. Leong ◽  
P.E. Hughes ◽  
M.A. Schwartz ◽  
M.H. Ginsberg ◽  
S.J. Shattil
Keyword(s):  
Protein Tyrosine Kinase ◽  
Cytoplasmic Tail ◽  
Cell Spreading ◽  
Cellular Responses ◽  
Distal Portion ◽  
Integrin Signaling ◽  
Adherent Cells ◽  
Wild Type ◽  
Truncation Mutant ◽  
Cytoplasmic Tails

pp125FAK (focal adhesion kinase) a protein tyrosine kinase that may mediate cellular responses to adhesion, is activated and tyrosine-phosphorylated when platelets adhere to fibrinogen via the integrin, alpha IIb beta 3. To determine whether either of the cytoplasmic tails of alpha IIb beta 3 regulates FAK phosphorylation, CHO cells were stably transfected with alpha IIb beta 3 or various cytoplasmic tail truncation mutants. Cells expressing wild-type alpha IIb beta 3 or alpha IIb beta 3 that lacked the COOH-terminal 13 or 18 residues of the 20 residue alpha IIb tail adhered to and spread on fibrinogen or on an anti-alpha IIb antibody, and FAK became tyrosine-phosphorylated. FAK also became phosphorylated in adherent cells lacking the COOH-terminal 35 or 39 residues of the 47 residue beta 3 tail, although the extent of phosphorylation was reduced by about 50% in the latter mutant. Little or no FAK phosphorylation was observed if 46 residues were deleted from the beta 3 tail. None of these beta 3 truncation mutants spread on the anti-alpha IIb antibody. When cells with wild-type alpha IIb beta 3 or truncated beta 3 were detached from a surface, FAK became rapidly dephosphorylated. In contrast, FAK remained phosphorylated in the two alpha IIb truncation mutants for up to 90 minutes in suspension. This persistent phosphorylation was not due to occupancy of alpha IIb beta 3 by adhesive ligands because it was also observed with an alpha IIb tail truncation mutant that contained an additional mutation in the extracellular portion of the receptor that prevents ligand binding. These studies demonstrate that: (1) the beta 3 cytoplasmic tail, including the membrane-proximal portion, is involved in initiation of FAK phosphorylation; (2) FAK phosphorylation can be initiated by cell adhesion in the absence of cell spreading; and (3) the membrane-distal portion of the alpha IIb cytoplasmic tail may normally function to dampen FAK phosphorylation in non-anchored cells.

scholarly journals Integration of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) into Cellular Structures

Antioxidants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1082
Author(s):  
Constanze Erdmann ◽  
Roua Hassoun ◽  
Sebastian Schmitt ◽  
Carlos Kikuti ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Mdck Cells ◽  
Cell System ◽  
Neonatal Rat ◽  
Actin Binding ◽  
Thin Filaments ◽  
Rat Cardiomyocytes ◽  
Actin Organization ◽  
Adaptive Processes ◽  
Microfilament System

The human mutant cardiac α-actins p.A295S or p.R312H and p.E361G, correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by the baculovirus/Sf21 insect cell system and purified to homogeneity. The purified cardiac actins maintained their native state but showed differences in Ca2+-sensitivity to stimulate the myosin-subfragment1 ATPase. Here we analyzed the interactions of these c-actins with actin-binding and -modifying proteins implicated in cardiomyocyte differentiation. We demonstrate that Arp2/3 complex and the formin mDia3 stimulated the polymerization rate and extent of the c-actins, albeit to different degrees. In addition, we tested the effect of the MICAL-1 monooxygenase, which modifies the supramolecular actin organization during development and adaptive processes. MICAL-1 oxidized these c-actin variants and induced their de-polymerization, albeit at different rates. Transfection experiments using MDCK cells demonstrated the preferable incorporation of wild type and p.A295S c-actins into their microfilament system but of p.R312H and p.E361G actins into the submembranous actin network. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding HA-tagged c-actin variants showed their incorporation into microfilaments after one day in culture and thereafter into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferentially incorporated at the minus ends.

scholarly journals Evidence that a 27-residue sequence is the actin-binding site of ABP-120

1991 ◽  
Vol 266 (20) ◽  
pp. 12989-12993
Author(s):  
A.R. Bresnick ◽  
P.A. Janmey ◽  
J. Condeelis
Keyword(s):  
Binding Site ◽  
Actin Binding ◽  
Actin Binding Site

scholarly journals Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin

1991 ◽  
Vol 266 (16) ◽  
pp. 10485-10489
Author(s):  
N. Yonezawa ◽  
E. Nishida ◽  
K. Iida ◽  
H. Kumagai ◽  
I. Yahara ◽  
...  
Keyword(s):  
Binding Site ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Actin Binding Site

scholarly journals Villidin, a Novel WD-repeat and Villin-related Protein fromDictyostelium,Is Associated with Membranes and the Cytoskeleton

2003 ◽  
Vol 14 (7) ◽  
pp. 2716-2727 ◽  
Author(s):  
Annika Gloss ◽  
Francisco Rivero ◽  
Nandkumar Khaire ◽  
Rolf Müller ◽  
William F. Loomis ◽  
...  
Keyword(s):  
Actin Binding ◽  
Similar Distribution ◽  
Fruiting Bodies ◽  
Targeted Deletion ◽  
C Terminus ◽  
Its Gene ◽  
Mutant Strains ◽  
Protein Functions ◽  
Wd Repeats ◽  
Actin Binding Site

Villidin is a novel multidomain protein (190 kDa) from Dictyostelium amoebae containing WD repeats at its N-terminus, three PH domains in the middle of the molecule, and five gelsolin-like segments at the C-terminus, followed by a villin-like headpiece. Villidin mRNA and protein are present in low amounts during growth and early aggregation, but increase during development and reach their highest levels at the tipped mound stage. The protein is present in the cytosol as well as in the cytoskeletal and membrane fractions. GFP-tagged full-length villidin exhibits a similar distribution as native villidin, including a distinct colocalization with Golgi structures. Interestingly, GFP fusions with the gelsolin/villin-like region are uniformly dispersed in the cytoplasm, whereas GFP fusions of the N-terminal WD repeats codistribute with F-actin and are associated with the Triton-insoluble cytoskeleton. Strains lacking villidin because of targeted deletion of its gene grow normally and can develop into fruiting bodies. However, cell motility is reduced during aggregation and phototaxis is impaired in the mutant strains. We conclude that villidin harbors a major F-actin binding site in the N-terminal domain and not in the villin-like region as expected; association of villidin with vesicular membranes suggests that the protein functions as a linker between membranes and the actin cytoskeleton.

Sign in / Sign up

Export Citation Format

Share Document