ABSTRACT
The CacyBP/SIP protein interacts with several targets, including actin. Since the majority of actin filaments are associated with tropomyosin, in this work we characterized binding of CacyBP/SIP to the actin-tropomyosin complex and examined the effects of CacyBP/SIP on actin filament functions. By using reconstituted filaments composed of actin and AEDANS-labeled tropomyosin, we observed that binding of CacyBP/SIP caused an increase in tropomyosin fluorescence intensity indicating the occurrence of conformational changes within the filament. We also found that CacyBP/SIP bound directly to tropomyosin and that these proteins did not compete with each other for binding to actin. Electron microscopy showed that in the absence of tropomyosin CacyBP/SIP destabilized actin filaments, but tropomyosin reversed this effect. Actin-activated myosin S1 ATPase activity assays, performed using a colorimetric method, indicated that CacyBP/SIP reduced ATPase activity and that the presence of tropomyosin enhanced this inhibitory effect. Thus, our results suggest that CacyBP/SIP, through its interaction with both actin and tropomyosin, regulates the organization and functional properties of the thin filament.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Calcium-Binding Proteins/metabolism , Myoblasts/metabolism , Myosin Subfragments/metabolism , Tropomyosin/metabolism , Animals , Blotting, Western , Calcium/metabolism , Cells, Cultured , Chickens , Chromatography, Affinity , Fluorescent Antibody Technique , Immunoenzyme Techniques , Intracellular Signaling Peptides and Proteins , Mice , Microscopy, Electron , Myoblasts/cytology , RatsABSTRACT
Myosin VI (MVI), the only known myosin that walks towards the minus end of actin filaments, is involved in several processes such as endocytosis, cell migration, and cytokinesis. It may act as a transporting motor or a protein engaged in actin cytoskeleton remodelling via its binding partners, interacting with its C-terminal globular tail domain. By means of pull-down technique and mass spectrometry, we identified Dock7 (dedicator of cytokinesis 7) as a potential novel MVI-binding partner in neurosecretory PC12 cells. Dock7, expressed mainly in neuronal cells, is a guanine nucleotide exchange factor (GEF) for small GTPases, Rac1 and Cdc42, which are the major regulators of actin cytoskeleton. MVI-Dock7 interaction was further confirmed by co-immunoprecipitation of endogenous MVI complexed with Dock7. In addition, MVI and Dock7 colocalized in interphase and dividing cells. We conclude that in PC12 cells MVI-Dock7 complexes may function at different cellular locations during the entire cell cycle. Of note, MVI and Dock7 colocalized in primary culture hippocampal neurons also, predominantly in the outgrowths. We hypothesize that this newly identified interaction between MVI and Dock7 may help explain a mechanism for MVI-dependent regulation of actin cytoskeleton organization.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Myosin Heavy Chains/metabolism , Neurons/metabolism , rho GTP-Binding Proteins/metabolism , Animals , Binding Sites , Immunoprecipitation , Microscopy, Confocal , Myosin Heavy Chains/genetics , PC12 Cells , Rats , Tandem Mass Spectrometry , rho GTP-Binding Proteins/geneticsABSTRACT
In previous studies we have demonstrated that prion protein (PrP) interacts with tubulin and disrupts microtubular cytoskeleton by inducing tubulin oligomerization. These observations may explain the molecular mechanism of toxicity of cytoplasmic PrP in transmissible spongiform encephalopathies (TSEs). Here, we check whether microtubule associated proteins (MAPs) that regulate microtubule stability, influence the PrP-induced oligomerization of tubulin. We show that tubulin preparations depleted of MAPs are more prone to oligomerization by PrP than those containing traces of MAPs. Tau protein, a major neuronal member of the MAPs family, reduces the effect of PrP. Importantly, phosphorylation of Tau abolishes its ability to affect the PrP-induced oligomerization of tubulin. We propose that the binding of Tau stabilizes tubulin in a conformation less susceptible to oligomerization by PrP. Since elevated phosphorylation of Tau leading to a loss of its function is observed in Alzheimer disease and related tauopathies, our results point at a possible molecular link between these neurodegenerative disorders and TSEs.
Subject(s)
Prions/pharmacology , Protein Multimerization/drug effects , Tubulin/metabolism , tau Proteins/pharmacology , Amino Acid Sequence , Animals , Cells, Cultured , Down-Regulation/drug effects , Humans , Microtubule-Associated Proteins/metabolism , Models, Biological , Protein Binding/drug effects , Protein Stability/drug effects , Swine , Tubulin Modulators/pharmacology , Up-Regulation/drug effects , tau Proteins/physiologyABSTRACT
Amebin [formerly termed as ApABP-FI; Sobczak et al. (2007) Biochem. Cell Biol. 85] is encoded in Amoeba proteus by two transcripts, 2672-nt and 1125-nt. A product of the shorter transcript (termed as C-amebin), comprising C-terminal 375 amino-acid-residue fragment of amebin, has been expressed and purified as the recombinant GST-fusion protein. GST-C-amebin bound both to monomeric and filamentous actin. The binding was Ca(2+)-independent and promoted filament bundling, as revealed with the transmission electron microscopy. GST-C-amebin significantly decreased MgATPase activity of rabbit skeletal muscle acto-S1. Removal with endoproteinase ArgC of a positively charged C-terminal region of GST-amebin containing KLASMWEQ sequence abolished actin-binding and bundling as well as the ATPase-inhibitory effect of C-amebin, indicating that this protein region was involved in the interaction with actin. Microinjection of amoebae with antibody against C-terminus of amebin significantly affected amoebae morphology, disturbed cell polarization and transport of cytoplasmic granules as well as blocked migration. These data indicate that amebin may be one of key regulators of the actin-cytoskeleton dynamics and actin-dependent motility in A. proteus.
Subject(s)
Actin Cytoskeleton/metabolism , Amoeba/chemistry , Amoeba/physiology , Myosins/antagonists & inhibitors , Protozoan Proteins/chemistry , Protozoan Proteins/physiology , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/ultrastructure , Amino Acid Sequence , Amoeba/genetics , Animals , In Vitro Techniques , Microscopy, Electron, Transmission , Molecular Sequence Data , Movement/physiology , Multiprotein Complexes , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/physiology , Peptide Fragments/ultrastructure , Protein Binding , Protozoan Proteins/genetics , Protozoan Proteins/ultrastructure , Rabbits , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/ultrastructureABSTRACT
CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1-179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178-229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.
