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The Journal of Biological Chemistry Jul 2004The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2...
The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2 (eIF2alpha) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn- phenotype) by suppressing eIF2alpha phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn- phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2alpha phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn- phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin.
Topics: Actins; Alleles; Amino Acids; Arginine; DNA-Binding Proteins; Eukaryotic Initiation Factor-2; Galactose; Gene Deletion; Genotype; Glutathione Transferase; Mass Spectrometry; Microfilament Proteins; Peptide Elongation Factors; Phenotype; Phosphorylation; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Protein Binding; Protein Kinases; Protein Serine-Threonine Kinases; RNA, Transfer; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcriptional Activation
PubMed: 15126500
DOI: 10.1074/jbc.M404009200 -
The Journal of Cell Biology May 1983Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and...
Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.
Topics: Actins; Animals; Carrier Proteins; Cytoskeleton; Gelsolin; Microcomputers; Microfilament Proteins; Microscopy, Electron; Models, Structural; Muscles; Rabbits
PubMed: 6682423
DOI: 10.1083/jcb.96.5.1400 -
Biochimica Et Biophysica Acta Feb 2010Formins represent a major branch of actin nucleators along with the Arp2/3 complex, Spire and Cordon-bleu. Formin-mediated actin nucleation requires the formin homology... (Review)
Review
Formins represent a major branch of actin nucleators along with the Arp2/3 complex, Spire and Cordon-bleu. Formin-mediated actin nucleation requires the formin homology 2 domain and, although the nucleation per se does not require additional factors, formin-binding proteins have been shown to be essential for the regulation of formin-dependent actin assembly in vivo. This regulation could be accomplished by formin-binding proteins being directly involved in formin-driven actin nucleation, by formin-binding proteins influencing the activated state of the formins, by linking formin-driven actin polymerization to Arp2/3 driven actin polymerization, or by influencing the subcellular localization of the formins. This review article will focus on mammalian formin-binding proteins and their roles during vital cellular processes, such as cell migration, cell division and intracellular trafficking.
Topics: Actin-Related Protein 2-3 Complex; Actins; Animals; Carrier Proteins; Cell Polarity; Fatty Acid-Binding Proteins; Fetal Proteins; Formins; Humans; Microfilament Proteins; Nuclear Proteins; Profilins; Signal Transduction; src-Family Kinases
PubMed: 19589360
DOI: 10.1016/j.bbamcr.2009.06.002 -
The Journal of Biological Chemistry Jan 1997SEK-1, a dual specificity protein kinase that serves as one of the immediate upstream activators of the stress-activated protein kinases (SAPKs), associates specifically...
Actin-binding protein-280 binds the stress-activated protein kinase (SAPK) activator SEK-1 and is required for tumor necrosis factor-alpha activation of SAPK in melanoma cells.
SEK-1, a dual specificity protein kinase that serves as one of the immediate upstream activators of the stress-activated protein kinases (SAPKs), associates specifically with the actin-binding protein, ABP-280, in vitro and in situ. SEK-1 binds to the carboxyl-terminal rod segment of ABP-280, upstream of the ABP carboxyl-terminal dimerization domain. Activation of SEK-1 in situ increases the SEK-1 activity bound to ABP-280 without changing the amount of SEK-1 polypeptide bound. The influence of ABP-280 on SAPK regulation was evaluated in human melanoma cells that lack ABP-280 expression, and in stable transformants of these cells expressing wild type ABP, or an actin-binding but dimerization-deficient mutant ABP (ABPDeltaCT109). ABP-280-deficient cells show an activation of SAPK in response to most stimuli that is comparable to that seen in ABP-280-replete cells; ABP-280-deficient cells, however, fail to show the brisk tumor necrosis factor-alpha (TNF-alpha) activation of SAPK seen in ABP-replete cells and have an 80% reduction in SAPK activation by lysophosphatidic acid. Expression of the dimerization-deficient mutant ABP-280 fails to correct the defective SAPK response to lysophosphatidic acid, but essentially normalizes the TNF-alpha activation of SAPK. Thus, a lack of ABP-280 in melanoma cells causes a defect in the regulation of SAPK that is selective for TNF-alpha and is attributable to the lack of ABP-280 polypeptide itself rather than to the disordered actin cytoskeleton that results therefrom. ABP-280 participates in TNF-alpha signal transduction to SAPKs, in part through the binding of SEK-1.
Topics: Actins; Animals; Anisomycin; B-Lymphocytes; Cell Line; Contractile Proteins; Dimerization; Enzyme Activation; Epidermal Growth Factor; Filamins; Glutathione Transferase; Humans; MAP Kinase Kinase 4; Melanoma; Mice; Microfilament Proteins; Mitogen-Activated Protein Kinase Kinases; Mutagenesis, Site-Directed; Phosphorylation; Protein Kinases; Recombinant Fusion Proteins; T-Lymphocytes; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha
PubMed: 9006895
DOI: 10.1074/jbc.272.5.2620 -
RNA (New York, N.Y.) Jun 2011Transfer RNAs contain various modified nucleotides that are introduced enzymatically at the post-transcriptional level. In Saccharomyces cerevisiae, 3-methylcytidine...
Transfer RNAs contain various modified nucleotides that are introduced enzymatically at the post-transcriptional level. In Saccharomyces cerevisiae, 3-methylcytidine (m³C) is found at position 32 of the tRNAs for Thr and Ser. We used a systematic reverse genetic approach combined with mass spectrometry (ribonucleome analysis), and identified the actin-binding protein ABP140 as the protein responsible for m³C formation in both tRNA(Thr1) and tRNA(Ser1). ABP140 consists of an N-terminal actin-binding sequence and a C-terminal S-adenosylmethionine (Ado-Met) binding motif. Deletion of the actin-binding sequence in ABP140 did not affect m³C formation, indicating that subcellular localization of ABP140 to actin filaments is not involved in tRNA modification. m³C formation in tRNA(Thr1) could be reconstituted using recombinant Abp140p in the presence of Ado-Met, whereas m³C did not form in tRNA(Ser1) in vitro, indicating the absence of a factor(s) required for tRNA(Ser1) m³C formation. Thus, ABP140 has been designated TRM140 according to the preferred nomenclature. In addition, we observed a specific reduction of m³C formation in HeLa cells by siRNA-mediated knock down of the human ortholog of TRM140.
Topics: Actins; Base Sequence; Binding Sites; Cytidine; HeLa Cells; Humans; Microfilament Proteins; Molecular Sequence Data; RNA, Transfer; S-Adenosylmethionine; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; tRNA Methyltransferases
PubMed: 21518805
DOI: 10.1261/rna.2653411 -
FEBS Letters Jan 2011Cell adhesion, spreading and migration on extracellular matrices are regulated by complex processes that involve the cytoskeleton and a large array of adhesion... (Review)
Review
Cell adhesion, spreading and migration on extracellular matrices are regulated by complex processes that involve the cytoskeleton and a large array of adhesion receptors, including the β1 integrin. Filamin A is a large, multi-domain, homodimeric actin binding protein that contributes to the mechanical stability of cells and interacts with several proteins that regulate cell adhesion including β1 integrin and several protein kinases. Here we review current data on the structure, mechanical properties and intracellular signaling functions of filamin that regulate cell adhesion. We also consider new data showing that interactions of filamin A with intermediate filaments and protein kinase C enable tight regulation of β1 integrin function and consequently early events in cell adhesion and migration on extracellular matrix proteins.
Topics: Cell Adhesion; Cell Movement; Contractile Proteins; Cytoskeletal Proteins; Filamins; Humans; Integrin beta1; Microfilament Proteins; Models, Biological; Protein Binding; Protein Kinase C; Signal Transduction
PubMed: 21095189
DOI: 10.1016/j.febslet.2010.11.033 -
Molecules (Basel, Switzerland) Oct 2020The ( and ) gene encodes multiple proteins, which together play crucial roles in modulating the assembly of the actin cytoskeleton. Splicing of the gene is complex,... (Review)
Review
The ( and ) gene encodes multiple proteins, which together play crucial roles in modulating the assembly of the actin cytoskeleton. Splicing of the gene is complex, with the two most studied TRIOBP protein isoforms sharing no overlapping amino acid sequence with each other. TRIOBP-1 (also known as TARA or TAP68) is a mainly structured protein that is ubiquitously expressed and binds to F-actin, preventing its depolymerization. It has been shown to be important for many processes including in the cell cycle, adhesion junctions, and neuronal differentiation. TRIOBP-1 has been implicated in schizophrenia through the formation of protein aggregates in the brain. In contrast, TRIOBP-4 is an entirely disordered protein with a highly specialized expression pattern. It is known to be crucial for the bundling of actin in the stereocilia of the inner ear, with mutations in it causing severe or profound hearing loss. Both of these isoforms are implicated in cancer. Additional longer isoforms of TRIOBP exist, which overlap with both TRIOBP-1 and 4. These appear to participate in the functions of both shorter isoforms, while also possessing unique functions in the inner ear. In this review, the structures and functions of all of these isoforms are discussed, with a view to understanding how they operate, both alone and in combination, to modulate actin and their consequences for human illness.
Topics: Actins; Amino Acid Sequence; Animals; Deafness; Humans; Mental Disorders; Microfilament Proteins; Neoplasms; Protein Isoforms; Protein Stability
PubMed: 33121024
DOI: 10.3390/molecules25214967 -
Structural plasticity of functional actin: pictures of actin binding protein and polymer interfaces.Structure (London, England : 1993) Oct 2003Actin is one of the most conserved and versatile proteins capable of forming homopolymers and interacting with numerous other proteins in the cell. We performed an...
Actin is one of the most conserved and versatile proteins capable of forming homopolymers and interacting with numerous other proteins in the cell. We performed an alanine mutagenesis scan covering the entire beta-actin molecule. Somewhat surprisingly, the majority of the mutants were capable of reaching a stable conformation. We tested the ability of these mutants to bind to various actin binding proteins, thereby mapping different interfaces with actin. Additionally, we tested their ability to copolymerize with alpha-actin in order to localize regions in actin that contact neighboring protomers in the filament. Hereby, we could discriminate between two existing models for filamentous actin and our data strongly support the right-handed double-stranded helix model. We present data corroborating this model in vivo. Mutants defective in copolymerization do not colocalize with the actin cytoskeleton and some impair its normal function, thereby disturbing cell shape.
Topics: Actins; Cell Cycle Proteins; Cytoskeletal Proteins; Microfilament Proteins; Mutation; Protein Structure, Tertiary; Thymosin
PubMed: 14527395
DOI: 10.1016/j.str.2003.09.002 -
European Journal of Cell Biology Apr 2022Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for...
Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constant of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-poly-L-proline (PLP) interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.
Topics: Actin Cytoskeleton; Actins; Amyotrophic Lateral Sclerosis; Formins; Humans; Microfilament Proteins; Profilins
PubMed: 35248815
DOI: 10.1016/j.ejcb.2022.151212 -
The Journal of Biological Chemistry Jan 1986Actophorin is a new actin-binding protein from Acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. The isoelectric point is...
Actophorin is a new actin-binding protein from Acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. The isoelectric point is 6.1, and amino acid analysis shows an excess of acidic residues over basic residues. The phosphate content is less than 0.2 mol/mol. There is 0.4 +/- 0.1 mg of actophorin/g of cells, so that the molar ratio of actin to actophorin is about 10:1 in the cell. Unique two-dimensional maps of tryptic and chymotryptic peptides and complete absence of antibody cross-reactivity show that Acanthamoeba actophorin, profilin, capping protein, and actin are separate gene products with minimal homology. Actophorin has features of both an actin monomer-binding protein and an actin filament-severing protein. Actophorin reduces the extent of actin polymerization at steady state in a concentration-dependent fashion and forms a complex with pyrene-labeled actin that has spectral properties of unpolymerized actin. During ultracentrifugation a complex of actophorin and actin sediments more rapidly than either actin monomers or actophorin. Although actophorin inhibits elongation at both ends of actin filaments, it accelerates the late stage of spontaneous polymerization like mechanical shearing and theoretical predictions of polymer fragmentation. Low concentrations of actophorin decrease the length and the low shear viscosity of actin filaments. High concentrations cause preformed filaments to shorten rapidly. Ca2+ is not required for any of these effects. Muscle and amoeba actin are equally sensitive to actophorin.
Topics: Actin Depolymerizing Factors; Actins; Amino Acids; Amoeba; Animals; Carrier Proteins; Chromatography, DEAE-Cellulose; Chymotrypsin; Destrin; Electrophoresis, Polyacrylamide Gel; Gelsolin; Microfilament Proteins; Microscopy, Electron; Molecular Weight; Peptide Fragments; Polymers; Proteins; Protozoan Proteins; Starfish; Viscosity
PubMed: 3941084
DOI: No ID Found