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 Domain structure in actin-binding proteins: expression and functional characterization of truncated severin.

1991 ◽  
Vol 112 (4) ◽  
pp. 665-676 ◽  
Author(s):  
L Eichinger ◽  
A A Noegel ◽  
M Schleicher
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Binding Sites ◽  
Actin Filaments ◽  
Functional Characterization ◽  
Tryptophan Fluorescence ◽  
Border Region ◽  
Actin Binding ◽  
Capping Proteins ◽  
Actin Binding Site

Severin from Dictyostelium discoideum is a Ca2(+)-activated actin-binding protein that severs actin filaments, nucleates actin assembly, and caps the fast growing ends of actin filaments. Sequence comparison with functionally related proteins, such as gelsolin, villin, or fragmin revealed highly conserved domains which are thought to be of functional significance. To attribute the different activities of the severin molecule to defined regions, progressively truncated severin polypeptides were constructed. The complete cDNA coding for 362 (DS362) amino acids and five 3' deletions coding for 277 (DS277), 177 (DS177), 151 (DS151), 117 (DS117), or 111 (DS111) amino acids were expressed in Escherichia coli. The proteins were purified to homogeneity and then characterized with respect to their effects on the polymerization or depolymerization kinetics of G- or F-actin solutions and their binding to G-actin. Furthermore, the Ca2+ binding of these proteins was investigated with a 45Ca-overlay assay and by monitoring Ca2(+)-dependent changes in tryptophan fluorescence. Bacterially expressed DS362 showed the same Ca2(+)-dependent activities as native severin. DS277, missing the 85 COOH-terminal amino acids of severin, had lost its strict Ca2+ regulation and displayed a Ca2(+)-independent capping activity, but was still Ca2+ dependent in its severing and nucleating activities. DS151 which corresponded to the first domain of gelsolin or villin had completely lost severing and nucleating properties. However, a residual severing activity of approximately 2% was detectable if 26 amino acids more were present at the COOH-terminal end (DS177). This locates similar to gelsolin the second actin-binding site to the border region between the first and second domain. Measuring the fluorescence enhancement of pyrene-labeled G-actin in the presence of DS111 showed that the first actin-binding site was present in the NH2-terminal 111 amino acids. Extension by six or more amino acids stabilized this actin-binding site in such a way that DS117 and even more pronounced DS151 became Ca2(+)-independent capping proteins. In comparison to many reports on gelsolin we draw the following conclusions. Among the three active actin-binding sites in gelsolin the closely neighboured sites one and two share the F-actin fragmenting function, whereas the actin-binding sites two and three, which are located in far distant domains, collaborate for nucleation. In contrast, severin contains two active actin-binding sites which are next to each other and are responsible for the severing as well as the nucleating function. The single actin-binding site near the NH2-terminus is sufficient for capping of actin filaments.

scholarly journals Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

2012 ◽  
Vol 444 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Keefe T. Chan ◽  
David W. Roadcap ◽  
Nicholas Holoweckyj ◽  
James E. Bear
Keyword(s):  
Binding Site ◽  
Leading Edge ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Unique Region ◽  
Equivalent Residue ◽  
Filament Networks ◽  
Actin Binding Site

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg30) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics.

scholarly journals Transcriptional Repression Mediated by LysR-Type Regulator CatR Bound at Multiple Binding Sites

1998 ◽  
Vol 180 (9) ◽  
pp. 2367-2372 ◽  
Author(s):  
Sudha A. Chugani ◽  
Matthew R. Parsek ◽  
A. M. Chakrabarty
Keyword(s):  
Binding Site ◽  
Structural Gene ◽  
Binding Sites ◽  
Transcriptional Start Site ◽  
Dnase I ◽  
Site Directed Mutagenesis ◽  
Dnase I Footprinting ◽  
Gel Shift

ABSTRACT The catBCA operon of Pseudomonas putidaencodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to thecatBCA promoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of thecatBCA promoter within the catB structural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured β-galactosidase activity ofcatB-lacZ transcriptional fusions in vivo. Our results suggest a probable cis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within the catBstructural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.

Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D

1993 ◽  
Vol 105 (3) ◽  
pp. 765-775 ◽  
Author(s):  
E. Friederich ◽  
T.E. Kreis ◽  
D. Louvard
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Binding Site ◽  
Actin Filaments ◽  
Living Cells ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Fast Growing ◽  
Actin Binding Site

Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca(2+)-dependent manner. In the absence of Ca2+, villin organizes microfilaments into bundles. Transfection of a villin-specific cDNA into cultured cells that do not produce this protein results in the growth of long surface microvilli and the reorganization of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plasma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the association of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structures, which were randomly distributed throughout the cytoplasm. The formation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy terminus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging revealed that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the important role of the plasma membrane in the organization of the actin cytoskeleton and suggest that the extension of the microvillar plasma membrane is dependent on the elongation of microfilaments at their fast-growing end. Inhibition of microfilament elongation near the plasma membrane by cytochalasin D may result in the ‘random’ nucleation of actin filaments throughout the cytoplasm. On the basis of the present data, we propose that villin is involved in the assembly of the microvillar actin bundle by a mechanism that does not prevent monomer association with the preferred end of microfilaments. For instance, villin may stabilize actin filaments by lateral interactions. The functional importance of the carboxy-terminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emphasize the observations that the effects of cytochalasin D in living cells can be modulated by actin-binding proteins.

scholarly journals Identification and characterization of an actin-binding site of CapZ.

1992 ◽  
Vol 116 (4) ◽  
pp. 923-931 ◽  
Author(s):  
C Hug ◽  
T M Miller ◽  
M A Torres ◽  
J F Casella ◽  
J A Cooper
Keyword(s):  
Binding Site ◽  
Primary Structure ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Terminal Region ◽  
Beta Subunit ◽  
In Vitro Translation ◽  
Actin Binding ◽  
Actin Binding Site

A mAb (1E5) that binds the COOH-terminal region of the beta subunit of chicken CapZ inhibits the ability of CapZ to bind the barbed ends of actin filaments and nucleate actin polymerization. CapZ prepared as fusion proteins in bacteria or nonfusion proteins by in vitro translation has activity similar to that of CapZ purified from muscle. Deletion of the COOH-terminus of the beta subunit of CapZ leads to a loss of CapZ's ability to bind the barbed ends of actin filaments. A peptide corresponding to the COOH-terminal region of CapZ beta, expressed as a fusion protein, binds actin monomers. The mAb 1E5 also inhibits the binding of this peptide to actin. These results suggest that the COOH-terminal region of the beta subunit of CapZ is an actin-binding site. The primary structure of this region is not similar to that of potential actin-binding sites identified in other proteins. In addition, the primary structure of this region is not conserved across species.

scholarly journals A Binding Site for Multiple Transcriptional Activators in the fushi tarazu Proximal Enhancer Is Essential for Gene Expression In Vivo

1998 ◽  
Vol 18 (6) ◽  
pp. 3384-3394 ◽  
Author(s):  
Wei Han ◽  
Yan Yu ◽  
Kai Su ◽  
Ronald A. Kohanski ◽  
Leslie Pick
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Binding Sites ◽  
Zinc Finger Protein ◽  
Regulatory Element ◽  
Site Directed Mutagenesis ◽  
Nuclear Proteins ◽  
Fushi Tarazu

ABSTRACT The Drosophila homeobox gene fushi tarazu(ftz) is expressed in a highly dynamic striped pattern in early embryos. A key regulatory element that controls theftz pattern is the ftz proximal enhancer, which mediates positive autoregulation via multiple binding sites for the Ftz protein. In addition, the enhancer is necessary for stripe establishment prior to the onset of autoregulation. We previously identified nine binding sites for multiple Drosophilanuclear proteins in a core 323-bp region of the enhancer. Three of these nine sites interact with the same cohort of nuclear proteins in vitro. We showed previously that the nuclear receptor Ftz-F1 interacts with this repeated module. Here we purified additional proteins interacting with this module from Drosophila nuclear extracts. Peptide sequences of the zinc finger protein Ttk and the transcription factor Adf-1 were obtained. While Ttk is thought to be a repressor of ftz stripes, we have shown that both Adf-1 and Ftz-F1 activate transcription in a binding site-dependent fashion. These two proteins are expressed ubiquitously at the timeftz is expressed in stripes, suggesting that either may activate striped expression alone or in combination with the Ftz protein. The roles of the nine nuclear factor binding sites were tested in vivo, by site-directed mutagenesis of individual and multiple sites. The three Ftz-F1–Adf-1–Ttk binding sites were found to be functionally redundant and essential for stripe expression in transgenic embryos. Thus, a biochemical analysis identifiedcis-acting regulatory modules that are required for gene expression in vivo. The finding of repeated binding sites for multiple nuclear proteins underscores the high degree of redundancy built into embryonic gene regulatory networks.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals PIN*POINT analysis on the endogenous MnSOD promoter: specific demonstration of Sp1 binding in vivo

2003 ◽  
Vol 284 (2) ◽  
pp. C528-C534 ◽  
Author(s):  
Shiuhyang Kuo ◽  
Ann L. Chokas ◽  
Richard J. Rogers ◽  
Harry S. Nick
Keyword(s):  
Binding Sites ◽  
Manganese Superoxide Dismutase ◽  
Consensus Sequence ◽  
Dimethyl Sulfate ◽  
Site Directed Mutagenesis ◽  
Cellular Respiration ◽  
Point Analysis ◽  
Specific Regulation

Manganese superoxide dismutase (MnSOD) is a critical antioxidant enzyme that protects against superoxide anion generated as a consequence of normal cellular respiration, as well as during the inflammatory response. By employing dimethyl sulfate in vivo footprinting, we have previously identified ten basal protein binding sites within the MnSODpromoter. On the basis of consensus sequence comparison and in vitro footprinting data, one would predict that Sp1 might occupy five of these binding sites. To address these findings in the context of the nucleoprotein environment, we first utilized chromatin immunoprecipitation (ChIP) to demonstrate the nuclear association of Sp1 with the MnSOD promoter region. To identify the precise location of Sp1 binding, we have modified the original protein position identification with nuclease tail (PIN*POINT) methodology, providing an approach to establish both the identity and binding occupancy of Sp1 in the context of the endogenous MnSOD promoter. These data, coupled with site-directed mutagenesis, demonstrate the functional importance of two of the Sp1 binding sites in the stimulus-specific regulation of MnSOD gene expression. We feel that the combination of ChIP and PIN*POINT analysis allows unequivocal identification and localization of protein/DNA interactions in vivo, specifically the demonstration of Sp1 with the MnSODpromoter.

scholarly journals Regulation of the Rhodobacter sphaeroides 2.4.1 hemA Gene by PrrA and FnrL

2008 ◽  
Vol 190 (20) ◽  
pp. 6769-6778 ◽  
Author(s):  
Britton Ranson-Olson ◽  
Jill H. Zeilstra-Ryalls
Keyword(s):  
Binding Site ◽  
Rhodobacter Sphaeroides ◽  
Binding Sites ◽  
Aminolevulinic Acid ◽  
Acid Synthesis ◽  
P2 Promoter ◽  
Indirect Action ◽  
Hema Gene

ABSTRACT Part of the oxygen responsiveness of Rhodobacter sphaeroides 2.4.1 tetrapyrrole production involves changes in transcription of the hemA gene, which codes for one of two isoenzymes catalyzing 5-aminolevulinic acid synthesis. Regulation of hemA transcription from its two promoters is mediated by the DNA binding proteins FnrL and PrrA. The two PrrA binding sites, binding sites I and II, which are located upstream of the more-5′ hemA promoter (P1), are equally important to transcription under aerobic conditions, while binding site II is more important under anaerobic conditions. By using phosphoprotein affinity chromatography and immunoblot analyses, we showed that the phosphorylated PrrA levels in the cell increase with decreasing oxygen tensions. Then, using both in vivo and in vitro methods, we demonstrated that the relative affinities of phosphorylated and unphosphorylated PrrA for the two binding sites differ and that phosphorylated PrrA has greater affinity for site II. We also showed that PrrA regulation is directed toward the P1 promoter. We propose that the PrrA component of anaerobic induction of P1 transcription is attributable to higher affinity of phosphorylated PrrA than of unphosphorylated PrrA for binding site II. Anaerobic activation of the more-3′ hemA promoter (P2) is thought to involve FnrL binding to an FNR consensuslike sequence located upstream of the P2 promoter, but the contribution of FnrL to P1 induction may be indirect since the P1 transcription start is within the putative FnrL binding site. We present evidence suggesting that the indirect action of FnrL works through PrrA and discuss possible mechanisms.

scholarly journals Aip1 and Cofilin Promote Rapid Turnover of Yeast Actin Patches and Cables: A Coordinated Mechanism for Severing and Capping Filaments

2006 ◽  
Vol 17 (7) ◽  
pp. 2855-2868 ◽  
Author(s):  
Kyoko Okada ◽  
Harini Ravi ◽  
Ellen M. Smith ◽  
Bruce L. Goode
Keyword(s):  
Close Correlation ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Interacting Protein ◽  
Rapid Turnover ◽  
Actin Cable ◽  
Actin Turnover ◽  
Actin Cables

Rapid turnover of actin structures is required for dynamic remodeling of the cytoskeleton and cell morphogenesis, but the mechanisms driving actin disassembly are poorly defined. Cofilin plays a central role in promoting actin turnover by severing/depolymerizing filaments. Here, we analyze the in vivo function of a ubiquitous actin-interacting protein, Aip1, suggested to work with cofilin. We provide the first demonstration that Aip1 promotes actin turnover in living cells. Further, we reveal an unanticipated role for Aip1 and cofilin in promoting rapid turnover of yeast actin cables, dynamic structures that are decorated and stabilized by tropomyosin. Through systematic mutagenesis of Aip1 surfaces, we identify two well-separated F-actin–binding sites, one of which contributes to actin filament binding and disassembly specifically in the presence of cofilin. We also observe a close correlation between mutations disrupting capping of severed filaments in vitro and reducing rates of actin turnover in vivo. We propose a model for balanced regulation of actin cable turnover, in which Aip1 and cofilin function together to “prune” tropomyosin-decorated cables along their lengths. Consistent with this model, deletion of AIP1 rescues the temperature-sensitive growth and loss of actin cable defects of tpm1Δ mutants.

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