Structural features of LIM kinase that control effects on the actin cytoskeleton.
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LIM kinase phosphorylates and inactivates the actin binding/depolymerizing factor cofilin and induces actin cytoskeletal changes. Several unique structural features within LIM kinase were investigated for their roles in regulation of LIM kinase activity. Disruption of the second LIM domain or the PDZ domain or deletion of the entire amino terminus increased activity in vivo measured as increasing aggregation of the actin cytoskeleton. A kinase-deleted alternate splice product was identified and characterized. This alternate splice product and a kinase inactive mutant inhibited LIM kinase in vivo, indicating that the amino terminus suppresses activity of the kinase domain. Mutation of threonine 508 in the activation loop to valine abolished activity whereas replacement with 2 glutamic acid residues resulted in a fully active enzyme. Dephosphorylation of LIM kinase inhibited cofilin phosphorylation. Mutation of the basic insert in the activation loop inhibited activity in vivo, but not in vitro. These results indicate phosphorylation is an essential regulatory feature of LIM kinase and indicate that threonine 508 and the adjacent basic insert sequences of the activation loop are required for this process. A combination of structural features are thus involved in receiving upstream signals that regulate LIM kinase-induced actin cytoskeletal reorganization. (+info)
Ischemia activates actin depolymerizing factor: role in proximal tubule microvillar actin alterations.
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Apical membrane of renal proximal tubule cells is extremely sensitive to ischemia, with structural alterations occurring within 5 min. These changes are felt secondary to actin cytoskeletal disruption, yet the mechanism responsible is unknown. Actin depolymerizing factor (ADF), a 19-kDa actin-binding protein, has recently been shown to play an important role in regulation of actin filament dynamics. Because ADF is known to mediate pH-dependent F-actin binding, depolymerization, and severing, and because ADF activation occurs by dephosphorylation, we questioned whether ADF played a role in microvilli microfilament disruption during ischemia. To test our hypothesis, we induced renal ischemia in the rat with the clamp model. Initial immunofluorescence and Western blot studies on cortical tissue documented the presence of ADF in proximal tubule cells. Under physiological conditions, ADF was distributed homogeneously throughout the cytoplasm, primarily in the Triton X-100-soluble fraction, and both phosphorylated (pADF) and nonphosphorylated forms were identified. During ischemia, marked alterations occurred. Intraluminal vesicle/bleb structures contained extremely high concentrations of ADF along with G-actin, but not F-actin. Western blot showed a rapidly occurring duration-dependent dephosphorylation of ADF. At 0-30 min of ischemia, total ADF levels were unchanged, whereas pADF decreased significantly to 72% and 19% of control levels, at 5 and 15 min, respectively. Urine collected under physiological conditions did not contain ADF or actin, whereas urine collected after 30 min of ischemia contained both ADF and actin. Reperfusion was associated with normalization of cellular pADF levels, pADF intracellular distribution, and repair of apical microvilli. These data suggest that activation of ADF during ischemia via dephosphorylation is, in part, responsible for apical actin disruption resulting in microvillar destruction and formation of intraluminal vesicles. (+info)
XAIP1: a Xenopus homologue of yeast actin interacting protein 1 (AIP1), which induces disassembly of actin filaments cooperatively with ADF/cofilin family proteins.
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We carried out affinity column chromatography using Xenopus ADF/cofilin (XAC), identified several polypeptides in oocytes specifically bound to this column with actin, and isolated a full-length cDNA clone for a 65 kDa protein in this fraction. The predicted amino acid sequence revealed that the 65 kDa protein has seven obvious WD repeats and exhibits striking homology with yeast actin interacting protein 1 (AIP1). Thus, we designated this protein Xenopus AIP1 (XAIP1). We purified XAIP1 from Xenopus oocytes, and its interaction with actin was characterized by a pelleting assay, photometrical analysis and electron microscopy. Although XAIP1 itself cosedimented with F-actin and increased unsedimented actin to some extent, it induced a rapid, drastic disassembly of actin filaments associated with XAC. Electron microscopic observation revealed that XAIP1 severs actin filaments in the presence of XAC. To elucidate the in vivo effects of XAIP1, the purified protein was injected into blastomeres at the two-cell stage. Although the localization of XAIP1 was similar to that of XAC, at the cortical cytoskeleton and diffusely in the cytoplasm, injection of a large amount of XAIP1 arrested development and abolished the strong cortical staining of both actin and XAC. From these results, we concluded that XAIP1 regulates the dynamics of the cortical actin cytoskeleton cooperatively with XAC in eggs. (+info)
Participation of cofilin in opsonized zymosan-triggered activation of neutrophil-like HL-60 cells through rapid dephosphorylation and translocation to plasma membranes.
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We studied the roles of cofilin, an actin-binding phosphoprotein, in superoxide production of neutrophil-like HL-60 cells triggered by opsonized zymosan (OZ). OZ caused dephosphorylation of cofilin as well as a transient increase of F-actin. Both reactions were complete within 30 s. Okadaic acid (OA) magnified the OZ-triggered O2--production 3.3-fold at 1 microM, but inhibited it completely at 5 microM. We used these critical concentrations to study the effects of OA on changes in phosphorylation and intracellular localization of cofilin. The OZ-induced dephosphorylation of cofilin was inhibited by 5 microM OA but not by 1 microM OA. Subcellular fractionation and immunoblotting revealed that 1 microM OA increased cofilin on the phagosomal membranous fraction but 5 microM OA decreased it. At 1 microM, OA increased translocation of p47phox to membranes, which may explain in part the enhancing effect of 1 microM OA. Confocal laser scanning microscopy showed that: (i) Cofilin diffused throughout the cytosol of resting cells, but accumulated at the plasma membranes forming phagocytic vesicles in activated cells. (ii) At 1 microM, OA had little effect on the OZ-evoked translocation of cofilin, whereas 5 microM OA suppressed it completely. (iii) OA alone, which could not trigger the phagocytic respiratory burst, did not cause any change in the distribution of cofilin at such concentrations. Furthermore, in a superoxide-producing cell-free system employing membranous and cytosolic fractions, affinity-purified anti-cofilin antibody showed an enhancing effect. These results suggest that cofilin participates in the superoxide production of the OZ-activated phagocytes through dephosphorylation and translocation. The roles of cofilin in the activated leukocytes will be discussed. (+info)
UNC-60B, an ADF/cofilin family protein, is required for proper assembly of actin into myofibrils in Caenorhabditis elegans body wall muscle.
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The Caenorhabditis elegans unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin that are implicated in myofibril assembly. Here, we show that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. We found that all homozygous viable unc-60 mutations resided in the unc-60B coding region, indicating that UNC-60B is responsible for the Unc-60 phenotype. Wild-type UNC-60B had F-actin binding, partial actin depolymerizing, and weak F-actin severing activities in vitro. However, mutations in UNC-60B caused various alterations in these activities. Three missense mutations resulted in weaker F-actin binding and actin depolymerizing activities and complete loss of severing activity. The r398 mutation truncated three residues from the COOH terminus and resulted in the loss of severing activity and greater actin depolymerizing activity. The s1307 mutation in a putative actin-binding helix caused greater activity in actin-depolymerizing and severing. Using a specific antibody for UNC-60B, we found varying protein levels of UNC-60B in mutant animals, and that UNC-60B was expressed in embryonic muscles. Regardless of these various molecular phenotypes, actin was not properly assembled into embryonic myofibrils in all unc-60 mutants to similar extents. We conclude that precise control of actin filament dynamics by UNC-60B is required for proper integration of actin into myofibrils. (+info)
Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.
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We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-1 s-1) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate. (+info)
Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia.
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The leading edge (approximately 1 microgram) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 micrometer++ of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181-6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex. (+info)
Aip1p interacts with cofilin to disassemble actin filaments.
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Actin interacting protein 1 (Aip1) is a conserved component of the actin cytoskeleton first identified in a two-hybrid screen against yeast actin. Here, we report that Aip1p also interacts with the ubiquitous actin depolymerizing factor cofilin. A two-hybrid-based approach using cofilin and actin mutants identified residues necessary for the interaction of actin, cofilin, and Aip1p in an apparent ternary complex. Deletion of the AIP1 gene is lethal in combination with cofilin mutants or act1-159, an actin mutation that slows the rate of actin filament disassembly in vivo. Aip1p localizes to cortical actin patches in yeast cells, and this localization is disrupted by specific actin and cofilin mutations. Further, Aip1p is required to restrict cofilin localization to cortical patches. Finally, biochemical analyses show that Aip1p causes net depolymerization of actin filaments only in the presence of cofilin and that cofilin enhances binding of Aip1p to actin filaments. We conclude that Aip1p is a cofilin-associated protein that enhances the filament disassembly activity of cofilin and restricts cofilin localization to cortical actin patches. (+info)