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At least 19 recordsLinked to original sources

PubMed2001

Cofilin-2, a novel type of cofilin, is expressed specifically at aggregation stage of Dictyostelium discoideum development.

BACKGROUND: A conventional cofilin, cofilin-1 in Dictyostelium discoideum plays significant roles in cell proliferation, phagocytosis, chemotactic movement and macropinocytosis. RESULTS: We identified a new member of the cofilin family, named cofilin-2 in D. discoideum. Cofilin-2 shows significant homology to a conventional Dictyostelium cofilin, cofilin-1, through its entire sequence, and contains residues conserved among the cofilin family that are responsible for actin-binding. On the other hand, several residues that are conserved among the cofilin family are missing from cofilin-2. Purified cofilin-2 depolymerized actin filaments in a dose- and pH-dependent manner and reduced the apparent viscosity of an actin solution, although they did not co-sediment with actin filaments at all. Cofilin-2 was not expressed in vegetative cells, but was transiently induced during the aggregation stage of development, whereas cofilin-1 was predominantly expressed in vegetative cells. Immunocytochemistry revealed that cofilin-2 localizes at substrate adhesion sites, where cofilin-1 is almost completely excluded. Disruption of the cofilin-2 gene caused an increase in actin accumulation at the substrate adhesion sites. We also found that cofilin-2 did not rescue Deltacof1 yeast cells, whereas cofilin-1 did. CONCLUSIONS: Cofilin-2 may play a distinct role from that of cofilin-1 in destabilization of the actin cytoskeleton during Dictyostelium development.

Actin Depolymerizing Factors↗

PubMed2006

Initiation of cofilin activity in response to EGF is uncoupled from cofilin phosphorylation and dephosphorylation in carcinoma cells.

It has been demonstrated that the actin-severing activity of cofilin can be downregulated by LIM kinase (LIMK)-dependent phosphorylation at residue Ser3. Chemotactic stimulation in various cell types induces cofilin dephosphorylation, suggesting that cofilin activation in these cells occurs by a dephosphorylation mechanism. However, resting metastatic carcinoma cells have the majority of their cofilin in a dephosphorylated but largely inactive state. Stimulation with epidermal growth factor (EGF) induces an increase in cofilin activity after 60 seconds together with an increase in phosphorylated cofilin (p-cofilin), indicating that cofilin dephosphorylation is not coupled to cofilin activation in these cells. Suppression of LIMK function by inhibiting Rho-associated protein kinase (ROCK) or LIMK siRNA inhibited the EGF-induced cofilin phosphorylation but had no effect on cofilin activity or cofilin-dependent lamellipod protrusion induced by EGF. Correlation analysis revealed that cofilin, p-cofilin and LIMK are not colocalized, and changes in the location of these proteins upon stimulation with EGF indicate that they are not functionally coupled. Phospholipase C, which has been implicated in cofilin activation following stimulation with EGF, does not regulate p-cofilin levels following stimulation with EGF. Therefore, our results do not support a model for the initial activation of cofilin by dephosphorylation in response to chemoattractant stimulation in metastatic carcinoma cells.

Actin Depolymerizing Factors↗

PubMed1995

Effects of cofilin on actin filamentous structures in cultured muscle cells. Intracellular regulation of cofilin action.

The previous investigation (Abe et al. (1989) J. Biochem. 106, 696-702) suggested that cofilin is deeply involved in the regulation of actin assembly in developing skeletal muscle. In this study, to examine further the function of cofilin in living myogenic cells in culture, recombinant cofilin having extra Cys residues at the N terminus was produced in Escherichia coli and was labeled with tetramethylrhodamine-iodoacetamide (IATMR). When the cofilin labeled with IATMR (IATMR-cofilin) was introduced into myogenic cells, actin filaments in the cytoplasm or nascent myofibrils were promptly disrupted, and many cytoplasmic rods which contained both IATMR-cofilin and actin were generated. Sarcomeric myofibrillar structures were not disrupted but tropomyosin was dissociated from the structures by the exogenous cofilin, and the IATMR-cofilin became localized in I-band regions. 24 hours after the injection, however, the actin-cofilin rods disappeared completely and the IATMR-cofilin became diffused in the cytoplasm as endogenous cofilin. Concomitantly, actin filaments were recovered and tropomyosin was re-associated with sarcomeric I-bands. At this point, the IATMR-cofilin in the cells still retained the functional activity to form intranuclear actin-cofilin rods in response to stimulation by DMSO just as endogenous cofilin. FITC-labeled actin introduced into myogenic cells at first failed to assemble into filamentous structures in the presence of the exogenous cofilin, but was gradually incorporated into myofibrils with time. The drastic effects of the exogenous cofilin on actin assembly were suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). These results indicate that the exogenous cofilin is active and alters actin dynamics remarkably in muscle cells, but its activity in the cytoplasm gradually becomes regulated by the action of some factors including PIP2-binding.

Actin Depolymerizing Factors↗

PubMed1996

Site-directed mutagenesis of the phosphorylation site of cofilin: its role in cofilin-actin interaction and cytoplasmic localization.

It has been demonstrated that the activity of ADF and cofilin, which constitute a functionally related protein family, is markedly altered by phosphorylation, and that the phosphorylation site is Ser 3 in their amino acid sequences [Agnew et al., 1995: J. Biol. Chem. 270:17582-17587; Moriyama et al., 1996: Genes Cells 1:73-86]. In order to clarify the function of the phosphorylated and unphosphorylated forms of cofilin in living cells especially in the process of cytokinesis, we generated analogs of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser 3) of cofilin to Ala and Asp, respectively. The mutated proteins were produced in an Escherichia coli expression system, and conjugated with fluorescent dyes. In in vitro functional assay, labeled A3-cofilin retained the authentic ability to bind to and sever F-actin, while labeled D3-cofilin failed to interact with actin. They were then injected into living cells to examine their cellular distribution. They exhibited distinct localization patterns in the cytoplasm; A3-cofilin was highly concentrated at the membrane ruffles and cleavage furrow, where endogenous cofilin is also known to be enriched. In contrast, D3-cofilin showed only diffuse distribution both in the cytoplasm and nucleus. These results suggest that the subcellular distribution of cofilin as well as its interacting with actin in vivo is regulated by its phosphorylation and dephosphorylation.

Actin Depolymerizing Factors↗

PubMed2001

The affinity of chick cofilin for actin increases when actin is complexed with DNase I: formation of a cofilin-actin-DNase I ternary complex.

Cofilin, an actin-binding protein, regulates the rate, nature and extent of assembly of the actin cytoskeleton. Native Phast gels show that the addition of cofilin to an actin-DNase I complex (74 kDa) results in the formation of a ternary complex of 94 kDa indicating an equimolar stoichiometry in the ternary complex. Furthermore, native gels show that the addition of cofilin to a solution containing free actin and actin-DNase I and run at pH 8.3 results in cofilin complexing preferentially to the actin-DNase I complex. Conversely, the addition of DNase I to a solution containing an actin-cofilin complex and free actin results in the preferential binding of DNase I to the actin-cofilin complex. These results show that the affinity of cofilin for actin can be increased when actin forms binary complexes. When native gels were run at pH 6.8 the affinity of cofilin for monomeric actin was greater than for the actin-DNase I complex indicating that the cofilin-actin interaction can be regulated by changes in pH. The addition of cofilin to actin resulted in the polymerisation of actin at pH 6.8 whereas at alkaline pH a stable cofilin-actin binary complex could be formed. The biological implications are discussed.

Actin Depolymerizing Factors↗

PubMed2005

Cooperative effects of cofilin (ADF) on actin structure suggest allosteric mechanism of cofilin function.

Using site-specific fluorescence probes and cross-linking we demonstrated that cofilin (ADF), a key regulator of actin cellular dynamics, weakens longitudinal contacts in F-actin in a cooperative manner. Differential scanning calorimetry detected a dual nature of cofilin effects on F-actin conformation. At sub-stoichiometric cofilin to actin ratios, cofilin stabilized sterically and non-cooperatively protomers at the points of attachment, and destabilized allosterically and cooperatively protomers in the cofilin-free parts of F-actin. This destabilizing effect had a long range, with one cofilin molecule affecting more than 100 protomers, and concentration-dependent amplitude that reached maximum at about 1:2 molar ratio of cofilin to actin. In contrast to existing models, our results suggest an allosteric mechanism of actin depolymerization by cofilin. We propose that cofilin is less likely to sever actin filaments at the points of attachment as thought previously. Instead, due to its dual structural effect, spontaneous fragmentation occurs most likely in cofilin-free segments of filaments weakened allosterically by nearby cofilin molecules.

Actin Depolymerizing Factors↗

PubMed1989

Dephosphorylation of cofilin accompanies heat shock-induced nuclear accumulation of cofilin.

Cofilin is a widely distributed 21-kDa actin-modulating protein that forms intranuclear actin/cofilin rods in cultured fibroblastic cells exposed to heat shock or 10% dimethyl sulfoxide. In this study, cofilin was shown to be phosphorylated on a serine residue in cultured rat fibroblastic 3Y1 cells. Two-dimensional gel electrophoresis revealed that about 50% of the cofilin was phosphorylated in 3Y1 cells at 37 degrees C. Exposure of the cells to heat shock at 43 degrees C induced dephosphorylation of cofilin. The dephosphorylation of cofilin was detected about 30 min after the temperature shift and was completed within 120 min. Moreover, treatment of cells with 10% dimethyl sulfoxide also caused the dephosphorylation of cofilin. However, incubation of the cells with an isotonic NaCl solution, which induced cytoplasmic actin/cofilin rods, did not induce dephosphorylation of cofilin. Other cellular stress agents such as 6% ethanol or 50 microM sodium arsenite, which caused some heat shock responses in cells, did not induce dephosphorylation of cofilin. Thus, cofilin dephosphorylation was closely correlated with its nuclear accumulation. Incubation of the enucleated 3Y1 cells at 43 degrees C still induced dephosphorylation of cofilin, suggesting that the dephosphorylation occurred mostly in the cytoplasm in intact cells. It is likely that cofilin is dephosphorylated in the cytoplasm prior to its nuclear accumulation.

Actin Depolymerizing Factors↗

PubMed2002

The actin-severing activity of cofilin is exerted by the interplay of three distinct sites on cofilin and essential for cell viability.

Cofilin/actin-depolymerizing factor is an essential and conserved modulator of actin dynamics. Cofilin binds to actin in either monomeric or filamentous form, severs and depolymerizes actin filaments, and speeds up their treadmilling. A high turnover rate of F-actin in actin-based motility seems driven largely by cofilin-mediated acceleration of directional subunit release, but little by fragmentation of the filaments. On the other hand, the filament-severing function of cofilin seems relevant for the healthy growth of cells. In this study, we have characterized three mutants of porcine cofilin to elucidate the molecular mechanism that underlies the filament-severing activity of cofilin. The first mutant could neither associate with actin filaments nor sever them, whereas it effectively accelerated their treadmilling and directional subunit release. The second mutant bound to actin filaments, but failed to sever them and to interfere with phalloidin binding to the filament. The third mutant could associate with actin filaments and sever them, although with a very reduced efficacy. Of these mutant proteins, only the last one was able to rescue Deltacof1 yeast cells and to induce thick actin bundles in mammalian cells upon overexpression. Therefore, the actin-severing activity of cofilin is an essential element in its vital function and suggested to be exerted by co-operation of at least three distinct sites of cofilin.

Actin Depolymerizing Factors↗

PubMed2005

Cofilin expression induces cofilin-actin rod formation and disrupts synaptic structure and function in Aplysia synapses.

Cofilin-actin rods are inclusion-like structures that are induced by certain chemical or physical stresses in cultured cells, and the rods formed in neurons are thought to be associated with neurodegeneration. Here, we cloned an Aplysia cofilin homolog and overexpressed it in cultured neurons. Overexpressed cofilin formed rod-like structures that included actin. The overall neuronal morphology was unaffected by cofilin overexpression; however, a decrease in number of synaptic varicosities was observed. Consistent with this structural change by cofilin overexpression, the synaptic strength was reduced, and furthermore, the long-term facilitation elicited by repeated pulses of 5-hydroxytryptamine was impaired in sensory-to-motor synapses. However, cofilin overexpression did not induce programmed cell death. These findings suggest that the formation of cofilin-actin rod-like structures can lead to neurodegeneration, and this might be a mechanism of rundown of neuronal and synaptic function without cell death in neurodegenerative diseases.

Actin Depolymerizing Factors↗

PubMed2004

A pathway of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilin in lamellipodia.

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, we show that cofilin-phosphatase activity of SSH1L increases approximately 10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. We also provide evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1beta induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin-rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1beta and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm.

14-3-3 Proteins↗

PubMed1992

Mutational analysis of an actin-binding site of cofilin and characterization of chimeric proteins between cofilin and destrin.

Cofilin and destrin are two related low molecular weight mammalian actin-binding proteins. Cofilin is an F-actin side-binding and pH-dependent actin-depolymerizing protein, and destrin is a pH-independent actin-depolymerizing protein. We have introduced a few point mutations within an actin-binding sequence of cofilin. Biochemical analyses of these mutant proteins have clearly shown that Lys112 and Lys114 of cofilin are crucially but differently involved in its interaction with actin and phosphatidylinositol 4,5-bisphosphate. This is the first example among actin-binding proteins whose point mutations inactivate their interaction with actin in vitro. We have also made and characterized a series of chimeric proteins between cofilin and destrin to identify the regions responsible for the pH dependence and the F-actin side binding activity of cofilin. Our results suggest that a central region consisting of 42 amino acid residues and a carboxyl-terminal quarter of cofilin are both involved in regulation of the pH-dependent actin depolymerizing activity and the activity to bind along F-actin.

Actin Depolymerizing Factors↗

PubMed1988

Cloning and characterization of porcine brain cofilin cDNA. Cofilin contains the nuclear transport signal sequence.

Cofilin is a widely distributed, pH-sensitive, actin-modulating protein with an apparent molecular mass of 21 kDa, which forms intranuclear and/or cytoplasmic actin/cofilin rods in cultured fibroblastic cells under specific conditions. In this study, a cDNA library from porcine brain mRNA was constructed, and full-length brain cofilin cDNA clones were isolated by screening with oligonucleotide probes. The deduced amino acid sequence of cofilin is 166 residues long and contains a sequence of Lys-Lys-Arg-Lys-Lys which is very similar to the nuclear transport signal sequence (Pro-Lys-Lys-Lys-Arg-Lys-Val) of SV40 large T antigen. The sequence may act as a signal capable of inducing nuclear accumulation of cofilin in cells exposed to heat shock or dimethyl sulfoxide. The cofilin sequence contains a hexapeptide (Asp-Ala-Ile-Lys-Lys-Lys) identical to the amino-terminal sequence (residues 2-7) of muscle and nonmuscle tropomyosin. Cofilin also has in the carboxyl-terminal portion a region homologous to the sequence shared by gelsolin, fragmin, and Acanthamoeba profilin. Furthermore, the overall amino acid sequence of cofilin shows weak homology with the rod portion of myosin and suggests a high alpha-helical content.

Actin Depolymerizing Factors↗

PubMed1996

Mapping of human non-muscle type cofilin (CFL1) to chromosome 11q13 and muscle-type cofilin (CFL2) to chromosome 14.

Cofilin is a widely-distributed, intracellular, actin binding protein which is involved in the translocation of actin-cofilin complex from cytoplasm to nucleus. We have cloned a non-muscle-type cofilin (CFL1) from a human promyelocytic cDNA library and mapped this to human chromosome 11 by PCR amplification of 3' untranslated sequence in a panel of rodent-human somatic cell hybrids, and to the interval 11q12-q13.2 in a chromosome 11 somatic cell hybrid mapping panel. Confirmation of regional localisation to 11q13 has been obtained by fluorescent in situ hybridisation of genomic cosmid clones, by demonstration of the presence of both SEA (the human homologue of avian retrovirus proviral tyrosine kinase, 11q13) and CFL1 in some of these clones and by close linkage of CFL1 to SEA in a panel of high-dose irradiation hybrids. We have identified human muscle-type cofilin sequences by comparison of human expressed sequence tags with M-type cofilins of other species and we have mapped the human M-type cofilin, CFL2, to chromosome 14.

Actin Depolymerizing Factors↗

PubMed2006

Mapping the phosphoinositide-binding site on chick cofilin explains how PIP2 regulates the cofilin-actin interaction.

Cofilin plays a key role in the choreography of actin dynamics via its ability to sever actin filaments and increase the rate of monomer dissociation from pointed ends. The exact manner by which phosphoinositides bind to cofilin and inhibit its interaction with actin has proven difficult to ascertain. We determined the structure of chick cofilin and used NMR chemical shift mapping and structure-directed mutagenesis to unambiguously locate its recognition site for phosphoinositides (PIs). This structurally unique recognition site requires both the acyl chain and head group of the PI for a productive interaction, and it is not inhibited by phosphorylation of cofilin. We propose that the interaction of cofilin with membrane-bound PIs abrogates its binding to both actin and actin-interacting protein 1, and facilitates spatiotemporal regulation of cofilin activity.

Actin Depolymerizing Factors↗

PubMed1985

Purification of cofilin, a 21,000 molecular weight actin-binding protein, from porcine kidney and identification of the cofilin-binding site in the actin sequence.

Cofilin, a 21,000 molecular weight protein originally purified from porcine brain that is capable of binding to actin filaments in a molar ratio of the protein to actin monomer of 1:1 in the filament (Nishida et al. (1984) Biochemistry 23, 5307-5313), was purified from porcine kidney in the present study. The two cofilins from brain and kidney were indistinguishable from each other with respect to the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the one-dimensional peptide map, and the mode of interaction with actin. Treatment of the actin-cofilin complex with a zero-length cross-linker, 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC), generated a cross-linked product with an apparent molecular weight of 63,000. Analysis of this product by peptide mapping (Sutoh (1982) Biochemistry 21, 3654-3661) showed that cofilin was cross-linked with the N-terminal segment of actin containing residues 1-12.

Actin Depolymerizing Factors↗

PubMed2006

Cofilin plays a critical role in IL-8-dependent chemotaxis of neutrophilic HL-60 cells through changes in phosphorylation.

Cofilin is a ubiquitous, actin-binding protein. Only unphosphorylated cofilin binds actin and severs or depolymerizes filamentous actin (F-actin), and the inactive form of cofilin is phosphorylated at Ser 3. We reported recently that cofilin plays a regulatory role in superoxide production and phagocytosis by leukocytes, and in the present study, we investigated the role of cofilin in the chemotaxis of neutrophilic HL-60 cells. IL-8 is a potent, physiological chemokine, and it triggers a rapid, transient increase in F-actin beneath the plasma membrane and rapid dephosphorylation and subsequent rephosphorylation of cofilin. In this study, cofilin phosphorylation was found to be inhibited by S3-R peptide, which consists of a peptide corresponding to part of the phosphorylation site of cofilin and a membrane-permeable arginine polymer. When S3-R peptide was introduced into the neutrophilic cells, their chemotactic activity was enhanced, whereas a control peptide that contained an inverted sequence of the phosphorylation site of cofilin had no enhancing effect. Cofilin small interfering RNA (siRNA) decreased cofilin expression by about half and inhibited chemotaxis. In IL-8-stimulated cells, unphosphorylated cofilin accumulated around F-actin, and colocalization of F-actin and phosphorylated cofilin was observed, but these changes in cofilin localization were less prominent in cofilin siRNA-treated cells. The inhibitors of PI-3K wortmannin and LY294002 inhibited the chemotaxis and suppressed IL-8-evoked dephosphorylation and rephosphorylation of cofilin. These results suggested that unphosphorylated cofilin plays a critical role in leukocyte chemotaxis and that PI-3K is involved in the control of the phosphorylation/dephosphorylation cycle of cofilin.

Actin Depolymerizing Factors↗

PubMed1996

Phosphorylation of Ser-3 of cofilin regulates its essential function on actin.

BACKGROUND: Cofilin is a low-molecular weight actin-modulating protein, and is structurally and functionally conserved in eucaryotes from yeast to mammals. The functions of cofilin appear to be regulated by phosphorylation and dephosphorylation. RESULTS: A proteolytic study of phosphorylated porcine cofilin and expression of a mutated cofilin in cultured cells revealed that Ser-3 is the unique phosphorylation site. Phosphorylated cofilin was found not to bind to either F- or G-actin while unphosphorylated cofilin binds to both. S3D-cofilin, in which Ser-3 was replaced with Asp, did not bind in vitro to actin while S3A-cofilin did. The transient over-expression of wild-type or S3A-cofilin in cultured cells caused disruption of preexisting actin structures and induced cytoplasmic actin bundles. Heat shock-induced nuclear or NaCl buffer-induced cytoplasmic actin/cofilin rods contained the expressed cofilin. In contrast, the over-expression of S3D-cofilin did not alter the actin structures. Induced actin rods did not contain S3D-cofilin. S3D-porcine cofilin did not complement the lethality associated with delta cof1 mutations in Saccharomyces cerevisiae while wild-type and S3A-cofilin did. Furthermore, we found that S2A/S4D- and S2D/S4D-yeast cofilin mutants were not viable. CONCLUSION: We conclude that the function of cofilin is negatively regulated in vivo by phosphorylation of Ser-3 and that cells require the function of unphosphorylated cofilin for viability.

Actin Depolymerizing Factors↗

PubMed1992

The KKRKK sequence is involved in heat shock-induced nuclear translocation of the 18-kDa actin-binding protein, cofilin.

The exposure of cultured mammalian cells to elevated temperatures induces the translocation of actin and cofilin into the nuclei and the formation of intranuclear bundles of actin filaments decorated by cofilin (actin/cofilin rods). Cofilin has a stretch of five basic amino acids, KKRKK, which was assumed to be the sequence involved in the heat shock-dependent accumulation of cofilin in nuclei. To examine this possibility, the site-directed mutagenesis technique was employed to alter the KKRKK sequence of cofilin to KTLKK and the mutated cofilin was expressed under the human beta-actin promoter in transfectants of mouse C3H-2K cell line. All the recombinants derived from porcine cofilin cDNA were constructed so as to possess an extra-nonapeptide at their N-termini when expressed; their intracellular distribution could, therefore, be discriminated from that of endogenous cofilin using the indirect immunofluorescence method with polyclonal antibodies directed against the extra-peptide. The results clearly showed that the mutated cofilin possessing KTLKK instead of KKRKK did not translocate into the nuclei in response to heat shock whereas a recombinant cofilin with the unaltered sequence of KKRKK responded to heat shock and formed intranuclear rods together with actin. Although in vitro actin binding experiments showed that KTLKK-cofilin has a weaker affinity to actin filaments than KKRKK-cofilin, KTLKK-cofilin was found to form cytoplasmic actin/cofilin rods when transformants were incubated in NaCl buffer. Furthermore, we have noted that endogenous cofilin present in cells expressing KTLKK-cofilin behaved normally, translocated into nuclei and formed intranuclear actin/cofilin rods upon heat shock. These results suggest that the KKRKK sequence of cofilin functions as a nuclear location signal upon heat shock.

Actin Depolymerizing Factors↗