Nucleotide sequence of mouse cofilin cDNA.
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Actin Depolymerizing Factors↗
Biomedical subjects
E Nishida
Publications and source records attributed to E Nishida.
Inhibition of the interactions of cofilin, destrin, and deoxyribonuclease I with actin by phosphoinositides.
Cofilin is a widely distributed actin-modulating protein that has the ability to bind along the side of F-actin and to depolymerize F-actin in a pH-dependent manner. We found that phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) inhibited both actions of cofilin in a dose-dependent manner, while inositol 1,4,5-triphosphate (IP3), 1-oleoyl-2-acetylglycerol (OAG), phosphatidylserine (PS), or phosphatidylcholine (PC) had little or no effect on them. Gel filtration analyses showed that PIP2 bound to cofilin and thereby inhibited the binding of cofilin to G-actin. Destrin is a mammalian, pH-independent actin-depolymerizing protein. The actin-depolymerizing activity of destrin was also inhibited by PI, PIP, and PIP2, but not by IP3, OAG, PS, or PC. In addition, we found further that an actin-depolymerizing activity of bovine pancreas deoxyribonuclease I, a G-actin-sequestering protein, was inhibited by PIP and PIP2, but not by PI, IP3, OAG, PS, or PC. These results together with previous findings (Lassing, I., and Lindberg, U. (1985) Nature 314, 472-474; Janmey, P. A., and Stossel, T. P. (1987) Nature 325, 362-364) suggest that the sensitivity to polyphosphoinositides may be a common feature in vitro among actin-binding proteins that can bind to G-actin and regulate the state of actin polymerization.
Actin Depolymerizing Factors↗
Destrin, a mammalian actin-depolymerizing protein, is closely related to cofilin. Cloning and expression of porcine brain destrin cDNA.
Destrin is a mammalian 19-kDa protein that rapidly depolymerizes F-actin in a stoichiometric manner. In this study, we isolated cDNA clones coding for destrin from a porcine brain cDNA library. The deduced amino acid sequence of destrin is 165 residues long and is very similar (71% identical) to that of cofilin, a widely distributed, pH-sensitive actin-modulating protein. Destrin contains a sequence nearly identical with the putative nuclear transport signal sequence of cofilin and a hexapeptide sequence identical with the amino-terminal sequence (residues 2-7) of tropomyosin, which is shown to be involved in cofilin binding to actin. Destrin, like cofilin, also has in its carboxyl-terminal portion a region homologous to the sequence shared by gelsolin, fragmin, and Acanthamoeba profilin. We have expressed destrin as well as cofilin in Escherichia coli, purified them, and examined their function in vitro. The two proteins were found to differ in their interaction with actin, like destrin and cofilin isolated from porcine brain. This suggests that the difference in the function of the two proteins results from the subtle difference in their amino acid sequence rather than possible differences in post-translational modifications. Northern blot analyses indicated that both destrin mRNA and cofilin mRNA are widely distributed in various tissues, but both mRNAs differ in their relative abundance among tissues.
Acanthamoeba↗
Characterization of interleukin 2 stimulated 65-kilodalton phosphoprotein in human T cells.
We have characterized the cellular proteins which are rapidly phosphorylated by interleukin 2 (IL 2) in a human IL 2 dependent cell line. When treated with IL 2, the phosphorylation of five proteins, 65, 50, 37, 24, and 21 kDa, was found in IL 2 dependent cell lines by two-dimensional gel electrophoretic analysis. After cell conversion from an IL 2 dependent state to an IL 2 independent state, one of the five phosphoproteins, the 65-kDa protein, became constitutively phosphorylated even without addition of IL 2. Also, in other IL 2 independent cell lines, such as KUT-2 and HUT-102, constitutive phosphorylation of the 65-kDa protein occurred without IL 2-stimulation. So our researchers were focused on biochemical characterization of the 65-kDa protein. It was found that the 65-kDa protein was one of the major cellular proteins by comparing the results of two-dimensional gel electrophoretic analysis of [32P]Pi-labeled and [3H]leucine-labeled cellular proteins and peptide mapping analysis. Subcellular fractionation studies indicated that the 65-kDa protein is a cytosol protein. The 65-kDa protein was purified from cytosol of a human T cell line, and its amino acid composition and amino acid sequences of its three oligopeptides were determined. It was found that the 65-kDa protein is identical with 1-plastin.
Amino Acid Sequence↗
[Actin and actin binding proteins in neuronal tissues].
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Actin Depolymerizing Factors↗
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↗
Characterization of a mitogen-activated, Ca2+-sensitive microtubule-associated protein-2 kinase.
We have previously found that treatment of quiescent mammalian fibroblast cells with several mitogenic factors activates in common a Ca2+-sensitive serine/threonine-specific protein kinase activity toward microtubule-associated protein 2 (MAP2) [Hoshi, M., Nishida, E. and Sakai, H. (1988) J. Biol. Chem. 263, 5396-5401]. Here, we characterized the mitogen-activated MAP2 kinase activity in rat 3Y1 cells. The activated kinase activity was detected in the cytosolic fraction but not in the membrane fraction. The inhibitory effect of Ca2+ on the kinase activity was reversible. Kinetic analyses revealed that the apparent Km values of the kinase activity for MAP2 and ATP were 1.6 microM and 30 microM, respectively. Free Ca2+ at 4 microM decreased apparent Vmax values for MAP2 and ATP without changing the apparent Km values. The MAP2 kinase had an apparent molecular mass of about 40 kDa as determined by gel filtration and by sucrose density gradient centrifugation. Myelin basic protein as well as MAP2 could serve as good substrates for this kinase, but 40S ribosomal protein S6, casein, histone, phosphorylase b, protamine, tubulin, actin and tau could not. These properties of the enzyme indicate that the Ca2+-sensitive MAP2 kinase may be a previously unidentified enzyme. Down-regulation of protein kinase C by prolonged phorbol ester treatment abolished the MAP2 kinase activation by phorbol ester, but did not prevent the MAP2 kinase activation by epidermal growth factor (EGF) or fresh serum. This suggests that the Ca2+-sensitive MAP2 kinase could be activated through protein-kinase-C-dependent and -independent pathways. Activation of the MAP2 kinase occurred shortly after the addition of EGF or phorbol ester even in the presence of protein synthesis inhibitors (cycloheximide, puromycin and emetin). Moreover, treatment of the EGF- or phorbol-ester-activated MAP2 kinase with acid phosphatase inactivated the kinase activity. Thus, the MAP2 kinase may be activated through phosphorylation.
Animals↗
Protein kinase C-dependent and -independent pathways in the growth factor-induced cytoskeletal reorganization.
Human epidermoid carcinoma KB cells exhibit rapid induction of membrane ruffling in response to epidermal growth factor (EGF), insulin, and insulin-like growth factor-I (IGF-I) (Kadowaki, T., Koyasu, S., Nishida, E., Sakai, H., Takaku, F., Yahara, I., and Kasuga, M. (1986) J. Biol. Chem. 261, 16141-16147). We have analyzed the role of protein kinase C (PKC) in this response. Treatment of KB cells with 4 beta-phorbol 12,13-dibutyrate (PDBu) (100 ng/ml) for 30 min caused translocation of PKC to the membrane. This treatment completely inhibited the induction of membrane ruffling by EGF, insulin, and IGF-I. Prolonged treatment with PDBu (200 ng/ml for 15 h) induced complete depletion of the PKC activity in the cells. Under these conditions, EGF binding to cells and autophosphorylation of the EGF receptor occurred normally, while EGF could not induce membrane ruffling. In contrast, insulin- or IGF-I-induced membrane ruffling occurred normally in the PKC-depleted cells. Moreover, H-7 (PKC inhibitor) inhibited only EGF-induced membrane ruffling in a dose-dependent manner. We further found that EGF, but not insulin/IGF-I, caused transient translocation of PKC to the membrane. All these results suggest that PKC is required for the membrane ruffling induced by EGF but not for that induced by insulin/IGF-I. Therefore, there are PKC-dependent and independent pathways in the growth factor-induced membrane ruffling. Furthermore, we propose dual roles of PKC in the EGF signaling, a signal transmitting role and a negative feedback role.
Cell Membrane↗
HSP100, a 100-kDa heat shock protein, is a Ca2+-calmodulin-regulated actin-binding protein.
The 100-kDa heat shock protein, HSP100, was purified from mouse lymphoma cells. Amino acid sequences of three peptide fragments which were obtained from the purified protein by lysylendopeptidase digestion were completely or nearly identical with those of a mouse endoplasmic reticulum protein, ERp99, of a hamster glucose-regulated protein, GRP94, and of a chicken heat shock protein, HSP108, all of which have been known to have strong homology with the 90-kDa heat shock protein, HSP90. HSP100 bound to actin filaments and an apparent Kd for the binding was determined to be 8 x 10(-7) M in 2 mM MgCl2 + 100 mM KCl. Calmodulin inhibited the binding in a Ca2+-dependent manner. Equilibrium gel filtration demonstrated that HSP100 has an ability to bind to calmodulin only in the presence of Ca2+. Moreover, HSP100 competed with HSP90 for binding to actin filaments. These results together with our previous findings that HSP90 and HSP100 have similar physicochemical properties (Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8054-8058) and HSP90 is a calmodulin-regulated actin-binding protein (Nishida, E., Koyasu, S., Sakai, H., and Yahara, I. (1986) J. Biol. Chem. 261, 16033-16036), strongly suggest that HSP100 is structurally and functionally related to HSP90.
Actin Cytoskeleton↗
Initiation of DNA synthesis by microtubule disruption in quiescent rat 3Y1 cells.
Disruption of cytoplasmic microtubules by colchicine, colcemid or vinblastine induced the initiation of DNA synthesis and cell division in quiescent cultures of rat embryo fibroblast cell line, 3Y1, in the absence of growth factors. The microtubule-disruption-induced DNA synthesis was so marked that we could initiate analysis of the mechanism. Incubation of quiescent 3Y1 cells with high concentrations of vinblastine or those of vinblastine plus colcemid, which formed large tubulin paracrystals concomitant with the depolymerization of cytoplasmic microtubules, initiated DNA synthesis in the cells. Because these treatments did not increase the free tubulin level in the cells, but rather decreased it, an increase in the free tubulin level may not be required for initiation of DNA synthesis induced by microtubule-disrupting agents. Culture fluid of the 3Y1 cells incubated with colchicine did not stimulate the initiation of DNA synthesis when added to other quiescent 3Y1 cells after conversion of the colchicine in the culture fluid to lumicolchicine by ultraviolet irradiation. This suggested that colchicine treatment did not induce secretion of any mitogenic factors from 3Y1 cells. These results suggest that disruption of normal microtubules may elicit intracellular signals leading to cell proliferation.
Animals↗
An actin-interacting heptapeptide in the cofilin sequence.
Cofilin, a 21-kDa actin-binding protein, has a hexapeptide sequence DAIKKK which is identical to the N-terminal portion (residues 2-7) of tropomyosin. The synthetic heptapeptide, DAIKKKL, corresponding to residues 122-128 of cofilin, inhibited the binding of cofilin to F-actin in a dose-dependent manner. The heptapeptide cosedimented with F-actin, decreased the fluorescence intensity of pyrene-labeled F-actin, and increased the rate of polymerization of G-actin. The hexapeptides, DIKKKL and DAIKKL, also inhibited the binding of cofilin to F-actin and affected the fluorescence intensity of pyrene-labeled F-actin and the rate of actin polymerization, like the heptapeptide. However, their effects were weaker than those of the heptapeptide. Moreover, the pentapeptide, DIKKL, had little or no effect. These results suggest that the heptapeptide sequence is specific for the interaction with actin and, therefore, may constitute part of the actin-binding domain of cofilin.
Acrylic Resins↗
Regulation by intracellular Ca2+ and cyclic AMP of the growth factor-induced ruffling membrane formation and stimulation of fluid-phase endocytosis and exocytosis.
Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) induce formation of ruffling membranes [T. Kadowaki et al. (1986) J. Biol. Chem. 261, 16,141-16,147] and stimulate the fluid-phase endocytosis and exocytosis [Y. Miyata et al. (1988) Exp. Cell Res. 178, 73-83] in human epidermoid carcinoma KB cells. An increase in intracellular Ca2+ concentration by treatment with A23187, a calcium ionophore, or an increase in intracellular cAMP level by treatment with dibutyryl cAMP or forskolin almost completely inhibited the insulin-, IGF-I-, or EGF-induced formation of ruffling membranes. Increases in Ca2+ or cAMP concentration also inhibited almost completely the stimulation of fluid-phase endocytosis and exocytosis elicited by these growth factors. These results suggest that the growth factor-induced ruffling membrane formation and the stimulation of fluid-phase endocytosis and exocytosis have a common regulatory mechanism involving intracellular concentrations of Ca2+ and cAMP. 125I-EGF binding assays and immunoprecipitation experiments with anti-phosphotyrosine antibody revealed that treatment of KB cells with A23187, dibutyryl cAMP, or forskolin did not inhibit the EGF binding to the cells nor subsequent tyrosine autophosphorylation of its receptors. These results indicate that Ca2+- and/or cAMP-sensitive intracellular reactions exist downstream from the receptor kinase activation in the process of these early cellular responses.
Bucladesine↗
[Growth factor-induced changes in the actin architecture].
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Actins↗
[Regulation of mitogenic signal transduction by microtubules].
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Agglutination↗
Purification and characterization of the 90-kDa heat-shock protein from mammalian tissues.
The 90-kDa heat-shock protein (HSP90) has been purified from mammalian tissues, mouse liver and porcine brain, with a good yield by a new method involving hydrophobic chromatography. Mouse liver HSP90 and porcine brain HSP90 were compared with mouse lymphoma HSP90 which was purified from T lymphoma cell line, L5178Y, by a modification of the previously reported method. These three HSP90s were indistinguishable from one another in amino acid composition, one-dimensional peptide mapping, elution pattern of proteolytic fragments (trypsin- or V8-protease-cleaved) in reverse-phase high-performance liquid chromatography, reactivity with the antibody against mouse T lymphoma HSP90 and the ability to bind to F-actin. The amino acid sequences of three portions (total 47 amino acid residues) of lymphoma HSP90 were determined and they were homologous to those of the corresponding portions of Drosophila HSP83A and yeast HSP90. These results suggest that HSP90 is a highly conserved protein during evolution.
Amino Acid Sequence↗
Colchicine acts as a progression factor to initiate DNA synthesis in quiescent Balb/c 3T3 cells.
In quiescent Balb/c 3T3 cells, competence factors such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and platelet-derived growth factor (PDGF) synergize with progression factors such as insulin to initiate DNA synthesis. In this study, we found that colchicine, a microtubule-disrupting agent, acted synergistically with TPA, but not with insulin, to induce the maximal stimulation of DNA synthesis. Colchicine also synergized with PDGF in the presence of epidermal growth factor to elicit nearly the optimal induction of DNA synthesis. Moreover, it acted synergistically with fibroblast growth factor, another competence factor. These results suggest that colchicine acts as a progression factor like insulin in quiescent Balb/c 3T3 cells.
Animals↗
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↗
Protein-kinase-C-catalyzed phosphorylation of the microtubule-binding domain of microtubule-associated protein 2 inhibits its ability to induce tubulin polymerization.
It has previously been demonstrated that microtubule-associated protein 2 (MAP2) is a good substrate for the purified protein kinase C [Tsuyama, S., Bramblett, G. T., Huang, K.-P. & Flavin, M. (1986) J. Biol. Chem. 261, 4110-4116; Akiyama, T., Nishida, E., Ishida, J., Saji, N., Ogawara, H., Hoshi, M., Miyata, Y. & Sakai, H. (1986) J. Biol. Chem. 261, 15648-15651]. We have shown here that phosphorylation of MAP2, catalyzed by protein kinase C, reduces the ability to induce tubulin polymerization. MAP2 is divided into two domains by digestion with alpha-chymotrypsin; the microtubule-binding and the non-binding (projection) domains. The limited chymotryptic digestion following phosphorylation of MAP2 by protein kinase C has shown that both the domains of MAP2 were phosphorylated by protein kinase C, 50-60% of the incorporated phosphates being detected in the microtubule-binding domain. Polypeptide fragments, containing the microtubule-binding domain of MAP2, were purified by DEAE-cellulose column chromatography after chymotryptic digestion of MAP2. The purified microtubule-binding fragments were competent to polymerize tubulin, and served as good substrates for protein kinase C. The phosphorylation of the microtubule-binding fragments by protein kinase C reduced their ability to induce tubulin polymerization. These results suggest that the ability of MAP2 to induce tubulin polymerization is inhibited by phosphorylation of the microtubule-binding domain catalyzed by protein kinase C.
Actins↗