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1.
The method of bimolecular fluorescence complementation (BiFC) enables selective visualization of protein interactions. While BiFC complex formation under in vitro conditions is considered to be essentially irreversible, there are hints that under in vivo conditions BiFC complex formation can be reversible. In the present study we used the BiFC method to visualize in vivo actin cytoskeleton dynamics. We demonstrate that in living cells formation of actin/actin BiFC complexes is reversible. Furthermore, we show heterologous binding between actin and protein kinase C delta (PKCdelta). Treatment with phorbol esters caused translocation of actin/PKCdelta complexes from the cytosol to the plasma membrane independent of an intact actin cytoskeleton. Our experiments demonstrate that the BiFC method might be a useful tool to investigate participation of the actin cytoskeleton in regulation of cell function. 相似文献
2.
Denis Riou Christos Roussakis Nelly Robillard Jean-Franois Biard Jean-Franois Verbist 《Biology of the cell / under the auspices of the European Cell Biology Organization》1993,77(3):261-264
Summary— Bistramide A, a new toxin isolated from a New Caledonian Urochordata, shows an antiproliferative effect on a non-small-cell lung carcinoma line in vitro and G1-blockade. In this work, the growth arrest induced by bistramide A was shown to be irreversible as assessed by growth kinetics of pretreated cells. Furthermore, the drug caused an underexpression of the nuclear antigen Ki67. These events are similar to a G1-differentiation cell cycle step blockage and a terminal maturation induction. 相似文献
3.
Lian JP Marks PG Wang JY Falls DL Badwey JA 《The Journal of biological chemistry》2000,275(4):2869-2876
Cofilin promotes the depolymerization of actin filaments, which is required for a variety of cellular responses such as the formation of lamellipodia and chemotaxis. Phosphorylation of cofilin on serine residue 3 is known to block these activities. We now report that neutrophils contain a protein kinase that selectively catalyzes the phosphorylation of cofilin on serine 3 (>/=70%) and a nonspecific kinase that recognizes multiple sites in this protein. The selective serine 3 cofilin kinase binds to a deoxyribonuclease I affinity column, whereas the nonspecific cofilin kinase does not. Deoxyribonuclease I forms a very tight complex with actin, and deoxyribonuclease affinity columns have been utilized to identify a variety of proteins that interact with the cytoskeleton. The serine 3 cofilin kinase did not react with antibodies to LIM kinase 1 or 2, which can catalyze the phosphorylation of cofilin in other cell types. The activity of the serine 3 cofilin kinase was insensitive to a variety of selective antagonists of protein kinases but was blocked by staurosporine. This pattern of inhibition is similar to that observed for the kinase that is active with cofilin in intact neutrophils. Thus, neutrophils contain a protein kinase distinct from LIM kinase-1/2 that selectively recognizes serine 3 in cofilin. 相似文献
4.
Actinis a 42-kDa protein which, due to its ability to polymerize into filaments (F-actin), is one of the major constituents of the cytoskeleton. It has been proposed that MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) proteins play an important role in regulating the structure and mechanical properties of the actin cytoskeleton by cross-linking actin filaments. We have recently reported that peptides corresponding to the effector domain of MARCKS proteins promote actin polymerization and cause massive bundling of actin filaments. We now investigate the effect of MARCKS-related protein, a 20-kDa member of the MARCKS family, on both filament structure and the kinetics of actin polymerization in vitro. Our experiments document that MRP binds to F-actin with micromolar affinity and that the myristoyl chain at the N-terminus of MRP is not required for this interaction. In marked contrast to the effector peptide, binding of MRP is not accompanied by an acceleration of actin polymerization kinetics, and we also could not reliably observe an actin cross-linking activity of MRP. 相似文献
5.
Plectin-controlled keratin cytoarchitecture affects MAP kinases involved in cellular stress response and migration
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Osmanagic-Myers S Gregor M Walko G Burgstaller G Reipert S Wiche G 《The Journal of cell biology》2006,174(4):557-568
Plectin is a major intermediate filament (IF)-based cytolinker protein that stabilizes cells and tissues mechanically, regulates actin filament dynamics, and serves as a scaffolding platform for signaling molecules. In this study, we show that plectin deficiency is a cause of aberrant keratin cytoskeleton organization caused by a lack of orthogonal IF cross-linking. Keratin networks in plectin-deficient cells were more susceptible to osmotic shock-induced retraction from peripheral areas, and their okadaic acid-induced disruption (paralleled by stress-activated MAP kinase p38 activation) proceeded faster. Basal activities of the MAP kinase Erk1/2 and of the membrane-associated upstream protein kinases c-Src and PKCdelta were significantly elevated, and increased migration rates, as assessed by in vitro wound-closure assays and time-lapse microscopy, were observed. Forced expression of RACK1, which is the plectin-binding receptor protein for activated PKCdelta, in wild-type keratinocytes elevated their migration potential close to that of plectin-null cells. These data establish a link between cytolinker-controlled cytoarchitecture/scaffolding functions of keratin IFs and specific MAP kinase cascades mediating distinct cellular responses. 相似文献
6.
The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain
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J Jongstra-Bilen P A Janmey J H Hartwig S Galea J Jongstra 《The Journal of cell biology》1992,118(6):1443-1453
The lymphocyte-specific phosphoprotein LSP1 associates with the cytoplasmic face of the plasma membrane and with the cytoskeleton. Mouse LSP1 protein contains 330 amino acids and contains an NH2-terminal acidic domain of approximately 177 amino acids. The COOH-terminal half of the LSP1 protein is rich in basic residues. In this paper we show that LSP1 protein which is immunoprecipitated with anti-LSP1 antibodies from NP-40-soluble lysates of the mouse B-lymphoma cell line BAL17 is associated with actin. In vitro binding experiments using recombinant LSP1 (rLSP1) protein and rabbit skeletal muscle actin show that LSP1 binds along the sides of F-actin but does not bind to G-actin. rLSP1 does not alter the initial polymerization kinetics of actin. The highly conserved COOH-terminal basic domains of mouse and human LSP1 share a significant homology with the 20-kD COOH-terminal F-actin binding fragment of caldesmon. A truncated rLSP1 protein containing the entire COOH-terminal basic domain from residue 179 to 330, but not the NH2-terminal acidic domain binds to F-actin at least as well as rLSP1. When LSP1/CAT fusion proteins are expressed in a LSP1-negative T-lymphoma cell line, only fusion proteins containing the basic COOH-terminal domain associate with the NP-40-insoluble cytoskeleton. These data show that LSP1 binds F-actin through its COOH-terminal basic domain and strongly suggest that LSP1 interacts with the cytoskeleton by direct binding to F-actin. We propose that LSP1 plays a role in mediating cytoskeleton driven responses in lymphocytes such as receptor capping, cell motility, or cell-cell interactions. 相似文献
7.
TRIP: a novel double stranded RNA binding protein which interacts with the leucine rich repeat of flightless I. 总被引:2,自引:1,他引:1
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A northwestern screen of a CHO-K1 cell line cDNA library with radiolabelled HIV-1 TAR RNA identified a novel TAR RNA interacting protein, TRIP. The human trip cDNA was also cloned and its expression is induced by phorbol esters. The N-terminus of TRIP shows high homology to the coiled coil domain of FLAP, a protein which binds the leucine-rich repeat (LRR) of Flightless I (FLI) and the interaction of TRIP with the FLI LRR has been confirmed in vitro . TRIP does not bind single stranded DNA or RNA significantly and binds double stranded DNA weakly. In contrast, TRIP binds double stranded RNA with high affinity and two molecules of TRIP bind the TAR stem. The RNA binding domain has been identified and encompasses a lysine-rich motif. A TRIP-GFP fusion is localised in the cytoplasm and excluded from the nucleus. FLI has a C-terminal gelsolin-like domain which binds actin and therefore the association of TRIP with the FLI LRR may provide a link between the actin cytoskeleton and RNA in mammalian cells. 相似文献
8.
Y Fu H W Liu S M Forsythe P Kogut J F McConville A J Halayko B Camoretti-Mercado J Solway 《Journal of applied physiology》2000,89(5):1985-1990
SM22 is a 201-amino acid actin-binding protein expressed at high levels in smooth muscle cells. It has structural homology to calponin, but how SM22 binds to actin remains unknown. We performed site-directed mutagenesis to generate a series of NH(2)-terminal histidine (His)-tagged mutants of human SM22 in Escherichia coli and used these to analyze the functional importance of potential actin binding domains. Purified full-length recombinant SM22 bound to actin in vitro, as demonstrated by cosedimentation assay. Binding did not vary with calcium concentration. The COOH-terminal domain of SM22 is required for actin affinity, because COOH terminally truncated mutants [SM22-(1-186) and SM22-(1-166)] exhibited markedly reduced cosedimentation with actin, and no actin binding of SM22-(1-151) could be detected. Internal deletion of a putative actin binding site (154-KKAQEHKR-161) partially prevented actin binding, as did point mutation to neutralize either or both pairs of positively charged residues at the ends of this region (KK154LL and/or KR160LL). Internal deletion of amino acids 170-180 or 170-186 also partially or almost completely inhibited actin cosedimentation, respectively. Of the three consensus protein kinase C or casein kinase II phosphorylation sites in SM22, only Ser-181 was readily phosphorylated by protein kinase C in vitro, and such phosphorylation greatly decreased actin binding. Substitution of Ser-181 to aspartic acid (to mimic serine phosphorylation) also reduced actin binding. Immunostains of transiently transfected airway myocytes revealed that full-length NH(2)-terminal FLAG-tagged SM22 colocalizes with actin filaments, whereas FLAG-SM22-(1-151) does not. These data confirm that SM22 binds to actin in vitro and in vivo and, for the first time, demonstrate that multiple regions within the COOH-terminal domain are required for full actin affinity. 相似文献
9.
Amlexanox reversibly inhibits cell migration and proliferation and induces the Src-dependent disassembly of actin stress fibers in vitro 总被引:2,自引:0,他引:2
Landriscina M Prudovsky I Mouta Carreira C Soldi R Tarantini F Maciag T 《The Journal of biological chemistry》2000,275(42):32753-32762
Amlexanox binds S100A13 and inhibits the release of fibroblast growth factor 1 (FGF1). Because members of the S100 gene family are known to be involved with the function of the cytoskeleton, we examined the ability of amlexanox to modify the cytoskeleton and report that amlexanox induces a dramatic reduction in the presence of actin stress fibers and the appearance of a random, non-oriented distribution of focal adhesion sites. Correspondingly, amlexanox induces the complete and reversible non-apoptotic inhibition of cell migration and proliferation, and although amlexanox does not induce either the down-regulation of F-actin levels or the depolymerization of actin filaments, it does induce the tyrosine phosphorylation of cortactin, a Src substrate known to regulate actin bundling. In addition, a dominant negative form of Src is able to partially rescue cells from the effect of amlexanox on both the actin cytoskeleton and cell migration. In contrast, the inhibition of cell proliferation by amlexanox correlates with the inhibition of cyclin D1 expression without interference of the receptor tyrosine kinase/mitogen-activated protein kinase signaling pathway. Last, the ability of amlexanox to inhibit FGF1 release is reversible and correlates with the restoration of the actin cytoskeleton, suggesting a role for the actin cytoskeleton in the FGF1 release pathway. 相似文献
10.
Frank Wohnsland Michel O. Steinmetz Ueli Aebi Guy Vergres 《Journal of structural biology》2000,131(3):217
Actinis a 42-kDa protein which, due to its ability to polymerize into filaments (F-actin), is one of the major constituents of the cytoskeleton. It has been proposed that MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) proteins play an important role in regulating the structure and mechanical properties of the actin cytoskeleton by cross-linking actin filaments. We have recently reported that peptides corresponding to the effector domain of MARCKS proteins promote actin polymerization and cause massive bundling of actin filaments. We now investigate the effect of MARCKS-related protein, a 20-kDa member of the MARCKS family, on both filament structure and the kinetics of actin polymerization in vitro. Our experiments document that MRP binds to F-actin with micromolar affinity and that the myristoyl chain at the N-terminus of MRP is not required for this interaction. In marked contrast to the effector peptide, binding of MRP is not accompanied by an acceleration of actin polymerization kinetics, and we also could not reliably observe an actin cross-linking activity of MRP. 相似文献
11.
Vetterkind S Illenberger S Kubicek J Boosen M Appel S Naim HY Scheidtmann KH Preuss U 《Experimental cell research》2005,305(2):392-408
Prostate apoptosis response-4 (Par-4) is a 38-kDa protein originally identified as a gene product upregulated in prostate cancer cells undergoing apoptosis. Cell death mediated by Par-4 and its interaction partner DAP like kinase (Dlk) is characterized by dramatic changes of the cytoskeleton. To uncover the role of the cytoskeleton in Par-4/Dlk-mediated apoptosis, we analyzed Par-4 for a direct association with cytoskeletal structures. Confocal fluorescence microscopy revealed that endogenous Par-4 is specifically associated with stress fibers in rat fibroblasts. In vitro cosedimentation analyses and in vivo FRET analyses showed that Par-4 directly binds to F-actin. Actin binding is mediated by the N-terminal 266 amino acids, but does not require the C-terminal region of Par-4 containing the leucine zipper and the death domain. Furthermore, the interaction of Par-4 with actin filaments leads to the formation of actin bundles in vitro and in vivo. In rat fibroblasts, this microfilament association is essential for the pro-apoptotic function of Par-4, since both disruption of the actin cytoskeleton by cytochalasin D treatment and overexpression of Par-4 constructs impaired in actin binding result in a significant decrease of apoptosis induction by Par-4 and Dlk. We propose a model, in which Par-4 recruits Dlk to stress fibers, leading to enhanced phosphorylation of the regulatory light chain of myosin II (MLC) and to the induction of apoptosis. 相似文献
12.
Sklyarova T De Corte V Meerschaert K Devriendt L Vanloo B Bailey J Cook LJ Goethals M Van Damme J Puype M Vandekerckhove J Gettemans J 《The Journal of biological chemistry》2002,277(42):39840-39849
We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity. Actin binding by fragmin60 requires higher calcium concentrations than does binding of actin by a fragmin60 mutant lacking the C2 domain, suggesting that the C2 domain secures the actin binding moiety in a conformation preventing actin binding at low calcium concentrations. The fragmin60 C2 domain does not bind phospholipids but interacts with the endogenous homologue of Saccharomyces cerevisiae S-phase kinase-associated protein (Skp1), as shown by pull-down assays and co-expression in mammalian cells. Recombinant fragmin60 promotes in vitro phosphorylation of actin Thr-203 by the actin-fragmin kinase. We further show that in vivo phosphorylation of actin in the fragmin60-actin complex occurs in sclerotia, a dormant stage of Physarum development, as well as in plasmodia. Our findings indicate that we have cloned a novel type of gelsolin-related actin-binding protein that is involved in controlling regulation of actin phosphorylation in vivo. 相似文献
13.
GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro
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Blader IJ Cope MJ Jackson TR Profit AA Greenwood AF Drubin DG Prestwich GD Theibert AB 《Molecular biology of the cell》1999,10(3):581-596
Recent cloning of a rat brain phosphatidylinositol 3,4, 5-trisphosphate binding protein, centaurin alpha, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin alpha, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Delta) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Delta was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae. 相似文献
14.
Palmgren S Ojala PJ Wear MA Cooper JA Lappalainen P 《The Journal of cell biology》2001,155(2):251-260
Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells. 相似文献
15.
Chen HC Chen CH Chuang NN 《Journal of experimental zoology. Part A, Comparative experimental biology》2004,301(4):307-316
Phosphotyrosyl protein phosphatase (PTPase) 1B was purified from human placenta. Immunoprecipitation analysis revealed that the isolated PTPase 1B appears as a complex with the receptor for protein kinase C (RACK1) and protein kinase C (PKC)delta. The abilities of PTPase 1B and PKCdelta to associate with RACK1 were reconfirmed by an in vitro reconstitution experiment. The E. coli expressed and biotinylated mice-RACK1-encoded fusion protein was capable of recruiting PTPase 1B and PKCdelta in the antibiotin immunoprecipitate as a complex of PTPase 1B/RACK1/PKCdelta. Thus PTPase 1B enzyme preparation was subjected to further purification by selective binding of PTPase 1B onto PEP(Taxol) affinity column in the absence of ATP. The purified PTPase 1B enzyme exihibited dose-dependent phosphatase activity towards [gamma-(32)P]-ATP labeled mice beta-tubulin-encoded fusion protein. The dephosphorylation reaction with PTPase 1B was enhanced with geranylgeranyl pyrophosphate, but not with farnesyl pyrophosphate. Interestingly, additional incubation of the purified PTPase 1B enzyme preparation with RACK1, geranylgeranyl pyrophosphate failed to modulate the dephosphorylation activity of PTPase 1B. In contrast, the enhancement effect of farnesyl pyrophosphate on the kinase activity of PKCdelta was sustained in the presence of RACK1. That is, farnesyl pyrophosphate may function as a signal to induce the kinase activity of PKCdelta in PTPase 1B/RACK1/PKCdelta complex but geranylgeranyl pyrophosphate may not for PTPase 1B. J. Exp. Zool. 301A:307-316, 2004. 相似文献
16.
Legendre-Guillemin V Metzler M Charbonneau M Gan L Chopra V Philie J Hayden MR McPherson PS 《The Journal of biological chemistry》2002,277(22):19897-19904
Huntingtin-interacting protein 1 (HIP1) and HIP12 are orthologues of Sla2p, a yeast protein with essential functions in endocytosis and regulation of the actin cytoskeleton. We now report that HIP1 and HIP12 are major components of the clathrin coat that interact but differ in their ability to bind clathrin and the clathrin adaptor AP2. HIP1 contains a clathrin-box and AP2 consensus-binding sites that display high affinity binding to the terminal domain of the clathrin heavy chain and the ear domain of the AP2 alpha subunit, respectively. These consensus sites are poorly conserved in HIP12 and correspondingly, HIP12 does not bind to AP2 nor does it demonstrate high affinity clathrin binding. Moreover, HIP12 co-sediments with F-actin in contrast to HIP1, which exhibits no interaction with actin in vitro. Despite these differences, both proteins efficiently stimulate clathrin assembly through their central helical domain. Interestingly, in both HIP1 and HIP12, this domain binds directly to the clathrin light chain. Our data suggest that HIP1 and HIP12 play related yet distinct functional roles in clathrin-mediated endocytosis. 相似文献
17.
18.
The Wiscott-Aldrich syndrome protein, WASP, is an effector through which cdc42, a Rho family GTPase, regulates the actin cytoskeleton in hematopoietic cells. We have found that WASP binds readily to a number of tyrosine protein kinases including the Src kinases and the Abl kinase when the proteins are coexpressed during transient transfection. Binding inhibited the activity of each of these kinases strikingly, both in vitro and in vivo. Surprisingly, the binding was not due to an interaction between the proline-rich domain of WASP and the SH3 domain of these kinases. Rather, residues 83-93 in WASP were found to bind to the catalytic domains of the kinases. Binding did not decrease the affinity of Src kinases for either ATP or a peptide substrate noticeably. Rather, the V(max) of substrate phosphorylation was reduced by the binding of the peptide. This inhibition represents a novel form of regulation of protein kinase activity and suggests that that the isolation of small molecules that exploit this inhibitory mechanism may be possible. 相似文献
19.
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. 相似文献
20.
Osmotic stress induces activation of an adaptive mitogen-activated protein kinase pathway in concert with disassembly of the actin cytoskeleton by a mechanism that is not understood. We have previously shown that the conserved actin-interacting MAP kinase kinase kinase Ssk2p/MEKK4, a member of the high-osmolarity glycerol (HOG) MAPK pathway of Saccharomyces cerevisiae, mediates recovery of the actin cytoskeleton following osmotic stress. In this study, we have employed in vitro kinase assays to show that Ssk2p kinase activity is activated for the actin recovery pathway via a noncanonical, Ssk1p-independent mechanism. Our work also shows that Ssk2p requires the polarisome proteins Bud6p and Pea2p to promote efficient, polarized actin reassembly but that this requirement can be bypassed by overexpression of Ssk2p. Formin (BNI1 or BNR1) and tropomyosin functions are also required for actin recovery but, unlike for Bud6p and Pea2p, these requirements cannot be bypassed by overexpression of Ssk2p. These results suggest that Ssk2p acts downstream of Bud6p and Pea2p and upstream of tropomyosin to drive actin recovery, possibly by upregulating the actin nucleation activity of the formins. 相似文献