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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Malignant transformation of the ovarian surface epithelium (OSE) accounts for most ovarian carcinoma. Detection of preneoplastic changes in the OSE leading to overt malignancy is important in prevention and management of ovarian cancer. We identified OSE proteins with altered expression derived from women with a family history (FH) of ovarian and/or breast cancer and mutations in the BRCA1 tumor suppressor gene. Proteins from SV-40-transformed FH-OSE cell lines and control OSE lines derived from women without such histories (non-family history) were separated by two-dimensional PAGE. Gels were analyzed, a protein data base was created, and proteins were characterized according to their molecular weight, isoelectric point, and relative abundance. Mass spectrometry was performed on tryptic protein digests, and data bases were searched for known proteins with the same theoretical tryptic peptide masses. Several proteins showed altered expression in the FH-OSE cells. Beta-tubulin and to a lesser extent ubiquitin carboxyl-terminal hydrolase and glyoxalase 1 appeared to be up-regulated. In contrast, proteins suppressed in FH lines include the 27-kDa heat shock protein, translationally controlled tumor protein, and several proteins associated with actin modification such as actin prepeptide, F-actin capping protein alpha subunit, and cofilin. Sequencing of several cofilin gel spots revealed phosphorylation of serine 3, a post-translational modification associated with decreased actin binding and cytoskeletal reorganization. Two-dimensional Western blots probed with cofilin antibody showed multiple protein spots with isoelectric points of 6-9 pH units. Blots of one-dimensional gels showed a significant reduction in cofilin expression in three FH lines when compared with three non-family history lines (p < or = 0.05). Identification of these and other OSE proteins may be useful in detecting changes suggestive of increased risk of developing preneoplastic disease and defining the possible role(s) of the BRCA1 gene in regulation of OSE cell function.
Mol Cell Proteomics 2005 Feb
PMID:Proteome changes in ovarian epithelial cells derived from women with BRCA1 mutations and family histories of cancer. 1559 24

Paxillin is a prominent focal adhesion docking protein that regulates cell adhesion and migration. Although numerous paxillin-binding proteins have been identified and paxillin is required for normal embryogenesis, the precise mechanism by which paxillin functions in vivo has not yet been determined. We identified an ortholog of mammalian paxillin in Drosophila (Dpax) and have undertaken a genetic analysis of paxillin function during development. Overexpression of Dpax disrupted leg and wing development, suggesting a role for paxillin in imaginal disc morphogenesis. These defects may reflect a function for paxillin in regulation of Rho family GTPase signaling as paxillin interacts genetically with Rac and Rho in the developing eye. Moreover, a gain-of-function suppressor screen identified a genetic interaction between Dpax and cdi in wing development. cdi belongs to the cofilin kinase family, which includes the downstream Rho target, LIM kinase (LIMK). Significantly, strong genetic interactions were detected between Dpax and Dlimk, as well as downstream effectors of Dlimk. Supporting these genetic data, biochemical studies indicate that paxillin regulates Rac and Rho activity, positively regulating Rac and negatively regulating Rho. Taken together, these data indicate the importance of paxillin modulation of Rho family GTPases during development and identify the LIMK pathway as a critical target of paxillin-mediated Rho regulation.
Mol Cell Biol 2005 Feb
PMID:Regulation of Rho and Rac signaling to the actin cytoskeleton by paxillin during Drosophila development. 1565 26

I have monitored equilibrium binding of human cofilin to rabbit skeletal muscle (alpha) and human non-muscle (85% beta, 15% gamma) actin filaments from the quenching of pyrene actin fluorescence. Filament binding is cooperative and stoichiometric (i.e. one cofilin molecule per actin subunit) for both actin isoforms. The Hill coefficient for binding to betagamma-actin filaments (n(H)=3.5) is greater than for muscle actin (n(H)=2.3). Analysis of equilibrium binding using a nearest-neighbor cooperativity model indicates that the intrinsic affinities for binding to an isolated site are comparable (10-14 microM) for both filament isoforms but the cooperative free energy is greater for binding betagamma-actin filaments. The predicted cofilin cluster sizes and filament binding densities are small at concentrations of cofilin where efficient filament severing is observed, indicating that a few bound cofilin molecules are sufficient to destabilize the filament lattice and promote fragmentation. The analysis used in this study provides a framework for evaluating proton and ion linkage and effects of regulatory proteins on cofilin binding and severing of actin filaments.
J Mol Biol 2005 Feb 18
PMID:Cofilin binding to muscle and non-muscle actin filaments: isoform-dependent cooperative interactions. 1567 Jun 4

Insulin-like growth factor I (IGF-I) is a potent stimulator of neuroblastoma cell motility. Cell motility requires lamellipodium extension at the leading edge of the cell through organized actin polymerization, and IGF-I stimulates lamellipodial elaboration in human neuroblastoma cells. Rac is a Rho GTPase that stimulates lamellipodial formation via the regulation of actin polymerization. In this study, we show that IGF-I-stimulated phosphatidylinositol 3-kinase (PI-3K) activity promotes rac activation and subsequent activation of the down- stream effectors LIM kinase and cofilin. Overexpression of wild-type LIM kinase and wild-type Xenopus ADF/cofilin (XAC) suppresses IGF-I-stimulated motility in SH-SY5Y cells, while expression of dominant negative LIM kinase and constitutively active XAC increases SH-SY5Y motility in the absence of IGF-I stimulation. These results suggest that regulation by cofilin of actin depolymerization is important in the process of neuroblastoma cell motility, and IGF-I regulates cofilin activity in part through PI-3K, rac, and LIM kinase.
Cell Mol Life Sci 2005 Feb
PMID:Cofilin activity during insulin-like growth factor I-stimulated neuroblastoma cell motility. 1571 72

Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.
Mol Biol Cell 2005 Jul
PMID:Structural and functional dissection of the Abp1 ADFH actin-binding domain reveals versatile in vivo adapter functions. 1587 87

The significance of actin cytoskeleton on cell growth was historically studied using toxic drugs, such as cytochalasin. However, it is possible that unpredictable effects of these agents may have influenced the reported observations. In our study, we have established a drug-free system using cofilin overexpression to investigate the relationship between actin filaments and cell cycle progression. Cofilin is a member of the actin depolymerization factor (ADF)/cofilin family, cofilin cDNA was cloned to a tetracycline-inducible gene expression vector and stably transfected to human lung cancer H1299 epithelial cells. Destabilization of actin filaments and morphological change was detected in cofilin overexpressing cells by actin analysis and microscopy, respectively. Measurements of growth rates showed that cell proliferation was retarded in cells with overexpressed cofilin. Also, cell cycle analysis showed that approx 90% of cofilin overexpressing cells were arrested in G1 phase, which is consistent with previous reports that drug-mediated disruption of actin filaments can cause G1 phase arrest. Taken together, cofilin overexpression cell model provides evidence that the effects of actin cytoskeletal destabilization on cell cycle progression can be studied using molecular approach instead of drug.
Mol Biotechnol 2005 Sep
PMID:Studying the effects of actin cytoskeletal destabilization on cell cycle by cofilin overexpression. 1611 10

We have measured the effects of cofilin on the conformation and dynamics of actin filaments labeled at Cys374 with erythrosin-iodoacetemide (ErIA), using time-resolved phosphorescence anisotropy (TPA). Cofilin quenches the phosphorescence intensity of actin-bound ErIA, indicating that binding changes the local environment of the probe. The cofilin concentration-dependence of the phosphorescence intensity is sigmoidal, consistent with cooperative actin filament binding. Model-independent analysis of the anisotropies indicates that cofilin increases the rates of the microsecond rotational motions of actin. In contrast to the reduction in phosphorescence intensity, the changes in the rates of rotational motions display non-nearest-neighbor cooperative interactions and saturate at substoichiometric cofilin binding densities. Detailed analysis of the TPA decays indicates that cofilin decreases the torsional rigidity (C) of actin, increasing the thermally driven root-mean-square torsional angle between adjacent filament subunits from approximately 4 degrees (C = 2.30 x 10(-27) Nm2 radian(-1)) to approximately 17 degrees (C = 0.13 x 10(-27) Nm2 radian(-1)) at 25 degrees C. We favor a mechanism in which cofilin binding shifts the equilibrium between thermal ErIA-actin filament conformers, and facilitates two distinct structural changes in actin. One is local in nature, which affects the structure of actin's C terminus and is likely to mediate nearest-neighbor cooperative binding and filament severing. The second is a change in the internal dynamics of actin, which displays non-nearest-neighbor cooperativity and increases the torsional flexibility of filaments. The long-range effects of cofilin on the torsional dynamics of actin may accelerate P(i) release from filaments and modulate interactions with other regulatory actin filament binding proteins.
J Mol Biol 2005 Nov 11
PMID:Cofilin increases the torsional flexibility and dynamics of actin filaments. 1621 21

Colon cancer is among the leading causes of cancer death in North America. Dysregulation of the crypt homeostasis is evident in the early stage of colon cancer. Moreover, cytoskeletal rearrangement of actin also plays a crucial role in morphological changes during apoptosis. CD44, an adhesion and anti-apoptotic molecule is overexpressed in colon cancer and is known to interact with certain cytoskeletal proteins. In this study, we used the human colon cancer cell line SW620, which does not express CD44 but stably transfected with standard, 3-10v and 8-10v variant isoforms of CD44. By two-dimensional isoelectric focusing (2DIEF), we found an increasing concentration of a 21-kDa protein in SW620 colon cancer cells transfected with CD44 3-10v, as compared to cells transfected with an empty vector. Mass spectrometry (MS) and proteomic analysis of this protein identified the peptide fragment (YALYDATYETK) of 11 amino acids in length spanning residue 82 to 92 of cofilin, a widely distributed 21-kDa actin-modulating protein. Western blot analysis of lysates from cells expressing CD44 variant isoforms 3-10v had increased level of expression of cofilin compared to vector control consistent with our finding by 2DIEF. Also, immunocytochemistry showed that cofilin expression in colonic epithelial cells was greater in cells transfected with CD44 3-10v, as compared to vector controls. We observed that the phosphorylated form of cofilin is downregulated in cells expressing the 3-10v isoform of CD44 both by Western blot and immunocytochemistry. Cofilin expression is thus mechanistically associated with CD44 expression and its 3-10v isoform. Dephosphorylation of cofilin could bring about directional motility of cells that could have important implications to the proliferation and motility of colonic epithelial cells in cancer.
Exp Mol Pathol 2005 Dec
PMID:Upregulation and dephosphorylation of cofilin: modulation by CD44 variant isoform in human colon cancer cells. 1622 33

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.
J Mol Biol 2006 Feb 17
PMID:Cooperative effects of cofilin (ADF) on actin structure suggest allosteric mechanism of cofilin function. 1637 20

Xin and nebulette are striated muscle-specific actin-binding proteins that both contain multiple actin-binding repeats. The nature of these repeats is different: nebulette has nebulin-like repeats, while Xin contains its own unique repeats. However, the suggestion was made from biochemical data that the Xin-repeats may bind to multiple sites on the actin molecule as was found for nebulin. We have used electron microscopy and the iterative helical real space reconstruction to visualize complexes of F-actin with Xin fragments containing either three or six Xin-repeats, and with the CN5-nebulette fragment, containing five nebulin-like repeats. Our results indicate that Xin and nebulette fragments bind to F-actin in a similar manner and in two distinct modes: in one mode actin subdomain 1 is bound, while in the second mode the binding bridges between a different site on actin subdomains 1/2 of one protomer and subdomains 3/4 of an adjacent actin protomer. Taken together with published data about nebulin, tropomyosin and ADF/cofilin, our results suggest that the ability to bind in multiple modes to the actin protomer is a general property of many actin-binding proteins.
J Mol Biol 2006 Feb 24
PMID:Xin-repeats and nebulin-like repeats bind to F-actin in a similar manner. 1638 82


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