Cell-Matrix Adhesions Differentially Regulate Fascin Phosphorylation

Abstract

Cell adhesion to individual macromolecules of the extracellular matrix has dramatic effects on the subcellular localization of the actin-bundling protein fascin and on the ability of cells to form stable fascin microspikes. The actin-binding activity of fascin is down-regulated by phosphorylation, and we used two differentiated cell types, C2C12 skeletal myoblasts and LLC-PK1 kidney epithelial cells, to examine the hypothesis that cell adhesion to the matrix components fibronectin, laminin-1, and thrombospondin-1 differentially regulates fascin phosphorylation. In both cell types, treatment with the PKC activator 12-tetradecanoyl phorbol 13-acetate (TPA) or adhesion to fibronectin led to a diffuse distribution of fascin after 1 h. C2C12 cells contain the PKC family members alpha, gamma, and lambda, and PKCalpha localization was altered upon cell adhesion to fibronectin. Two-dimensional isoelectric focusing/SDS-polyacrylamide gels were used to determine that fascin became phosphorylated in cells adherent to fibronectin and was inhibited by the PKC inhibitors calphostin C and chelerythrine chloride. Phosphorylation of fascin was not detected in cells adherent to thrombospondin-1 or to laminin-1. LLC-PK1 cells expressing green fluorescent protein (GFP)-fascin also displayed similar regulation of fascin phosphorylation. LLC-PK1 cells expressing GFP-fascin S39A, a nonphosphorylatable mutant, did not undergo spreading and focal contact organization on fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive negative charge spread more extensively than wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-1, and cells that expressed fascin S39D attached to thrombospondin-1 but did not form microspikes. Blockade of PKCalpha activity by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also evident in the presence of an inhibitory antibody to integrin alpha5 subunit. These novel results establish matrix-initiated PKC-dependent regulation of fascin phosphorylation at serine 39 as a mechanism whereby matrix adhesion is coupled to the organization of cytoskeletal structure.

Independent prognostic value of fascin immunoreactivity in stage I nonsmall cell lung cancer

“…Fascin immunoreactivity may thus be considered a more reliable tool for better assessing the biological aggressiveness and clinical course of NSCLC. Furthermore, the inhibition of fascin activity by phosphorylation (Yamakita et al, 1996, Ono et al, 1997, Tilney et al, 1998Adams et al, 1999, Cohan et al, 2001 or antisense oligonucleotide strategy (Al-Alwan et al, 2001) could represent potentially novel therapeutic options in the treatment of lung cancer. The validation of differentially expressed fascin levels in NSCLC could open new diagnostic and therapeutic perspectives, such as the development of radiologic imaging systems based on tagged antibody strategy to detect small lung cancers, either primary or metastatic, or the strategy of cell-mediated or antibody-based immunotherapy using fascin for targeting more aggressive lung carcinomas.…”

Dual Actin-bundling and Protein Kinase C-binding Activities of Fascin Regulate Carcinoma Cell Migration Downstream of Rac and Contribute to Metastasis

“…All antibodies, antibiotics and pharmacological reagents are listed in Table 1. GFP-fascin plasmids for Homo sapiens fascin-1 were as described previously (Adams et al, 1999;Adams and Schwartz, 2000). A GFP-Xenopus tropicalis fascin-1 expression plasmid was prepared by subcloning X. tropicalis fascin-1 cDNA (clone Thda 017B16; obtained from Cambridge University and The Wellcome Trust Sanger Institute and Wellcome Trust/Cancer Research UK Institute X. tropicalis EST project; Gilchrist et al, 2004) into pEGFP-C (Clontech, Mountain View, CA) by using the polymerase chain reaction (PCR) primers listed in Table 2.…”

“…In vitro, fascin cross-links filamentous actin (Factin) into tightly packed, parallel bundles in cooperation with Arp2/3 complex and Wiskott-Aldrich syndrome protein Haviv et al, 2006). In intact cells, fascin-and-actin bundles support cortical cell protrusions and growth cone filopodia Adams et al, 1999;Cohan et al, 2001, Adams andAnilkumar et al, 2003;Svitkina et al, 2003;Vignjevic et al, 2006). Fascin contains N-and C-terminal actin-binding sites and the actin cross-linking activity of fascin is negatively regulated by a protein kinase C (PKC) phosphorylation site (serine-39 in human fascin) that lies within the amino-terminal actin-binding site (Ono et al, 1997).…”

Fascin, an Actin-Bundling Protein, Modulates Colonic Epithelial Cell Invasiveness and Differentiation in Vitro

“…12 Ethical approval was obtained from the local research ethics committee of the United Bristol Health Care Trust. Rabbit polyclonal antiserum to fascin 17 was used for Western blotting and immunoprecipitation and a mouse monoclonal antibody obtained from DAKO was used for indirect immunofluorescence as previously described. 18,19 Mouse monoclonal antibodies to E-cadherin, and ␣-, ␤-, and ␥-catenin were purchased from Transduction Laboratories, Exeter, UK.…”

“…17 For transfection, a single cell suspension of the colonic epithelial carcinoma cell line SW1222, was prepared by trypsinization under calciumfree conditions. The cell suspension was passed through a 23-gauge needle three times and the formation of a single cell suspension confirmed by phase-contrast microscopy.…”