Organization of pp60src and selected cytoskeletal proteins within adhesion plaques and junctions of Rous sarcoma virus-transformed rat cells.

Abstract

The localization of pp60sr°within adhesion structures of epithelioid rat kidney cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus was compared to the organization of actin, a-actinin, vinculin (a 130,000-dalton protein), tubulin, and the 58,000-dalton intermediate filament protein . The adhesion structures included both adhesion plaques and previously uncharacterized adhesive regions formed at cell-cell junctions. We have termed these latter structures "adhesion junctions." Both adhesion plaques and adhesion junctions were identified by interference-reflection microscopy and compared to the location of pp60à nd the various cytoskeletal proteins by double fluorescence . The results demonstrated that the src gene product was found within both adhesion plaques and adhesion junctions. In addition, actin, a-actinin, and vinculin were also localized within the same pp60 sr°-containing adhesion structures . In contrast, tubulin and the 58,000-dalton intermediate filament protein were not associated with either adhesion plaques or adhesion junctions.Both adhesion plaques and adhesion junctions were isolated as substratum-bound structures and characterized by scanning electron microscopy. Immunofluorescence revealed that pp60grc, actin, a-actinin, and vinculin were organized within specific regions of the adhesion junctions. Heavy accumulations of actin and a-actinin were found on both sides of the junctions with a narrow gap of unstained material at the midline, whereas pp60 ,r, stain was more intense in this central region . Antibody to vinculin stained double narrow lines defining the periphery of the junctional complexes but was excluded from the intervening region . In addition, the distribution of vinculin relative to pp60 ,r, within adhesion plaques suggested an inverse relationship between the presence of these two proteins .Overall, these results establish a close link between the src gene product and components of the cytoskeleton and implicate the adhesion plaques and adhesion junctions in the mechanism of Rous sarcoma virus-induced transformation .Infection of susceptible chicken and mammalian cells with Rous sarcoma virus (RSV) leads to the genetically controlled expression of viral functions and the transformation of these cells to the neoplastic state. This transforming event is mediated by a single genetic locus within the viral genome called the sre gene (46). The product of the src gene is a 60,000-dalton phosphoprotein (pp60-; 3, 29, 34, 39) possessing a unique kinase activity capable of phosphorylating substrate proteins on tyrosine residues (21). Because of this enzymatic activity, transformation in this system has been proposed to involve phosphorylation of host cell proteins (8,21), and recent evidence has indicated that RSV-transformed cells possess ele-

N-WASP Involvement in Dorsal Ruffle Formation in Mouse Embryonic Fibroblasts

“…Tyrosine phosphorylation and hyperactivation of Src targets, such as dynamin and cortactin, could potentially compensate for N-WASP loss and enable highly abnormal dorsal ruffles to form. The similarities between dorsal ruffles and podosomes support this hypothesis, because podosomes spontaneously form in Src-transformed cells (Shriver and Rohrschneider, 1981). Interestingly, the wiskostatin treated MEFs and N-WASP null FLCs were still able to recruit Arp2/3 complex and form normal peripheral ruffles.…”

Suppression of src-induced transformed phenotype by expression of tropomyosin-1

“…V-SRC tyrosine kinase is one of the best-studied oncoproteins (Erpel and Courtneidge, 1995;Parsons and Parsons, 1997;Thomas and Brugge, 1997;Superti-Furga, 1995), and it is biochemically distinct from Ras, which is a GTPase. Moreover, SRC is closely associated with the reorganization of the cytoskeleton (Chang et al, 1995;Shriver and Rohrschneider, 1981) and its targets include many cytoskeletal proteins (Flynn et al, 1993;Nigg et al, 1986). Oncogenic forms of src have been reported to associate preferentially with the cytoskeletal compartment (Okamura and Resh, 1994;Kellie et al, 1993).…”

The interplay between the proteolytic, invasive, and adhesive domains of invadopodia and their roles in cancer invasion

“…6 Five years later, these v-src induced structures were defined by Tarone and Marchisio as "podosomes" 7 , due to their foot-like morphology. Later, more of their structural and signaling components, such as actin, 8 fimbrin, 9 and the oncogenic v-src itself, 10,11 were identified, and RSV-induced podosomes were shown by Parsons et al to be ECM contacts that also serve as sites of local matrix degradation. 11 Their capacity to invade and degrade the ECM led Chen, in 1989, to name these structures "invadopodia."…”