The Bovine Papillomavirus E6 Protein Binds to the LD Motif Repeats of Paxillin and Blocks Its Interaction with Vinculin and the Focal Adhesion Kinase

Abstract

The bovine papillomavirus type 1 (BPV-1) E6 oncoprotein can transform fibroblasts and induce anchorage-independent growth and disassembly of the actin stress fibers. We have previously shown that the E6 protein interacts with the focal adhesion protein, paxillin, suggesting a direct role of E6 in the disruption of the actin cytoskeleton. We have now mapped the E6 binding sites on paxillin to the LD motif repeats region, which has been implicated in mediating paxillin binding to two other focal adhesion proteins, vinculin and the focal adhesion kinase. The five LD motif repeats identified in paxillin do not contribute equally to its interaction with E6. The first LD repeat is most critical for paxillin binding to E6 both in vitro and in vivo. Furthermore, the binding of recombinant wild-type E6 protein to paxillin blocked the interaction of several cellular proteins with paxillin, including vinculin and the focal adhesion kinase. A mutant E6 protein (H105) which does not bind to paxillin had no effect on the binding of these cellular proteins to paxillin. These data suggest that E6 disruption of the actin stress fibers occurs through blocking the interaction of paxillin with its cellular effectors such as vinculin and the focal adhesion kinase.Cellular adhesion to the extracellular matrix involves signals that are essential in many processes including cell morphology, cell division, cell motility, and tumor metastasis (for review, see Refs. 1-4). Focal adhesions are specialized structures where cells adhere to the extracellular matrix through a network of actin cytoskeleton (for review, see Refs. 5 and 6). Focal adhesions contain integrins, tensin, vinculin, the focal adhesion kinase (FAK), 1 and paxillin. Paxillin is tyrosine phosphorylated in response to a variety of stimuli including cell adhesion, alterations in the actin cytoskeleton, and treatment with growth factors (7). Tyrosine phosphorylation of focal adhesion proteins is closely associated with changes in the structure of the actin cytoskeleton, although the precise downstream molecular consequences of such phosphorylation are currently unknown. Several lines of evidence have suggested that FAK is, at least in part, responsible for the tyrosine phosphorylation of paxillin observed in vivo (8, 9). Paxillin also binds to vinculin (10), an abundant cytoskeletal protein that is important in the assembly of the actin cytoskeleton, possibly through its interactions with other focal adhesion proteins, ␣-actinin, and actin (for review, see Ref. 6). The importance of a role for paxillin in cell signaling is suggested by the fact that paxillin is the target of several viral oncoproteins including v-Src (11), v-Crk (12), v-Abl (13), and the E6 oncoprotein of bovine papillomavirus type-1 (BPV-1) from our previous work (14). The interaction of paxillin with BPV-1 E6 is specific for transformation competent E6 proteins, and the expression of E6 results in the disruption of the actin stress fibers (14), suggesting that the binding of BPV-1 E6 to paxillin may be impor...

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…This sequence homology translates into a leucine and aspartic acid-rich consensus sequence, LDXLLXXL, after which this domain was named [66]. This sequence has been found repeated four times within the N-terminus of the paxillin protein, defining LD motifs 1, 2, 4 and 5 [66,100,101]. The fifth LD domain, denoted by LD3 and encoded by the highly divergent exon 5, was later proposed by Tong et al in 1997 [66].…”

The bovine papillomavirus type 1 E6 oncoprotein sensitizes cells to tumor necrosis factor alpha-induced apoptosis

“…However, a candidate cellular target for a potential BPV-1 E6/E6-AP complex has not been identi®ed. More recently, an interaction between the BPV-1 E6 protein and the focal adhesion associated protein paxillin as well as the trans-Golgi network-speci®c clathrin adaptor (AP-1) complex has been described (Tong and Howley, 1997;Tong et al, 1998;Vande Pol et al, 1997), although the biological relevance of these interactions remains to be established.…”

Bovine papillomaviruses, papillomas and cancer in cattle

“…E6 can transform fibroblasts and induce anchorage-independent growth and disassembly of the actin stress fibers by interacting with the focal adhesion protein, paxillin. E6 disruption of the actin stress fibers occurs by blocking the interaction of paxillin with its cellular effectors, such as vinculin and the focal adhesion kinase [135]. Finally, E6 interacts with AP-1, the TGN (trans-Golgi network)-specific clathrin adaptor complex.…”