Paxillin: A New Vinculin-binding Protein Present in Focal Adhesions Christopher E. Turner,* John R. Glermey, Jr.,~ and Keith Burridge* * Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, North Carolina 27599-7090; ~Department of Biochemistry, The Markey Cancer Center, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0084 Abstract. The 68-kD protein (paxillin) is a cytoskele- other focal adhesion protein, vinculin. Cleavage of tal component that localizes to the focal adhesions at vinculin with Staphylococcus aureus V8 protease the ends of actin stress fibers in chicken embryo results in the generation of two fragments of ~85 and fibroblasts. It is also present in the focal adhesions of 27 kD. Unlike talin, which binds to the large vinculin Madin-Darby bovine kidney (MDBK) epithelial cells fragment, paxillin was found to bind to the small vin- but is absent, like talin, from the cell-cell adherens culin fragment, which represents the rod domain of junctions of these cells. Paxillin purified from chicken the molecule. Together with the previous observation gizzard smooth muscle migrates as a diffuse band on that paxillin is a major substrate of pp60 src in Rous SDS-PAGE gels with a molecular mass of 65-70 kD. sarcoma virus-transformed ceils (Glenney, J. R., and It is a protein of multiple isoforms with pls ranging L. Zokas. 1989. J. Cell Biol. 108:2401-2408), this in- from 6.31 to 6.85. Using purified paxillin, we have teraction with vinculin suggests paxillin may be a key demonstrated a specific interaction in vitro with an- component in the control of focal adhesion organization. r'll~riE linkage between actin filaments and the plasma ity present in partially purified vinculin preparations (Wil- | membrane of cells spread on plastic or glass in tissue kins and Lin, 1986; Wilkins et al., 1986). Unfortunately, lit- .A. culture has been demonstrated previously to involve tle is known with respect to the functional contribution of the recruitment of a variety of cytoskeletal proteins (for re- these and other as yet uncharacterized proteins present at view see Burridge et al., 1988), including talin (Burridge these sites. Similarly, the regulation of focal adhesion integ- and Connell, 1983), vinculin (Geiger, 1979; Burridge and rity is poorly understood. Feramisco, 1980) and ot-actinin (Lazarides and Burridge, Much interest has been directed towards a variety of reg- 1975). These specializations, where the cells come in closest ulatory proteins which localize to focal adhesions. Potential proximity to the underlying substratum, have been variously regulatory proteins include the calcium-dependent protease, referred to as focal adhesions, focal contacts or adhesion calpain II, which has been shown to be concentrated in the plaques. In vitro assays have enabled an apparent link be- focal adhesions of epithelial cells (Beckerle et al., 1987), and tween actin through these proteins to a family of glycopro- a number of kinases. The loss of focal adhesion organization teins, the integrins (for review see Hynes, 1987), to be sug- after the activation of protein kinase C by tumor promoters gested. In turn, the integrins span the plasma membrane and (Schliwa el al., 1984; Kellie et al., 1985; Meigs and Wang, interact with a variety of extracellular matrix proteins, such 1986) and the presence of protein kinase C at focal adhesions as fibronectin and vitronectin (Buck and Horwitz, 1987; Oaken et al., 1989) have led to a number of studies focusing Ruoslahti and Pierschbacher, 1987). on the possible role played by serine and threonine phosphor- Undoubtedly, other links to the membrane exist and a ylation of focal adhesion proteins, via the activation of pro- number of additional proteins, which localize to focal adhe- tein kinase C, in the reorganization of these structures (Werth sions, have been described. These include fimbrin (Bretscher et al., 1983; Werth and Pastan, 1984; Litchfield and Ball, and Weber, 1980), an actin-bundling protein (Bretscher, 1986; Turner et al., 1989; Beckerle, 1990). However, the 1981; Glenney et al., 1981), also present in large quantities stoichiometry of phosphorylation observed on many of these in intestinal microvilli (Bretscher, 1981; Glenney et al., proteins is very low (Turner et al., 1989), and it is difficult 1981), although its absolute requirement for focal adhesion to invoke a model for their involvement in the control of focal stability has recently been questioned (De Pasquale, J. A., adhesion integrity without envisaging rapid phosphorylation/ and C. S. Izzard, 1989. J. Cell Biol. 109:267a). An 82-kD dephosphorylation events or phosphorylation of specific sub- protein (Beckerle, 1986) is also present in focal adhesions populations of the proteins in question. Similarly, when chick in low abundance, as is tensin (Risinger, M. A., J. A. Wil- embryo fibroblasts (CEFs) ~are transformed by the R~us sar- kins, and S. Lin. 1987. J. Cell Biol. 105:130a), which has 1. Abbreviations used in this paper: CEF, chick embryo fibroblast; HAP, been proposed to be responsible for the actin-capping activ- hYdroxylapatite; ZA, zonula adherens. © The Rockefeller University Press, 0021-9525/90/09/1059/10 $2.00 The Journal of Cell Biology, Volume 111, September 1990 1059-1068 1059 coma viral tyrosine kinase pp60'rL the proteins talin, vin- in a Waring blender before centrifugation at 16000 g for 10 win in a 13SA culin, and integrin are substrates for phosphorylation (Scfton rotor (DuPont Corp., Wilmington, DE). The supernetant Was saved and the pellet resnspended at medium speed three times for 5 s each in a further et al., 1981; Hirst et al., 1986; Pasquale ct al., 1986; DeCluc 800 ml of the above buffer. The extract was centrifug~ as above and the and Martin, 1987). A direct correlation between these post- supernatants from the two washes were combined and filtered through six translational modifications and altered cell morphology has layers of fine cheesecloth. The volume was measured and paxillin was been questioned, since comparable levels of phosphorylation precipitated by adding solid (NHa)2SO4 (13.4 g/100 nil), with stirring for 60 rain. The precipitate was collected by centrifugation at 12,000 g for 10 of vinculin, for example, have been observed in cells con- rain and re.suspended in buffer B (20 mM Tris/acetate pH 7.6, 20 mM NaCI, talning a mutant variant of pp60 s~ lacking the membrane 0.1 mM EDTA, 0.1%/~-mercaptoethanol) plus 0.5 mM PMSF and 5 pM binding domain which does not give rise to a transformed leupeptin and dialyzed overnight against buffer B. Precipitated protein (mostly phenotype (Kamps et al., 1986). myosin) was removed by centrifugation at 100,000 g for 30 rain (45 Ti rotor; Recently, a number of mAbs have been generated against Beckman Instruments, Inc., FuUerton, CA) and the clarified supernatant was loaded onto a 100 ml DEAE-cellulose (DE 52; Whatman Biosystems phosphotyrosinc-containing proteins from RSV-transformed Ltd., Kent, UK) anion exchange column preequilibrated in buffer B. After CEFs (Glenncy and Zokas, 1989). One of these proteins, loading, the column was washed with 300 ml of buffer B and proteins were with a molecular mass of ,x,65-76 kD, localizes to focal eluted with a 650-ml salt gradient going from buffer B to buffer B containing adhesions in nontransformed cells. Upon transformation by 325 mM NaCi. Fractions containing protein were assayed by SDS-PAGE and Western blotting with the antipaxillin antibody. Relevant fractions were RSV, 20-30% of this protein is phosphorylated on tyrosinc pooled and loaded directly onto a 25-ml hydroxylapatite (HAP) column and its cellular distribution becomes more diffuse (Glenney equilibrated in buffer B. The proteins were eluted from the column with a and Zokas, 1989), suggesting that phosphorylation of this 15-240 mM potassium phosphate, pH 7.5 gradient (120 ml) containing protein may have a role in the disassembly of focal adhesions 0.1%/3-mercaptoethanol. Paxillin enriched fractions were pooled and dia- and stress fibers during transformation. In an effort to deter- lyzed against TBS and then loaded onto an antipaxillin antibody affinity column. Paxillin antibody was coupled to Affi-Gel 10 (Bio-Rad Laborato- mine the contribution of the 68-kD protein in focal adhesion ties, Cambridge, MA) according to the manufacturers' instructions. The organization we describe herein the purification and initial column was washed extensively with PBS and then prewasbed with 10 mM biochemical characterization of this protein from chicken sodium phosphate pH 6.8 before elution of the paxillin with 100 mM gly- gizzard smooth muscle. In addition, its interactions with cine, HCI, pH 2.5 into tubes containing sufficient Tris/HCl pH 10 to neu- other known focal adhesion proteins were investigated. In tralize the acid and 0.2% ~-mereaptoethanol. view of its localization to focal adhesions in nontransformcd CEFs we propose the name paxillin after the Latin "paxillus" Iodination meaning a small stake or peg, to fit in with the idea of pro- 60 td of a 1 mg/ml stock of Iodogen (Pierce Chemical Co., Rockford, IL) teins being tethered to the membrane at focal adhesions. in chloroform was dried onto the walls of an Eppendorf tube using a stream of nitrogen gas. 100-~d aliquots of paxillin or vincniin dialyzed into TBS were added to the tube in addition to 1 mCi carrier-free 125I (DuPont Materials and Methods Corp.) in 0.1 M potassium phosphate pH 7.2. The reaction was allowed to proceed for 7 min on ice before excess free iodine was quenched by the addi- tion of 20 ~tl saturated tyrosine solution and incubation for a further 5 rain Cell Culture on ice. Labeled protein was separated from free iodine by passage over a Chick embryo dermal fibroblasts taken from 10-d embryos were grown in 0.7 x 15 cm G 50 column (Pharmacia Fine Chemicals, Piscatawey, NJ), DME (Gibeo Laboratories, Grand Island, NY) supplemented with 10% preblocked with buffer B containing 0.3% BSA, and eluted with the same FBS (Gibeo Laboratories), 50/tl/mi streptomycin and 50 U/ml penicillin buffer.