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. 1996 Nov 2;135(4):1109–1123. doi: 10.1083/jcb.135.4.1109

Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding

PMCID: PMC2133378  PMID: 8922390

Abstract

Paxillin is a 68-kD focal adhesion phosphoprotein that interacts with several proteins including members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal proteins vinculin and the tyrosine kinase, focal adhesion kinase (FAK). This suggests a function for paxillin as a molecular adaptor, responsible for the recruitment of structural and signaling molecules to focal adhesions. The current study defines the vinculin- and FAK-interaction domains on paxillin and identifies the principal paxillin focal adhesion targeting motif. Using truncation and deletion mutagenesis, we have localized the vinculin-binding site on paxillin to a contiguous stretch of 21 amino acids spanning residues 143-164. In contrast, maximal binding of FAK to paxillin requires, in addition to the region of paxillin spanning amino acids 143-164, a carboxyl-terminal domain encompassing residues 265-313. These data demonstrate the presence of a single binding site for vinculin, and at least two binding sites for FAK that are separated by an intervening stretch of 100 amino acids. Vinculin- and FAK-binding activities within amino acids 143-164 were separable since mutation of amino acid 151 from a negatively charged glutamic acid to the uncharged polar residue glutamine (E151Q) reduced binding of vinculin to paxillin by >90%, with no reduction in the binding capacity for FAK. The requirement for focal adhesion targeting of the vinculin- and FAK-binding regions within paxillin was determined by transfection into CHO.K1 fibroblasts. Significantly and surprisingly, paxillin constructs containing both deletion and point mutations that abrogate binding of FAK and/or vinculin were found to target effectively to focal adhesions. Additionally, expression of the amino-terminal 313 amino acids of paxillin containing intact vinculin- and FAK-binding domains failed to target to focal adhesions. This indicated other regions of paxillin were functioning as focal adhesion localization motifs. The carboxyl-terminal half of paxillin (amino acids 313-559) contains four contiguous double zinc finger LIM domains. Transfection analyses of sequential carboxyl-terminal truncations of the four individual LIM motifs and site-directed mutagenesis of LIM domains 1, 2, and 3, as well as deletion mutagenesis, revealed that the principal mechanism of targeting paxillin to focal adhesions is through LIM3. These data demonstrate that paxillin localizes to focal adhesions independent of interactions with vinculin and/or FAK, and represents the first definitive demonstration of LIM domains functioning as a primary determinant of protein subcellular localization to focal adhesions.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Arber S., Caroni P. Specificity of single LIM motifs in targeting and LIM/LIM interactions in situ. Genes Dev. 1996 Feb 1;10(3):289–300. doi: 10.1101/gad.10.3.289. [DOI] [PubMed] [Google Scholar]
  3. Avraham S., London R., Fu Y., Ota S., Hiregowdara D., Li J., Jiang S., Pasztor L. M., White R. A., Groopman J. E. Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain. J Biol Chem. 1995 Nov 17;270(46):27742–27751. doi: 10.1074/jbc.270.46.27742. [DOI] [PubMed] [Google Scholar]
  4. Backer J. M., Shoelson S. E., Weiss M. A., Hua Q. X., Cheatham R. B., Haring E., Cahill D. C., White M. F. The insulin receptor juxtamembrane region contains two independent tyrosine/beta-turn internalization signals. J Cell Biol. 1992 Aug;118(4):831–839. doi: 10.1083/jcb.118.4.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Belkin A. M., Ornatsky O. I., Glukhova M. A., Koteliansky V. E. Immunolocalization of meta-vinculin in human smooth and cardiac muscles. J Cell Biol. 1988 Aug;107(2):545–553. doi: 10.1083/jcb.107.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bellis S. L., Miller J. T., Turner C. E. Characterization of tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase. J Biol Chem. 1995 Jul 21;270(29):17437–17441. doi: 10.1074/jbc.270.29.17437. [DOI] [PubMed] [Google Scholar]
  7. Birge R. B., Fajardo J. E., Reichman C., Shoelson S. E., Songyang Z., Cantley L. C., Hanafusa H. Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts. Mol Cell Biol. 1993 Aug;13(8):4648–4656. doi: 10.1128/mcb.13.8.4648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burridge K., Fath K., Kelly T., Nuckolls G., Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol. 1988;4:487–525. doi: 10.1146/annurev.cb.04.110188.002415. [DOI] [PubMed] [Google Scholar]
  9. Burridge K., Turner C. E., Romer L. H. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol. 1992 Nov;119(4):893–903. doi: 10.1083/jcb.119.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen W. J., Goldstein J. L., Brown M. S. NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J Biol Chem. 1990 Feb 25;265(6):3116–3123. [PubMed] [Google Scholar]
  11. Clark E. A., Brugge J. S. Integrins and signal transduction pathways: the road taken. Science. 1995 Apr 14;268(5208):233–239. doi: 10.1126/science.7716514. [DOI] [PubMed] [Google Scholar]
  12. Coll J. L., Ben-Ze'ev A., Ezzell R. M., Rodríguez Fernández J. L., Baribault H., Oshima R. G., Adamson E. D. Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9161–9165. doi: 10.1073/pnas.92.20.9161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Crawford A. W., Michelsen J. W., Beckerle M. C. An interaction between zyxin and alpha-actinin. J Cell Biol. 1992 Mar;116(6):1381–1393. doi: 10.1083/jcb.116.6.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feuerstein R., Wang X., Song D., Cooke N. E., Liebhaber S. A. The LIM/double zinc-finger motif functions as a protein dimerization domain. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10655–10659. doi: 10.1073/pnas.91.22.10655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guan J. L., Shalloway D. Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation. Nature. 1992 Aug 20;358(6388):690–692. doi: 10.1038/358690a0. [DOI] [PubMed] [Google Scholar]
  16. Hanks S. K., Calalb M. B., Harper M. C., Patel S. K. Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8487–8491. doi: 10.1073/pnas.89.18.8487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hemmings L., Barry S. T., Critchley D. R. Cell-matrix adhesion: structure and regulation. Biochem Soc Trans. 1995 Aug;23(3):619–626. doi: 10.1042/bst0230619. [DOI] [PubMed] [Google Scholar]
  18. Hildebrand J. D., Schaller M. D., Parsons J. T. Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase. Mol Biol Cell. 1995 Jun;6(6):637–647. doi: 10.1091/mbc.6.6.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  20. Ilić D., Furuta Y., Kanazawa S., Takeda N., Sobue K., Nakatsuji N., Nomura S., Fujimoto J., Okada M., Yamamoto T. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature. 1995 Oct 12;377(6549):539–544. doi: 10.1038/377539a0. [DOI] [PubMed] [Google Scholar]
  21. Juliano R. L., Haskill S. Signal transduction from the extracellular matrix. J Cell Biol. 1993 Feb;120(3):577–585. doi: 10.1083/jcb.120.3.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kornberg L., Earp H. S., Parsons J. T., Schaller M., Juliano R. L. Cell adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase. J Biol Chem. 1992 Nov 25;267(33):23439–23442. [PubMed] [Google Scholar]
  23. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lev S., Moreno H., Martinez R., Canoll P., Peles E., Musacchio J. M., Plowman G. D., Rudy B., Schlessinger J. Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature. 1995 Aug 31;376(6543):737–745. doi: 10.1038/376737a0. [DOI] [PubMed] [Google Scholar]
  25. Li J., Avraham H., Rogers R. A., Raja S., Avraham S. Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes. Blood. 1996 Jul 15;88(2):417–428. [PubMed] [Google Scholar]
  26. Lo S. H., Chen L. B. Focal adhesion as a signal transduction organelle. Cancer Metastasis Rev. 1994 Mar;13(1):9–24. doi: 10.1007/BF00690415. [DOI] [PubMed] [Google Scholar]
  27. Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
  28. Miyamoto S., Teramoto H., Coso O. A., Gutkind J. S., Burbelo P. D., Akiyama S. K., Yamada K. M. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J Cell Biol. 1995 Nov;131(3):791–805. doi: 10.1083/jcb.131.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. O'Toole T. E., Ylanne J., Culley B. M. Regulation of integrin affinity states through an NPXY motif in the beta subunit cytoplasmic domain. J Biol Chem. 1995 Apr 14;270(15):8553–8558. doi: 10.1074/jbc.270.15.8553. [DOI] [PubMed] [Google Scholar]
  30. Okano I., Hiraoka J., Otera H., Nunoue K., Ohashi K., Iwashita S., Hirai M., Mizuno K. Identification and characterization of a novel family of serine/threonine kinases containing two N-terminal LIM motifs. J Biol Chem. 1995 Dec 29;270(52):31321–31330. doi: 10.1074/jbc.270.52.31321. [DOI] [PubMed] [Google Scholar]
  31. Rabbitts T. H., Boehm T. LIM domains. Nature. 1990 Aug 2;346(6283):418–418. doi: 10.1038/346418a0. [DOI] [PubMed] [Google Scholar]
  32. Ren R., Mayer B. J., Cicchetti P., Baltimore D. Identification of a ten-amino acid proline-rich SH3 binding site. Science. 1993 Feb 19;259(5098):1157–1161. doi: 10.1126/science.8438166. [DOI] [PubMed] [Google Scholar]
  33. Reszka A. A., Hayashi Y., Horwitz A. F. Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal association. J Cell Biol. 1992 Jun;117(6):1321–1330. doi: 10.1083/jcb.117.6.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rodríguez Fernández J. L., Geiger B., Salomon D., Ben-Ze'ev A. Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells. J Cell Biol. 1993 Sep;122(6):1285–1294. doi: 10.1083/jcb.122.6.1285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sadler I., Crawford A. W., Michelsen J. W., Beckerle M. C. Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton. J Cell Biol. 1992 Dec;119(6):1573–1587. doi: 10.1083/jcb.119.6.1573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Samuels M., Ezzell R. M., Cardozo T. J., Critchley D. R., Coll J. L., Adamson E. D. Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell. J Cell Biol. 1993 May;121(4):909–921. doi: 10.1083/jcb.121.4.909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sasaki H., Nagura K., Ishino M., Tobioka H., Kotani K., Sasaki T. Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily. J Biol Chem. 1995 Sep 8;270(36):21206–21219. doi: 10.1074/jbc.270.36.21206. [DOI] [PubMed] [Google Scholar]
  38. Schaller M. D., Borgman C. A., Parsons J. T. Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK. Mol Cell Biol. 1993 Feb;13(2):785–791. doi: 10.1128/mcb.13.2.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schaller M. D., Otey C. A., Hildebrand J. D., Parsons J. T. Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains. J Cell Biol. 1995 Sep;130(5):1181–1187. doi: 10.1083/jcb.130.5.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schaller M. D., Parsons J. T. pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk. Mol Cell Biol. 1995 May;15(5):2635–2645. doi: 10.1128/mcb.15.5.2635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shibanuma M., Mashimo J., Kuroki T., Nose K. Characterization of the TGF beta 1-inducible hic-5 gene that encodes a putative novel zinc finger protein and its possible involvement in cellular senescence. J Biol Chem. 1994 Oct 28;269(43):26767–26774. [PubMed] [Google Scholar]
  42. Sánchez-García I., Rabbitts T. H. The LIM domain: a new structural motif found in zinc-finger-like proteins. Trends Genet. 1994 Sep;10(9):315–320. doi: 10.1016/0168-9525(94)90034-5. [DOI] [PubMed] [Google Scholar]
  43. Tachibana K., Sato T., D'Avirro N., Morimoto C. Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK. J Exp Med. 1995 Oct 1;182(4):1089–1099. doi: 10.1084/jem.182.4.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Turner C. E., Glenney J. R., Jr, Burridge K. Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol. 1990 Sep;111(3):1059–1068. doi: 10.1083/jcb.111.3.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Turner C. E., Miller J. T. Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region. J Cell Sci. 1994 Jun;107(Pt 6):1583–1591. doi: 10.1242/jcs.107.6.1583. [DOI] [PubMed] [Google Scholar]
  47. Turner C. E. Paxillin: a cytoskeletal target for tyrosine kinases. Bioessays. 1994 Jan;16(1):47–52. doi: 10.1002/bies.950160107. [DOI] [PubMed] [Google Scholar]
  48. Turner C. E., Pietras K. M., Taylor D. S., Molloy C. J. Angiotensin II stimulation of rapid paxillin tyrosine phosphorylation correlates with the formation of focal adhesions in rat aortic smooth muscle cells. J Cell Sci. 1995 Jan;108(Pt 1):333–342. doi: 10.1242/jcs.108.1.333. [DOI] [PubMed] [Google Scholar]
  49. Turner C. E., Schaller M. D., Parsons J. T. Tyrosine phosphorylation of the focal adhesion kinase pp125FAK during development: relation to paxillin. J Cell Sci. 1993 Jul;105(Pt 3):637–645. doi: 10.1242/jcs.105.3.637. [DOI] [PubMed] [Google Scholar]
  50. Volberg T., Geiger B., Kam Z., Pankov R., Simcha I., Sabanay H., Coll J. L., Adamson E., Ben-Ze'ev A. Focal adhesion formation by F9 embryonal carcinoma cells after vinculin gene disruption. J Cell Sci. 1995 Jun;108(Pt 6):2253–2260. doi: 10.1242/jcs.108.6.2253. [DOI] [PubMed] [Google Scholar]
  51. Weng Z., Taylor J. A., Turner C. E., Brugge J. S., Seidel-Dugan C. Detection of Src homology 3-binding proteins, including paxillin, in normal and v-Src-transformed Balb/c 3T3 cells. J Biol Chem. 1993 Jul 15;268(20):14956–14963. [PubMed] [Google Scholar]
  52. Wood C. K., Turner C. E., Jackson P., Critchley D. R. Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin. J Cell Sci. 1994 Feb;107(Pt 2):709–717. [PubMed] [Google Scholar]
  53. Wu R. Y., Gill G. N. LIM domain recognition of a tyrosine-containing tight turn. J Biol Chem. 1994 Oct 7;269(40):25085–25090. [PubMed] [Google Scholar]

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