Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Sep 1;126(5):1299–1309. doi: 10.1083/jcb.126.5.1299

Altered localization and cytoplasmic domain-binding properties of tyrosine-phosphorylated beta 1 integrin

PMCID: PMC2120155  PMID: 7520449

Abstract

We describe a novel approach to study tyrosine-phosphorylated (PY) integrins in cells transformed by virally encoded tyrosine kinases. We have synthesized a peptide (PY beta 1 peptide) that represents a portion of the cytoplasmic domain of the beta 1 integrin subunit and is phosphorylated on the tyrosine residue known to be the target of oncogenic tyrosine kinases. Antibodies prepared against the PY beta 1 peptide, after removal of cross-reacting antibodies by absorption and affinity purification, recognized the PY beta 1 peptide and the tyrosine-phosphorylated form of the intact beta 1 subunit, but did not bind the nonphosphorylated beta 1 peptide, the nonphosphorylated beta 1 subunit or other unrelated tyrosine-phosphorylated proteins. The anti- PY beta 1 antibodies labeled the podosomes of Rous sarcoma virus- transformed fibroblasts, but did not detectably stain nontransformed fibroblasts. The localization of the tyrosine phosphorylated beta 1 subunits appeared distinct from that of the beta 1 subunit. Adhesion plaques were stained by the anti-beta 1 subunit antibodies in Rous sarcoma virus-transformed fibroblasts plated on fibronectin, whereas neither podosomes nor adhesion plaques were labeled on vitronectin or on uncoated plates. Anti-phosphotyrosine antibodies labeled podosomes, adhesion plaques and cell-cell boundaries regardless of the substratum. One of the SH2 domains of the p85 subunit of phosphatidylinositol-3- kinase bound to the PY beta 1 peptide, but not to the non- phosphorylated beta 1 cytoplasmic peptide. Other SH2 domains did not bind to the PY beta 1 peptide. These results show that the phosphorylated form of the beta 1 integrin subunit is detected in a different subcellular localization than the nonphosphorylated form and suggest that the phosphorylation on tyrosine of the beta 1 subunit cytoplasmic domain may affect cellular signaling pathways.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Argraves W. S., Suzuki S., Arai H., Thompson K., Pierschbacher M. D., Ruoslahti E. Amino acid sequence of the human fibronectin receptor. J Cell Biol. 1987 Sep;105(3):1183–1190. doi: 10.1083/jcb.105.3.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bangalore L., Tanner A. J., Laudano A. P., Stern D. F. Antiserum raised against a synthetic phosphotyrosine-containing peptide selectively recognizes p185neu/erbB-2 and the epidermal growth factor receptor. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11637–11641. doi: 10.1073/pnas.89.23.11637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Behrens J., Vakaet L., Friis R., Winterhager E., Van Roy F., Mareel M. M., Birchmeier W. Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-sensitive v-SRC gene. J Cell Biol. 1993 Feb;120(3):757–766. doi: 10.1083/jcb.120.3.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benoit R., Böhlen P., Ling N., Briskin A., Esch F., Brazeau P., Ying S. Y., Guillemin R. Presence of somatostatin-28-(1-12) in hypothalamus and pancreas. Proc Natl Acad Sci U S A. 1982 Feb;79(3):917–921. doi: 10.1073/pnas.79.3.917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  7. Chen W. T., Chen J. M., Parsons S. J., Parsons J. T. Local degradation of fibronectin at sites of expression of the transforming gene product pp60src. Nature. 1985 Jul 11;316(6024):156–158. doi: 10.1038/316156a0. [DOI] [PubMed] [Google Scholar]
  8. Chen W. T., Wang J., Hasegawa T., Yamada S. S., Yamada K. M. Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides. J Cell Biol. 1986 Nov;103(5):1649–1661. doi: 10.1083/jcb.103.5.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Czernik A. J., Girault J. A., Nairn A. C., Chen J., Snyder G., Kebabian J., Greengard P. Production of phosphorylation state-specific antibodies. Methods Enzymol. 1991;201:264–283. doi: 10.1016/0076-6879(91)01025-w. [DOI] [PubMed] [Google Scholar]
  10. David-Pfeuty T., Singer S. J. Altered distributions of the cytoskeletal proteins vinculin and alpha-actinin in cultured fibroblasts transformed by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6687–6691. doi: 10.1073/pnas.77.11.6687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Davis S., Lu M. L., Lo S. H., Lin S., Butler J. A., Druker B. J., Roberts T. M., An Q., Chen L. B. Presence of an SH2 domain in the actin-binding protein tensin. Science. 1991 May 3;252(5006):712–715. doi: 10.1126/science.1708917. [DOI] [PubMed] [Google Scholar]
  12. Fitzgerald L. A., Steiner B., Rall S. C., Jr, Lo S. S., Phillips D. R. Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. Identity with platelet glycoprotein IIIa and similarity to "integrin". J Biol Chem. 1987 Mar 25;262(9):3936–3939. [PubMed] [Google Scholar]
  13. Freed E., Gailit J., van der Geer P., Ruoslahti E., Hunter T. A novel integrin beta subunit is associated with the vitronectin receptor alpha subunit (alpha v) in a human osteosarcoma cell line and is a substrate for protein kinase C. EMBO J. 1989 Oct;8(10):2955–2965. doi: 10.1002/j.1460-2075.1989.tb08445.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Giancotti F. G., Comoglio P. M., Tarone G. A 135,000 molecular weight plasma membrane glycoprotein involved in fibronectin-mediated cell adhesion. Immunofluorescence localization in normal and RSV-transformed fibroblasts. Exp Cell Res. 1986 Mar;163(1):47–62. doi: 10.1016/0014-4827(86)90557-4. [DOI] [PubMed] [Google Scholar]
  15. Giancotti F. G., Ruoslahti E. Elevated levels of the alpha 5 beta 1 fibronectin receptor suppress the transformed phenotype of Chinese hamster ovary cells. Cell. 1990 Mar 9;60(5):849–859. doi: 10.1016/0092-8674(90)90098-y. [DOI] [PubMed] [Google Scholar]
  16. Glenney J. R., Jr, Zokas L. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol. 1989 Jun;108(6):2401–2408. doi: 10.1083/jcb.108.6.2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Haimovich B., Aneskievich B. J., Boettiger D. Cellular partitioning of beta-1 integrins and their phosphorylated forms is altered after transformation by Rous sarcoma virus or treatment with cytochalasin D. Cell Regul. 1991 Apr;2(4):271–283. doi: 10.1091/mbc.2.4.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hamaguchi M., Matsuyoshi N., Ohnishi Y., Gotoh B., Takeichi M., Nagai Y. p60v-src causes tyrosine phosphorylation and inactivation of the N-cadherin-catenin cell adhesion system. EMBO J. 1993 Jan;12(1):307–314. doi: 10.1002/j.1460-2075.1993.tb05658.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hirst R., Horwitz A., Buck C., Rohrschneider L. Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6470–6474. doi: 10.1073/pnas.83.17.6470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Horvath A. R., Elmore M. A., Kellie S. Differential tyrosine-specific phosphorylation of integrin in Rous sarcoma virus transformed cells with differing transformed phenotypes. Oncogene. 1990 Sep;5(9):1349–1357. [PubMed] [Google Scholar]
  21. Horwitz A., Duggan K., Buck C., Beckerle M. C., Burridge K. Interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage. Nature. 1986 Apr 10;320(6062):531–533. doi: 10.1038/320531a0. [DOI] [PubMed] [Google Scholar]
  22. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Kanner S. B., Reynolds A. B., Wang H. C., Vines R. R., Parsons J. T. The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110. EMBO J. 1991 Jul;10(7):1689–1698. doi: 10.1002/j.1460-2075.1991.tb07693.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  26. LaFlamme S. E., Akiyama S. K., Yamada K. M. Regulation of fibronectin receptor distribution. J Cell Biol. 1992 Apr;117(2):437–447. doi: 10.1083/jcb.117.2.437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Larsson E., Lüning B., Heinegård D. Synthesis and function of an O-phosphorylated peptide corresponding to the cell adhesion sequence of bone sialoprotein (BSP). Acta Chem Scand. 1993 Jun;47(6):565–569. doi: 10.3891/acta.chem.scand.47-0565. [DOI] [PubMed] [Google Scholar]
  28. Maher P. A., Pasquale E. B., Wang J. Y., Singer S. J. Phosphotyrosine-containing proteins are concentrated in focal adhesions and intercellular junctions in normal cells. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6576–6580. doi: 10.1073/pnas.82.19.6576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Marchisio P. C., Cirillo D., Teti A., Zambonin-Zallone A., Tarone G. Rous sarcoma virus-transformed fibroblasts and cells of monocytic origin display a peculiar dot-like organization of cytoskeletal proteins involved in microfilament-membrane interactions. Exp Cell Res. 1987 Mar;169(1):202–214. doi: 10.1016/0014-4827(87)90238-2. [DOI] [PubMed] [Google Scholar]
  30. Matsuyoshi N., Hamaguchi M., Taniguchi S., Nagafuchi A., Tsukita S., Takeichi M. Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts. J Cell Biol. 1992 Aug;118(3):703–714. doi: 10.1083/jcb.118.3.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Miura O., Nakamura N., Ihle J. N., Aoki N. Erythropoietin-dependent association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated erythropoietin receptor. J Biol Chem. 1994 Jan 7;269(1):614–620. [PubMed] [Google Scholar]
  32. Mueller S. C., Chen W. T. Cellular invasion into matrix beads: localization of beta 1 integrins and fibronectin to the invadopodia. J Cell Sci. 1991 Jun;99(Pt 2):213–225. doi: 10.1242/jcs.99.2.213. [DOI] [PubMed] [Google Scholar]
  33. Otey C. A., Pavalko F. M., Burridge K. An interaction between alpha-actinin and the beta 1 integrin subunit in vitro. J Cell Biol. 1990 Aug;111(2):721–729. doi: 10.1083/jcb.111.2.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Otey C. A., Vasquez G. B., Burridge K., Erickson B. W. Mapping of the alpha-actinin binding site within the beta 1 integrin cytoplasmic domain. J Biol Chem. 1993 Oct 5;268(28):21193–21197. [PubMed] [Google Scholar]
  35. Pasquale E. B., Maher P. A., Singer S. J. Talin is phosphorylated on tyrosine in chicken embryo fibroblasts transformed by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5507–5511. doi: 10.1073/pnas.83.15.5507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pytela R., Pierschbacher M. D., Ruoslahti E. Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell. 1985 Jan;40(1):191–198. doi: 10.1016/0092-8674(85)90322-8. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Ruoslahti E., Giancotti F. G. Integrins and tumor cell dissemination. Cancer Cells. 1989 Dec;1(4):119–126. [PubMed] [Google Scholar]
  39. Ruoslahti E. Integrins. J Clin Invest. 1991 Jan;87(1):1–5. doi: 10.1172/JCI114957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sarin V. K., Kent S. B., Tam J. P., Merrifield R. B. Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction. Anal Biochem. 1981 Oct;117(1):147–157. doi: 10.1016/0003-2697(81)90704-1. [DOI] [PubMed] [Google Scholar]
  41. Sefton B. M., Hunter T., Ball E. H., Singer S. J. Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus. Cell. 1981 Apr;24(1):165–174. doi: 10.1016/0092-8674(81)90512-2. [DOI] [PubMed] [Google Scholar]
  42. Sefton B. M., Hunter T., Beemon K., Eckhart W. Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus. Cell. 1980 Jul;20(3):807–816. doi: 10.1016/0092-8674(80)90327-x. [DOI] [PubMed] [Google Scholar]
  43. Singer I. I., Scott S., Kawka D. W., Kazazis D. M., Gailit J., Ruoslahti E. Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation. J Cell Biol. 1988 Jun;106(6):2171–2182. doi: 10.1083/jcb.106.6.2171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Solowska J., Guan J. L., Marcantonio E. E., Trevithick J. E., Buck C. A., Hynes R. O. Expression of normal and mutant avian integrin subunits in rodent cells. J Cell Biol. 1989 Aug;109(2):853–861. doi: 10.1083/jcb.109.2.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tamkun J. W., DeSimone D. W., Fonda D., Patel R. S., Buck C., Horwitz A. F., Hynes R. O. Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell. 1986 Jul 18;46(2):271–282. doi: 10.1016/0092-8674(86)90744-0. [DOI] [PubMed] [Google Scholar]
  46. Tapley P., Horwitz A., Buck C., Duggan K., Rohrschneider L. Integrins isolated from Rous sarcoma virus-transformed chicken embryo fibroblasts. Oncogene. 1989 Mar;4(3):325–333. [PubMed] [Google Scholar]
  47. Tarone G., Cirillo D., Giancotti F. G., Comoglio P. M., Marchisio P. C. Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Exp Cell Res. 1985 Jul;159(1):141–157. doi: 10.1016/s0014-4827(85)80044-6. [DOI] [PubMed] [Google Scholar]
  48. Vogel B. E., Tarone G., Giancotti F. G., Gailit J., Ruoslahti E. A novel fibronectin receptor with an unexpected subunit composition (alpha v beta 1). J Biol Chem. 1990 Apr 15;265(11):5934–5937. [PubMed] [Google Scholar]
  49. Volberg T., Zick Y., Dror R., Sabanay I., Gilon C., Levitzki A., Geiger B. The effect of tyrosine-specific protein phosphorylation on the assembly of adherens-type junctions. EMBO J. 1992 May;11(5):1733–1742. doi: 10.1002/j.1460-2075.1992.tb05225.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wang J. Y. Generation and use of anti-phosphotyrosine antibodies raised against bacterially expressed abl protein. Methods Enzymol. 1991;201:53–65. doi: 10.1016/0076-6879(91)01008-p. [DOI] [PubMed] [Google Scholar]
  51. Wu H., Reynolds A. B., Kanner S. B., Vines R. R., Parsons J. T. Identification and characterization of a novel cytoskeleton-associated pp60src substrate. Mol Cell Biol. 1991 Oct;11(10):5113–5124. doi: 10.1128/mcb.11.10.5113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yatohgo T., Izumi M., Kashiwagi H., Hayashi M. Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell Struct Funct. 1988 Aug;13(4):281–292. doi: 10.1247/csf.13.281. [DOI] [PubMed] [Google Scholar]
  53. Zhang Z., Morla A. O., Vuori K., Bauer J. S., Juliano R. L., Ruoslahti E. The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin. J Cell Biol. 1993 Jul;122(1):235–242. doi: 10.1083/jcb.122.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES