Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

β2-syntrophin and Par-3 promote an apicobasal Rac activity gradient at cell–cell junctions by differentially regulating Tiam1 activity

Nature Cell Biology volume 14, pages 1169–1180 (2012)Cite this article

Subjects

Abstract

Although Rac and its activator Tiam1 are known to stimulate cell–cell adhesion, the mechanisms regulating their activity in cell–cell junction formation are poorly understood. Here, we identify β2-syntrophin as a Tiam1 interactor required for optimal cell–cell adhesion. We show that during tight-junction (TJ) assembly β2-syntrophin promotes Tiam1–Rac activity, in contrast to the function of the apical determinant Par-3 whose inhibition of Tiam1–Rac activity is necessary for TJ assembly. We further demonstrate that β2-syntrophin localizes more basally than Par-3 at cell–cell junctions, thus generating an apicobasal Rac activity gradient at developing cell–cell junctions. Targeting active Rac to TJs shows that this gradient is required for optimal TJ assembly and apical lumen formation. Consistently, β2-syntrophin depletion perturbs Tiam1 and Rac localization at cell–cell junctions and causes defects in apical lumen formation. We conclude that β2-syntrophin and Par-3 fine-tune Rac activity along cell–cell junctions controlling TJ assembly and the establishment of apicobasal polarity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Tiam1 interacts with the β2-syntrophin PDZ domain using an internal PBM.
Figure 2: β2-syntrophin regulates TJ assembly in MDCKII cells.
Figure 3: Tiam1-induced Rac activity impedes TJ assembly in MDCKII cells.
Figure 4: β2-syntrophin promotes Tiam1–Rac activity during TJ assembly.
Figure 5: β2-syntrophin, Par-3 and Tiam1 localize differently along the apicobasal junctional axis.
Figure 6: An apicobasal Rac activity gradient at cell–cell junctions.
Figure 7: The cell–cell junction Rac activity gradient promotes TJ assembly and apicobasal polarity.
Figure 8: β2-syntrophin promotes apicobasal polarity.

Similar content being viewed by others

References

  1. Matter, K., Aijaz, S., Tsapara, A. & Balda, M. S. Mammalian tight junctions in the regulation of epithelial differentiation and proliferation. Curr. Opin. Cell Biol. 17, 453–458 (2005).

    Article  CAS  Google Scholar 

  2. Steed, E., Balda, M. S. & Matter, K. Dynamics and functions of tight junctions. Trends Cell Biol. 20, 142–149 (2010).

    Article  CAS  Google Scholar 

  3. Assemat, E., Bazellieres, E., Pallesi-Pocachard, E., Le Bivic, A. & Massey-Harroche, D. Polarity complex proteins. Biochim. Biophys. Acta 1778, 614–630 (2008).

    Article  CAS  Google Scholar 

  4. Feigin, M. E. & Muthuswamy, S. K. Polarity proteins regulate mammalian cell–cell junctions and cancer pathogenesis. Curr. Opin. Cell Biol. 21, 694–700 (2009).

    Article  CAS  Google Scholar 

  5. McCaffrey, L. M. & Macara, I. G. Epithelial organization, cell polarity and tumorigenesis. Trends Cell Biol. 21, 727–735 (2011).

    Article  CAS  Google Scholar 

  6. Royer, C. & Lu, X. Epithelial cell polarity: a major gatekeeper against cancer? Cell Death Differ. 18, 1470–1477 (2011).

    Article  CAS  Google Scholar 

  7. Mack, N. A., Whalley, H. J., Castillo-Lluva, S. & Malliri, A. The diverse roles of Rac signaling in tumorigenesis. Cell Cycle 10, 1571–1581 (2011).

    Article  CAS  Google Scholar 

  8. Chen, X. & Macara, I. G. Par-3 controls tight junction assembly through the Rac exchange factor Tiam1. Nat. Cell Biol. 7, 262–269 (2005).

    Article  CAS  Google Scholar 

  9. Mertens, A. E., Rygiel, T. P., Olivo, C., van der Kammen, R. & Collard, J. G. The Rac activator Tiam1 controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex. J. Cell Biol. 170, 1029–1037 (2005).

    Article  CAS  Google Scholar 

  10. Guillemot, L., Paschoud, S., Jond, L., Foglia, A. & Citi, S. Paracingulin regulates the activity of Rac1 and RhoA GTPases by recruiting Tiam1 and GEF-H1 to epithelial junctions. Mol. Biol. Cell 19, 4442–4453 (2008).

    Article  CAS  Google Scholar 

  11. Woodcock, S. A. et al. SRC-induced disassembly of adherens junctions requires localized phosphorylation and degradation of the rac activator tiam1. Mol. Cell 33, 639–653 (2009).

    Article  CAS  Google Scholar 

  12. Malliri, A., van Es, S., Huveneers, S. & Collard, J. G. The Rac exchange factor Tiam1 is required for the establishment and maintenance of cadherin-based adhesions. J. Biol. Chem. 279, 30092–30098 (2004).

    Article  CAS  Google Scholar 

  13. Hordijk, P. L. et al. Inhibition of invasion of epithelial cells by Tiam1–Rac signaling. Science 278, 1464–1466 (1997).

    Article  CAS  Google Scholar 

  14. Malliri, A. et al. Mice deficient in the Rac activator Tiam1 are resistant to Ras-induced skin tumours. Nature 417, 867–871 (2002).

    Article  CAS  Google Scholar 

  15. Malliri, A. et al. The rac activator Tiam1 is a Wnt-responsive gene that modifies intestinal tumor development. J. Biol. Chem. 281, 543–548 (2006).

    Article  CAS  Google Scholar 

  16. Woodcock, S. A. et al. Tiam1–Rac signaling counteracts Eg5 during bipolar spindle assembly to facilitate chromosome congression. Curr. Biol. 20, 669–675 (2010).

    Article  CAS  Google Scholar 

  17. Otsuki, Y. et al. Guanine nucleotide exchange factor, Tiam1, directly binds to c-Myc and interferes with c-Myc-mediated apoptosis in rat-1 fibroblasts. J. Biol. Chem. 278, 5132–5140 (2003).

    Article  CAS  Google Scholar 

  18. Rygiel, T. P., Mertens, A. E., Strumane, K., van der Kammen, R. & Collard, J. G. The Rac activator Tiam1 prevents keratinocyte apoptosis by controlling ROS-mediated ERK phosphorylation. J. Cell Sci. 121, 1183–1192 (2008).

    Article  CAS  Google Scholar 

  19. Qi, H. et al. Caspase-mediated cleavage of the TIAM1 guanine nucleotide exchange factor during apoptosis. Cell Growth Differ. 12, 603–611 (2001).

    CAS  PubMed  Google Scholar 

  20. Minard, M. E., Ellis, L. M. & Gallick, G. E. Tiam1 regulates cell adhesion, migration and apoptosis in colon tumor cells. Clin. Exp. Metastasis 23, 301–313 (2006).

    Article  CAS  Google Scholar 

  21. Joneson, T. & Bar-Sagi, D. Suppression of Ras-induced apoptosis by the Rac GTPase. Mol. Cell Biol. 19, 5892–5901 (1999).

    Article  CAS  Google Scholar 

  22. Woodcock, S. A., Jones, R. C., Edmondson, R. D. & Malliri, A. A modified tandem affinity purification technique identifies that 14-3-3 proteins interact with Tiam1, an interaction which controls Tiam1 stability. J. Proteome Res. 8, 5629–5641 (2009).

    Article  CAS  Google Scholar 

  23. Fleming, I. N., Elliott, C. M., Buchanan, F. G., Downes, C. P. & Exton, J. H. Ca2+/calmodulin-dependent protein kinase II regulates Tiam1 by reversible protein phosphorylation. J. Biol. Chem. 274, 12753–12758 (1999).

    Article  CAS  Google Scholar 

  24. Stafford, R. L., Ear, J., Knight, M. J. & Bowie, J. U. The molecular basis of the Caskin1 and Mint1 interaction with CASK. J. Mol. Biol. 412, 3–13 (2011).

    Article  CAS  Google Scholar 

  25. Kachinsky, A. M., Froehner, S. C. & Milgram, S. L. A PDZ-containing scaffold related to the dystrophin complex at the basolateral membrane of epithelial cells. J. Cell Biol. 145, 391–402 (1999).

    Article  CAS  Google Scholar 

  26. Gee, S. H. et al. Cyclic peptides as non-carboxyl-terminal ligands of syntrophin PDZ domains. J. Biol. Chem. 273, 21980–21987 (1998).

    Article  CAS  Google Scholar 

  27. Noren, N. K., Niessen, C. M., Gumbiner, B. M. & Burridge, K. Cadherin engagement regulates Rho family GTPases. J. Biol. Chem. 276, 33305–33308 (2001).

    Article  CAS  Google Scholar 

  28. McNeil, E., Capaldo, C. T. & Macara, I. G. Zonula occludens-1 function in the assembly of tight junctions in Madin-Darby canine kidney epithelial cells. Mol. Biol. Cell 17, 1922–1932 (2006).

    Article  CAS  Google Scholar 

  29. Zheng, B. & Cantley, L. C. Regulation of epithelial tight junction assembly and disassembly by AMP-activated protein kinase. Proc. Natl Acad. Sci. USA 104, 819–822 (2007).

    Article  CAS  Google Scholar 

  30. Timpson, P. et al. Spatial regulation of RhoA activity during pancreatic cancer cell invasion driven by mutant p53. Cancer Res. 71, 747–757 (2011).

    Article  CAS  Google Scholar 

  31. Yoshizaki, H. et al. Activity of Rho-family GTPases during cell division as visualized with FRET-based probes. J. Cell Biol. 162, 223–232 (2003).

    Article  CAS  Google Scholar 

  32. Bertazzi, D. T., Kurtulmus, B. & Pereira, G. The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4. J. Cell Biol. 193, 1033–1048 (2011).

    Article  CAS  Google Scholar 

  33. Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 3, 887–889 (2006).

    Article  CAS  Google Scholar 

  34. Rothbauer, U. et al. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol. Cell. Proteomics 7, 282–289 (2008).

    Article  CAS  Google Scholar 

  35. Schornack, S. et al. Protein mislocalization in plant cells using a GFP-binding chromobody. Plant J. 60, 744–754 (2009).

    Article  CAS  Google Scholar 

  36. Ferrari, A., Veligodskiy, A., Berge, U., Lucas, M. S. & Kroschewski, R. ROCK-mediated contractility, tight junctions and channels contribute to the conversion of a preapical patch into apical surface during isochoric lumen initiation. J. Cell Sci. 121, 3649–3663 (2008).

    Article  CAS  Google Scholar 

  37. Delous, M. et al. Nephrocystin-1 and nephrocystin-4 are required for epithelial morphogenesis and associate with PALS1/PATJ and Par6. Hum. Mol. Genet. 18, 4711–4723 (2009).

    Article  CAS  Google Scholar 

  38. Horikoshi, Y. et al. Interaction between PAR-3 and the aPKC-PAR-6 complex is indispensable for apical domain development of epithelial cells. J. Cell Sci. 122, 1595–1606 (2009).

    Article  CAS  Google Scholar 

  39. Schluter, M. A. & Margolis, B. Apical lumen formation in renal epithelia. J. Am. Soc. Nephrol. 20, 1444–1452 (2009).

    Article  Google Scholar 

  40. Miyoshi, J. & Takai, Y. Molecular perspective on tight-junction assembly and epithelial polarity. Adv. Drug Deliv. Rev. 57, 815–855 (2005).

    Article  CAS  Google Scholar 

  41. Balda, M. S. & Matter, K. Tight junctions and the regulation of gene expression. Biochim. Biophys. Acta 1788, 761–767 (2009).

    Article  CAS  Google Scholar 

  42. Brennan, K., Offiah, G., McSherry, E. A. & Hopkins, A. M. Tight junctions: a barrier to the initiation and progression of breast cancer? J. Biomed. Biotechnol. 2010, 460607 (2010).

    Article  Google Scholar 

  43. Engers, R. et al. Prognostic relevance of Tiam1 protein expression in prostate carcinomas. Br. J. Cancer 95, 1081–1086 (2006).

    Article  CAS  Google Scholar 

  44. Kobayashi, T. et al. Activation of Rac1 is closely related to androgen-independent cell proliferation of prostate cancer cells both in vitro and in vivo. Mol. Endocrinol. 24, 722–734 (2010).

    Article  CAS  Google Scholar 

  45. Engers, R. et al. Prognostic relevance of increased Rac GTPase expression in prostate carcinomas. Endocr. Relat. Cancer 14, 245–256 (2007).

    Article  CAS  Google Scholar 

  46. Dahlman, A. et al. Evaluation of the prognostic significance of MSMB and CRISP3 in prostate cancer using automated image analysis. Mod. Pathol. 24, 708–719 (2011).

    Article  CAS  Google Scholar 

  47. Hillier, B. J., Christopherson, K. S., Prehoda, K. E., Bredt, D. S. & Lim, W. A. Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex. Science 284, 812–815 (1999).

    Article  CAS  Google Scholar 

  48. Harris, B. Z., Hillier, B. J. & Lim, W. A. Energetic determinants of internal motif recognition by PDZ domains. Biochemistry 40, 5921–5930 (2001).

    Article  CAS  Google Scholar 

  49. Penkert, R. R., DiVittorio, H. M. & Prehoda, K. E. Internal recognition through PDZ domain plasticity in the Par-6-Pals1 complex. Nat. Struct. Mol. Biol. 11, 1122–1127 (2004).

    Article  CAS  Google Scholar 

  50. Wong, H. C. et al. Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled. Mol. Cell 12, 1251–1260 (2003).

    Article  CAS  Google Scholar 

  51. London, T. B., Lee, H. J., Shao, Y. & Zheng, J. Interaction between the internal motif KTXXXI of Idax and mDvl PDZ domain. Biochem. Biophys. Res. Commun. 322, 326–332 (2004).

    Article  CAS  Google Scholar 

  52. Lemaire, J. F. & McPherson, P. S. Binding of Vac14 to neuronal nitric oxide synthase: Characterisation of a new internal PDZ-recognition motif. FEBS Lett. 580, 6948–6954 (2006).

    Article  CAS  Google Scholar 

  53. Georgiou, M. & Baum, B. Polarity proteins and Rho GTPases cooperate to spatially organise epithelial actin-based protrusions. J. Cell Sci. 123, 1089–1098 (2010).

    Article  CAS  Google Scholar 

  54. Yagi, S., Matsuda, M. & Kiyokawa, E. Suppression of Rac1 activity at the apical membrane of MDCK cells is essential for cyst structure maintenance. EMBO Rep. (2012).

  55. Masuda-Hirata, M. et al. Intracellular polarity protein PAR-1 regulates extracellular laminin assembly by regulating the dystroglycan complex. Genes Cells 14, 835–850 (2009).

    Article  CAS  Google Scholar 

  56. O’Brien, L. E. et al. Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly. Nat. Cell Biol. 3, 831–838 (2001).

    Article  Google Scholar 

  57. Hamelers, I. H. et al. The Rac activator Tiam1 is required for (alpha)3(beta)1-mediated laminin-5 deposition, cell spreading, and cell migration. J. Cell Biol. 171, 871–881 (2005).

    Article  CAS  Google Scholar 

  58. Lane, J., Martin, T. A., Mansel, R. E. & Jiang, W. G. The expression and prognostic value of the guanine nucleotide exchange factors (GEFs) Trio, Vav1 and TIAM-1 in human breast cancer. Int. Semin. Surg. Oncol. 5, 23 (2008).

    Article  Google Scholar 

  59. Yang, W. et al. Up-regulation of Tiam1 and Rac1 correlates with poor prognosis in hepatocellular carcinoma. Jpn. J. Clin. Oncol. (2010).

  60. Ding, Y. et al. Overexpression of Tiam1 in hepatocellular carcinomas predicts poor prognosis of HCC patients. Int. J. Cancer 124, 653–658 (2009).

    Article  CAS  Google Scholar 

  61. Qi, Y. et al. Prognostic value of Tiam1 and Rac1 overexpression in nasopharyngeal carcinoma. ORL J. Otorhinolaryngol. Relat. Spectrosc. 71, 163–171 (2009).

    Article  CAS  Google Scholar 

  62. Liu, L., Wu, D. H. & Ding, Y. Q. Tiam1 gene expression and its significance in colorectal carcinoma. World J. Gastroenterol. 11, 705–707 (2005).

    Article  CAS  Google Scholar 

  63. Zhao, L., Liu, Y., Sun, X., He, M. & Ding, Y. Overexpression of T lymphoma invasion and metastasis 1 predict renal cell carcinoma metastasis and overall patient survival. J. Cancer Res. Clin. Oncol. (2010).

  64. Li, Y. et al. UTRN on chromosome 6q24 is mutated in multiple tumors. Oncogene 26, 6220–6228 (2007).

    Article  CAS  Google Scholar 

  65. Nelson, W. J. Remodeling epithelial cell organization: transitions between front-rear and apical-basal polarity. Cold Spring Harb. Perspect. Biol. 1, a000513 (2009).

    Article  Google Scholar 

  66. Lambert, J. M. et al. Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism. Nat. Cell Biol. 4, 621–625 (2002).

    Article  CAS  Google Scholar 

  67. Tassidis, H. et al. Immunohistochemical detection of tyrosine phosphatase SHP-1 predicts outcome after radical prostatectomy for localized prostate cancer. Int. J. Cancer 126, 2296–2307 (2010).

    CAS  PubMed  Google Scholar 

  68. Syed Khaja, A. S. et al. Elevated level of Wnt5a protein in localized prostate cancer tissue is associated with better outcome. PLoS One 6, e26539 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by Cancer Research UK grant number C147/A6058 and Medical Research Council grant number MR/J00104X/1. We thank colleagues mentioned in the Methods for providing reagents; E. Nilsson for immunohistochemical stainings; S. Bagley and A. Dunne for help with microscopy and image analysis; C. O’Neill, M. Balda, S. Ohno and V. Braga for technical advice; A. Hurlstone, I. Hagan, D. Garrod, J. Brognard and members of the Cell Signalling Group for helpful discussions and critical reading of the manuscript.

Author information

Authors and Affiliations

  1. Cell Signalling Group, Cancer Research UK Paterson Institute for Cancer Research, The University of Manchester, Manchester M20 4BX, UK

    Natalie A. Mack, Andrew P. Porter, Helen J. Whalley & Angeliki Malliri

  2. Tumour Cell Migration Group, Cancer Research UK Beatson Institute for Cancer Research, Glasgow G61 1BD, UK

    Juliane P. Schwarz & Kurt I. Anderson

  3. MS Bioworks, 3950 Varsity Drive, Ann Arbor, Michigan 48108, USA

    Richard C. Jones

  4. Department of Clinical Sciences, Division of Urological Cancers, Lund University, Skåne University Hospital, Malmö, SE-205 02, Sweden

    Azharuddin Sajid Syed Khaja & Anders Bjartell

Authors
  1. Natalie A. Mack
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew P. Porter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Helen J. Whalley
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Juliane P. Schwarz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Richard C. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Azharuddin Sajid Syed Khaja
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Anders Bjartell
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kurt I. Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Angeliki Malliri
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.A.M. performed most of the experimental work, data analysis and manuscript preparation. A.P.P. contributed extensively to experiments and preparation of the manuscript. H.J.W. contributed to experiments and analysis. R.C.J. performed the mass spectrometry analysis. J.P.S. and K.I.A. contributed to and supervised the FLIM–FRET analysis. A.S.S.K. and A.B. performed the TMA staining and scoring together with N.A.M. A.M. was the grant holder and principal investigator who supervised the study and manuscript preparation and made intellectual contributions throughout.

Corresponding author

Correspondence to Angeliki Malliri.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 3201 kb)

Supplementary Table 1

Supplementary Information (XLS 15 kb)

Supplementary Table 2

Supplementary Information (XLS 24 kb)

Supplementary Table 3

Supplementary Information (XLS 13 kb)

Supplementary Table 4

Supplementary Information (XLS 15 kb)

Supplementary Table 5

Supplementary Information (XLS 13 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mack, N., Porter, A., Whalley, H. et al. β2-syntrophin and Par-3 promote an apicobasal Rac activity gradient at cell–cell junctions by differentially regulating Tiam1 activity. Nat Cell Biol 14, 1169–1180 (2012). https://doi.org/10.1038/ncb2608

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2608

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing