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The PDZ2 domain of zonula occludens-1 and -2 is a phosphoinositide binding domain

  • Kris Meerschaert
  • Moe Phyu Tun
  • Eline Remue
  • Ariane De Ganck
  • Ciska Boucherie
  • Berlinda Vanloo
  • Gisèle Degeest
  • Joël Vandekerckhove
  • Pascale Zimmermann
  • Nitin Bhardwaj
  • Hui Lu
  • Wonhwa Cho
  • Jan GettemansEmail author
Research Article

Abstract

Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction. Furthermore, peptide and PtdInsP binding of ZO PDZ2 domains are mutually exclusive. Although lipid binding does not seem to be required for plasma membrane localisation of ZO-1, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2) binding to the PDZ2 domain of ZO-2 regulates ZO-2 recruitment to nuclear speckles. Knockdown of ZO-2 expression disrupts speckle morphology, indicating that ZO-2 might play an active role in formation and stabilisation of these subnuclear structures. This study shows for the first time that ZO isoforms bind PtdInsPs and offers an alternative regulatory mechanism for the formation and stabilisation of protein complexes in the nucleus.

Keywords

Tight junction Phospholipid Post synaptic density-discs large-zonula occludens Nucleus Cell polarity 

Notes

Acknowledgments

We thank Dr. E. Mortier for helpful discussions during the course of this project and Dr. L. Van Troys and Dr. G. Hammond for useful advice on the lipid stainings. This work was supported by the Fund for Scientific Research-Flanders (FWO-Vlaanderen), the Concerted Actions Programme of Ghent University (GOA), the Interuniversity attraction poles (IUAP06), the Human Frontier Science Program (HFSP), a NIH grant (GM68849) (for W.C.), and the Catalyst Award from Chicago Biomedical Consortium (for W.C. and H.L.). K.M. was supported by a Postdoctoral Fellowship of the Fund for Scientific Research-Flanders (Belgium) (FWO-Vlaanderen). E.R. is supported by a fellowship from the research council of Ghent University (BOF).

Supplementary material

18_2009_156_MOESM1_ESM.pdf (40 kb)
Supplementary Figure S1. ELISA PtdInsP binding assay with PDZ domains 1-3 of ZO-2. Each well of a ‘PIP specificity’ microtiter plate (Echelon Biosciences) was overlaid with GST, GST- PH-PLC-d1 (positive control) and GST-ZO2 PDZ1-3 at a final concentration of 10 nM. Data represent means ± STDEV (n = 3). A.U. = absorbance unit (PDF 39 kb)
18_2009_156_MOESM2_ESM.pdf (42 kb)
Supplementary Figure S2. Gel filtration chromatography of GST-ZO1-PDZ2 (red line). Molecular weight standards (blue line) include blue dextran, immunoglobin G (IgG; 150 kDa), bovine serum albumin (BSA; 67 kDa) and ovalbumin (43 kDa). 100 nM to 5 µM GST-ZO1-PDZ2 (and GST-ZO2-PDZ2) showed the same elution pattern. The estimated molecular weight of the protein was 78 kDa, which approximates that of the GST-ZO1-PDZ2 dimer (PDF 42 kb)
18_2009_156_MOESM3_ESM.pdf (79 kb)
Supplementary material 3Supplementary Figure S3. A) PtdInsP selectivity of ZO-2 PDZ2 determined by kinetic SPR measurements. 1 µM of protein was added to POPC/POPE/POPS/PtdInsP (37:40:20:3) vesicles containing 7 different PtdInsPs. (B) Effects of mutations of basic residues on binding of the ZO-2 PDZ2 to POPC/POPE/POPS/PtdIns(4,5)P 2 (37:40:20:3) vesicles measured by kinetic SPR analysis. Protein concentrations were kept at 1 µM. Kd values were determined by equilibrium SPR analysis as shown in Figure 2B and 2C and listed in Table II. (PDF 79 kb)
18_2009_156_MOESM4_ESM.pdf (642 kb)
Supplementary Figure S4. Expression of wild-type and mutant ZO constructs in MDCK cells. (A) Expression of various GFP-tagged ZO-1 mutants defective in lipid and/or peptide binding does not affect plasma membrane localisation of ZO-1. (B) Similar experiments for ZO-2. WT=wild type. Scale bar = 10µm. (PDF 641 kb)

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Copyright information

© Birkhäuser Verlag, Basel/Switzerland 2009

Authors and Affiliations

  • Kris Meerschaert
    • 1
    • 2
    • 6
  • Moe Phyu Tun
    • 3
  • Eline Remue
    • 1
    • 2
  • Ariane De Ganck
    • 1
    • 2
  • Ciska Boucherie
    • 1
    • 2
  • Berlinda Vanloo
    • 1
    • 2
  • Gisèle Degeest
    • 4
  • Joël Vandekerckhove
    • 1
    • 2
  • Pascale Zimmermann
    • 4
  • Nitin Bhardwaj
    • 5
  • Hui Lu
    • 5
  • Wonhwa Cho
    • 3
    • 8
  • Jan Gettemans
    • 1
    • 2
    • 7
    Email author
  1. 1.Department of Medical Protein ResearchVIBGhentBelgium
  2. 2.Department of Biochemistry, Faculty of Medicine and Health SciencesGhent UniversityGhentBelgium
  3. 3.Departments of ChemistryUniversity of IllinoisChicagoUSA
  4. 4.Department of Human GeneticsK.U.LeuvenBelgium
  5. 5.Departments of BioengineeringUniversity of IllinoisChicagoUSA
  6. 6.Ablynx nvGhent/ZwijnaardeBelgium
  7. 7.Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Flanders Interuniversity Institute for BiotechnologyGhent UniversityGhentBelgium
  8. 8.Department of Chemistry (M/C 111)University of Illinois at ChicagoChicagoUSA

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