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E-cadherin and Src associate with extradesmosomal Dsg3 and modulate desmosome assembly and adhesion

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A Letters and Comments to this article was published on 24 December 2015

Abstract

Desmosomes provide strong intercellular cohesion essential for the integrity of cells and tissues exposed to continuous mechanical stress. For desmosome assembly, constitutively synthesized desmosomal cadherins translocate to the cell–cell border, cluster and mature in the presence of Ca2+ to stable cell contacts. As adherens junctions precede the formation of desmosomes, we investigated in this study the relationship between the classical cadherin E-cadherin and the desmosomal cadherin Desmoglein 3 (Dsg3), the latter of which is indispensable for cell–cell adhesion in keratinocytes. By using autoantibodies from patients with the blistering skin disease pemphigus vulgaris (PV), we showed in loss of function studies that E-cadherin compensates for effects of desmosomal disassembly. Overexpression of E-cadherin reduced the loss of cell cohesion induced by PV autoantibodies and attenuated activation of p38 MAPK. Silencing of E-cadherin abolished the localization of Dsg3 at the membrane and resulted in a shift of Dsg3 from the cytoskeletal to the non-cytoskeletal protein pool which conforms to the notion that E-cadherin regulates desmosome assembly. Mechanistically, we identified a complex consisting of extradesmosomal Dsg3, E-cadherin, β-catenin and Src and that the stability of this complex is regulated by Src. Moreover, Dsg3 and E-cadherin are phosphorylated on tyrosine residues in a Src-dependent manner and Src activity is required for recruiting Dsg3 to the cytoskeletal pool as well as for desmosome maturation towards a Ca2+-insensitive state. Our data provide new insights into the role of E-cadherin and the contribution of Src signaling for formation and maintenance of desmosomal junctions.

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Abbreviations

Dsg:

Desmogleins

Dsc:

Desmocollin

PV:

Pemphigus vulgaris

PG:

Plakoglobin

DP:

Desmoplakin

E-cad:

E-cadherin

Ab:

Antibody

GFP:

Green fluorescent protein

PKC:

Protein kinase C

p38 MAPK:

p38 mitogen-activated protein kinase

p-Tyr:

Phospho-tyrosine

Src:

Rous sarcoma (Src) kinase

Pkp:

Plakophilin

AJ:

Adherens junction

IgG:

Immunoglobulin G

EGF:

Epidermal growth factor

EGFR:

Epidermal growth factor receptor

PBS:

Phosphate buffered saline

Hbss:

Hank’s buffered saline solution

IP:

Immunoprecipitation

siRNA:

Small interfering RNA

n.t.:

Non-targeting

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Acknowledgments

We thank Dr. Yasushi Hanakawa for providing the pEGFP-N1-Ecad construct. Furthermore, we thank Veronika Heimbach, Martina Hitzenbichler, Claudia Meyerhofer, Linda Jakobi and Andrea Wehmeyer for their skillful technical assistance. This work was supported by Deutsche Forschungsgemeinschaft Grant DFG SP1300/1-1 and 1-3 (to V.S. and J.W.)

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Authors and Affiliations

  1. Institute of Anatomy and Cell Biology, Department I, Ludwig-Maximilians-Universität, Munich, Germany

    Vera Rötzer, Eva Hartlieb, Franziska Vielmuth, Volker Spindler & Jens Waschke

  2. Department of Ophthalmology, University of Bonn, Bonn, Germany

    Martin Gliem

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  1. Vera Rötzer
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  2. Eva Hartlieb
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  3. Franziska Vielmuth
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  4. Martin Gliem
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  5. Volker Spindler
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Correspondence to Volker Spindler or Jens Waschke.

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18_2015_1977_MOESM1_ESM.tif

S1 Src signaling is inhibited by specific c-Src inhibitor pp2. (a) Pp2 treatment for 2 h prevented EGF-mediated loss of cell cohesion, whereas pp2 alone had no effect compared to controls (n = 6, *p < 0.05). (b) In Western blot analysis, pp2 treatment for 2 h blocked EGF-induced activation of EGFR (n = 3) (TIFF 7917 kb)

18_2015_1977_MOESM2_ESM.tif

S2 Efficiency of the Ca2+ switch assay. Staining of Dsg3 (purple), together with antibodies against the intracellular (clone 36; green) and the extracellular domain of E-cadherin (ECCD-2; red) served to monitor the efficiency of Ca2+ depletion and following repletion. ECCD-2 detects homophilic binding of E-cadherin [11] and thus in controls, Dsg3 and the two antibodies against E-cadherin co-localized at the cell cortex. After 1 h of Ca2+ depletion ECCD-2 staining disappeared and both Dsg3 and E-cadherin (clone 36) fluorescence signals were weakened. Replacement of Ca2+ (repletion) for 5 h restored nearby completely localization of Dsg3 and E-cadherin at the cell membrane (n = 3) (TIFF 7657 kb)

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Rötzer, V., Hartlieb, E., Vielmuth, F. et al. E-cadherin and Src associate with extradesmosomal Dsg3 and modulate desmosome assembly and adhesion. Cell. Mol. Life Sci. 72, 4885–4897 (2015). https://doi.org/10.1007/s00018-015-1977-0

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