Plakoglobin phosphorylation at serine 665 is capable of stabilizing cadherin-mediated adhesion in keratinocytes
Franziska Vielmuth, Anna M. Sigmund, Desalegn T. Egu, Matthias Hiermaier, Letyfee S. Steinert, Sina Moztarzadeh, Mariia Klimkina, Margarethe E.C. Schikora, Paulina M. Rion, Thomas Schmitt, Katharina Meier, Kamran Ghoreschi, Anja K.E. Horn, Mariya Y. Radeva, Daniela Kugelmann, Jens Waschke
Franziska Vielmuth, Anna M. Sigmund, Desalegn T. Egu, Matthias Hiermaier, Letyfee S. Steinert, Sina Moztarzadeh, Mariia Klimkina, Margarethe E.C. Schikora, Paulina M. Rion, Thomas Schmitt, Katharina Meier, Kamran Ghoreschi, Anja K.E. Horn, Mariya Y. Radeva, Daniela Kugelmann, Jens Waschke
View: Text | PDF
Research Article Cell biology

Plakoglobin phosphorylation at serine 665 is capable of stabilizing cadherin-mediated adhesion in keratinocytes

Abstract

In pemphigus, autoantibodies against the desmosomal cadherins desmoglein (DSG) DSG1 and DSG3 cause intraepidermal blistering. Recently, we found that increasing cAMP with the phosphodiesterase-4 inhibitor apremilast stabilizes keratinocyte cohesion in pemphigus. This effect is paralleled by phosphorylation of the desmosomal plaque protein plakoglobin (PG) at serine 665 (S665). Here, we investigated the relevance of PG phosphorylation at S665 for stabilization of keratinocyte cohesion and further characterized the underlying mechanisms. Ultrastructural analysis of a recently established PG-S665 phospho-deficient mouse model (PG-S665A) showed diminished keratin insertion. Accordingly, the protective effect of apremilast against pemphigus autoantibody-induced skin blistering was diminished, and apremilast failed to restore alterations of the keratin cytoskeleton in PG-S665A mice. Keratinocytes derived from PG-S665A mice revealed a disorganized keratin cytoskeleton and reduced single-molecule binding strength of DSG3. In line with this, in ex vivo human skin, increased cAMP augmented keratin insertion into desmosomal plaques. Additionally, PG phosphorylated at S665 colocalized with desmoplakin and keratin filaments anchoring to desmosomes and increased cAMP-accelerated assembly of desmosomes. Taken together, phosphorylation of PG at S665 was crucial for protective effects of apremilast in pemphigus and for maintenance of DSG3 binding and keratin filament anchorage to desmosomes.

Authors

Franziska Vielmuth, Anna M. Sigmund, Desalegn T. Egu, Matthias Hiermaier, Letyfee S. Steinert, Sina Moztarzadeh, Mariia Klimkina, Margarethe E.C. Schikora, Paulina M. Rion, Thomas Schmitt, Katharina Meier, Kamran Ghoreschi, Anja K.E. Horn, Mariya Y. Radeva, Daniela Kugelmann, Jens Waschke

×

Figure 6

PG phosphorylation at S665 affects DSG3-dependent binding properties.

Options: View larger image (or click on image) Download as PowerPoint
PG phosphorylation at S665 affects DSG3-dependent binding properties. (A...
(A) AFM experiments in PG-WT and PG-S665A keratinocytes at 48 hours Ca2+ differentiation. In topography images, PG-S665A keratinocytes revealed a smaller and more roundish cell shape. Cell borders revealed elevated structures with a higher slope in both cell lines. Small areas (2 × 4 μm) perpendicular to cell borders were selected for adhesion measurements. In adhesion maps, every dot represents 1 force-distance curve, and every green dot depicts 1 DSG3-dependent adhesion event. Representative of n = 6. Scale bars: 1 μm and 10 μm. (B) Quantification of binding frequency reveals a significant higher DSG3-dependent binding frequency in PG-S665A keratinocytes. (C) Quantification of unbinding forces showed a significantly lower binding strength of DSG3-dependent single-molecule interactions in PG-S665A keratinocytes. In every single experiment (n), unbinding force distribution was evaluated with peak fit using the extreme fit model. Dots represent peaks of unbinding force distribution. (D) Evaluation of step position revealed an increased step position in PG-S665A keratinocytes. (B–D) n = 6, 2 cell-borders/n, 800 force-distance curves/cell border. *P < 0.05, unpaired 2-tailed t test. (E) Schematic of cAMP signaling and effects of PG phosphorylation at S665. Schematic created with BioRender.