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c-Src mediated tyrosine phosphorylation of plakophilin 3 as a new mechanism to control desmosome composition in cells exposed to oxidative stress

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Abstract

Plakophilins (PKP1 to PKP3) are essential for the structure and function of desmosomal junctions as demonstrated by the severe skin defects observed as a result of loss-of-function mutations in mice and men. PKPs play additional roles in cell signaling processes, such as those controlling the cellular stress response and cell proliferation. A key post-translational process controlling PKP function is phosphorylation. We have discovered that reactive oxygen species (ROS) trigger the c-Src kinase-mediated tyrosine (Tyr)-195 phosphorylation of PKP3. This modification is associated with a change in the subcellular distribution of the protein. Specifically, PKP3 bearing phospho-Tyr-195 is released from the desmosomes, suggesting that phospho-Tyr-195 is relevant for the control of desmosome disassembly and function, at least in cells exposed to ROS. Tyr-195 phosphorylation is transient under normal physiological conditions and seems to be strictly regulated, as the activation of particular growth factor receptors results in a modification at this site only when tyrosine phosphatases are inactivated by pervanadate. We have identified Tyr-195 of PKP3 as a phosphorylation target of epidermal growth factor receptor signaling. Interestingly, this PKP3 phosphorylation also occurs in certain poorly differentiated adenocarcinomas of the prostate, suggesting a possible role in tumor progression. Our study thus identifies a new mechanism controlling PKP3 and hence desmosome function in epithelial cells.

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  1. Stephanie Jäger

    Present address: , Lindengasse 4a, 35325, Mücke, Germany

Authors and Affiliations

  1. Institute of Pathology, Philipps University of Marburg, Baldingerstrasse, 35033, Marburg, Germany

    Steffen Neuber, Stephanie Jäger, Merle Meyer, Viktoria Wischmann, Roland Moll & Ansgar Schmidt

  2. Department of Dermatology, Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado School of Medicine, Aurora, Colo., USA

    Peter J. Koch

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  1. Steffen Neuber
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Correspondence to Ansgar Schmidt.

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This study was supported by a grant from the Deutsche Krebshilfe e.V. (German Cancer Aid, no. 108 513) to A.S. and R.M.

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Suppl. Figure 1

Isoelectric variants of PKP3 as detected by 2D gel electrophoresis in extracts from HaCaT cells. a Proteins from HaCaT cells were subjected to two-dimensional gel electrophoresis by using isoelectric focusing (IEF) in the first dimension (from pH 7 to pH 10) and SDS-PAGE in the second dimension. Western blotting with PKP3-specific antibodies (P3-3) revealed several isoelectric variants of PKP3. b To access general Tyr-phosphorylation of PKP3, immunoprecipitated PKP3 (IP: P3-3) from either untreated (−PV) or pervanadate (+PV)-treated HaCaT cells was analyzed by using monoclonal antibodies against PKP3 (P3) or phospho-Tyr-specific antibody p-Y20 (pY). Only weak or frequently no reaction was noticed for phospho-Tyr antibody when proteins were isolated from cells not exposed to phosphatase inhibitors. In the presence of tyrosine phosphatase inhibitor pervanadate, Tyr-phosphorylated PKP3 is clearly detectable. In (a), abscissa pH, ordinate apparent molecular weight in kDa (GIF 13 kb)

High Resolution Image (TIFF 1016 kb)

Suppl. Figure 2

Double-immunofluorescence microscopy of HaCaT cells treated with low concentrations of hydrogen peroxide. HaCaT cells were incubated with hydrogen peroxide (1 mM H2O2) for 1 h followed by staining with antibodies against G3BP1 (a, a’’, G3BP) or PKP3 (a’, a’’, PKP3). HaCaT cells do not form large stress granules as seen in previous experiments with sodium arsenite, but a change of G3BP1 distribution occurs from fine and diffusely distributed granules to somewhat larger aggregates. PKP3 localizes mainly to the desmosomes and the cytoplasm. Neither activation of c-Src kinase (b, b’’, p-Src) nor phosphorylation of Tyr-195 (b’, b’’, p-Y195) can be detected under these conditions. Inset in a Magnification of a typical detail showing small aggregates of G3BP1 in the cytoplasm probably reflecting non-canonical stress granules that form under these conditions. Bars 10 μm (GIF 182 kb)

High Resolution Image (TIFF 7397 kb)

Suppl. Figure 3

Double-immunofluorescence microscopy of sodium-arsenite-treated HaCaT cells. HaCaT cells were treated with 1 mM sodium arsenite for the indicated times (0, 10, 20, and 30 min Ars) and then analyzed with antibodies against G3BP1 (a–d, G3BP) and PKP3 (a’–d’, PKP3) by immunofluorescence microscopy. Without sodium arsenite treatment, PKP3 localizes to the desmosomes and also diffusely in the cytoplasm (a’). G3BP1 is diffusely distributed throughout the cytoplasm (a). After 10 min of sodium arsenite incubation, cells started to develop stress granules (b, arrow), a process that eventually becomes detectable in all cells (c, d). PKP3 also appears in stress granules (arrows in insets in c–c’’). Inset in a Magnification of a typical detail showing finely distributed cytoplasmic staining of G3BP1 of untreated cells with no obvious formation of stress granules. Bars: 10 μm (GIF 523 kb)

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Neuber, S., Jäger, S., Meyer, M. et al. c-Src mediated tyrosine phosphorylation of plakophilin 3 as a new mechanism to control desmosome composition in cells exposed to oxidative stress. Cell Tissue Res 359, 799–816 (2015). https://doi.org/10.1007/s00441-014-2063-x

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