Abstract
Cell-cell communication in multicellular organisms depends on the dynamic and reversible phosphorylation of protein tyrosine residues. The receptor-linked protein tyrosine phosphatases (RPTPs) receive cues from the extracellular environment and are well placed to influence cell signaling. However, the direct events downstream of these receptors have been challenging to resolve. We report here that the homophilic receptor PTPRK is stabilized at cell-cell contacts in epithelial cells. By combining interaction studies, quantitative tyrosine phosphoproteomics, proximity labeling and dephosphorylation assays we identify high confidence PTPRK substrates. PTPRK directly and selectively dephosphorylates at least five substrates, including Afadin, PARD3 and δ-catenin family members, which are all important cell-cell adhesion regulators. In line with this, loss of PTPRK phosphatase activity leads to disrupted cell junctions and increased invasive characteristics. Thus, identifying PTPRK substrates provides insight into its downstream signaling and a potential molecular explanation for its proposed tumor suppressor function.
Highlights
Multicellular organisms have evolved elaborate mechanisms of intercellular communication in order to organize cells into functioning tissues
In order to detect endogenous PTPRK by immunoblot and immunofluorescence, we generated and characterized monoclonal antibodies against the purified PTPRK extracellular domain (ECD) (Figure 1—figure supplement 1B–D)
Using one of our antibodies for structured illumination microscopy, we found PTPRK localized to puncta at basal cell-cell contacts that partially overlap with the adherens junction (AJ) protein E-Cadherin in MCF10A epithelial cells (Figure 1B)
Summary
Multicellular organisms have evolved elaborate mechanisms of intercellular communication in order to organize cells into functioning tissues. The phosphorylation of protein tyrosine residues is an essential feature of cell-cell communication and effectively coordinates diverse cell behaviors such as cell adhesion and motility in response to external stimuli. Kinases and phosphatases dynamically regulate phosphotyrosine levels, such that cells are primed to acutely respond to developmental cues or changes to their local environment. Receptor tyrosine kinases (RTKs) dimerize upon ligand binding leading to trans-autophosphorylation, which recruits phosphotyrosine-binding proteins that propagate a variety of signaling cascades (Hunter, 2009). Protein tyrosine phosphatases (PTPs) are often thought to function in terminating or thresholding such signals (Agazie and Hayman, 2003). It is increasingly apparent that phosphatases themselves can propagate signals in response to growth factors; with the best example being PTPN11/SHP2; a key therapeutic target in cancer (Brown and Cooper, 1996).
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