Tyrosine-Based Signals Regulate the Assembly of Daple⋅PARD3 Complex at Cell-Cell Junctions

Abstract

Polarized distribution of organelles and molecules inside a cell is vital for a range of cellular processes and its loss is frequently encountered in disease. Polarization during planar cell migration is a special condition in which cellular orientation is triggered by cell-cell contact. We demonstrate that the protein Daple (CCDC88C) is a component of cell junctions in epithelial cells which serves like a cellular ''compass'' for establishing and maintaining contact-triggered planar polarity. Furthermore, these processes may be mediated through interaction with the polarity regulator PARD3. This interaction, mediated by Daple's PDZ-binding motif (PBM) and the third PDZ domain of PARD3, is fine-tuned by tyrosine phosphorylation on Daple's PBM by receptor and non-receptor tyrosine kinases, such as Src. Hypophosphorylation strengthens the interaction, whereas hyperphosphorylation disrupts it, thereby revealing an unexpected role of Daple as a platform for signal integration and gradient sensing for tyrosine-based signals within the planar cell polarity pathway.

Evolution of Modularity, Interactome and Functions of GIV/Girdin (CCDC88A) from Invertebrates to Vertebrates

“…A notable feature of the C-terminal PBM on GIV-L is its highly conserved sequence, which, like that in Daple (Figure 3A), includes two tyrosine residues. Those sites in Daple serve as substrates for multiple tyrosine kinases and their phosphorylation has been shown to modulate PDZ•PBM interactions with ParD3 and Dvl ( Figure 4A) (Aznar, Midde et al 2015, Ear, Saklecha et al 2020. In coimmunoprecipitation experiments between GIV-L and ParD3, we observed a specific interaction between GIV-L and the third PDZ domain on ParD3 (Figure 4B), mirroring exactly what was shown previously for Daple (Ear, Saklecha et al 2020).…”

“…CCDC88C/Daple also features a C-terminal PBM motif that is similar but not identical in sequence to that in GIV-L, raising the possibility that GIV-L has a unique PDZ interactome. Analysis of our own BioID proximity labeling studies, and others, reveal that GIV-L indeed binds to several PDZ domain-containing proteins, which only partially overlaps with that of Daple (Ear, Saklecha et al 2020). Interaction assays confirmed that GIV-L and Daple can indeed bind to the some common PDZ-proteins (e.g., ParD3 and Dvl).…”

“…The cell polarity regulator, ParD3, and the Wnt signaling regulator, Dvl, were chosen as the PDZ proteins because they have been identified as interactors of GIV in prior studies without a clear understanding of the mode of these interactions (Ohara, Enomoto et al 2012, Sasaki, Kakuwa et al 2015, Boldt, van Reeuwijk et al 2016. In addition, these two PDZ proteins have been demonstrated to bind to Daple's PBM (Oshita, Kishida et al 2003, Aznar, Midde et al 2015, Siletti, Tarchini et al 2017, Ear, Saklecha et al 2020. Finally, because of the high sequence similarity between GIV-L's and Daple's PBM, we rationalized that GIV-L's PBM can also bind to ParD3 and Dvl.…”

“…Prior work showed that binding of some PDZ proteins to Daple's PBM modulates the ability of the latter to bind G proteins ( Figure 4F)--while Dvl competes with Gai3 for binding Daple, ParD3 does not; the latter is capable of co-existing in ternary ParD3•Daple•Gai complexes (Aznar, Ear et al 2018, Ear, Saklecha et al 2020). In the case of GIV-L, coimmunoprecipitation assays from cell lysates overexpressing Gai3 and either GIV or GIV-L (wt or DPBM) showed that Dvl can also precipitate with the Gai3•GIV complex ( Figure 4D).…”

“…Those sites in Daple serve as substrates for multiple tyrosine kinases and their phosphorylation has been shown to modulate PDZ•PBM interactions with ParD3 and Dvl ( Figure 4A) (Aznar, Midde et al 2015, Ear, Saklecha et al 2020. In coimmunoprecipitation experiments between GIV-L and ParD3, we observed a specific interaction between GIV-L and the third PDZ domain on ParD3 (Figure 4B), mirroring exactly what was shown previously for Daple (Ear, Saklecha et al 2020). This ability to bind multiple PDZs (apparent promiscuity), and yet, preferentially doing so via one but not the other PDZ module on the same protein (i.e., specificity) highlights a key property of the conserved PBM binding modality shared by GIV-L and Daple.…”