WW, PH and C-Terminal Domains Cooperate to Direct the Subcellular Localizations of PLEKHA5, PLEKHA6 and PLEKHA7.

Sophie Sluysmans,Lionel Jond,Sandra Citi,Isabelle Méan

doi:10.3389/fcell.2021.729444

Sophie Sluysmans, Lionel Jond + Show 2 more

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https://doi.org/10.3389/fcell.2021.729444

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Abstract

PLEKHA5, PLEKHA6, and PLEKHA7 (WW-PLEKHAs) are members of the PLEKHA family of proteins that interact with PDZD11 through their tandem WW domains. WW-PLEKHAs contribute to the trafficking and retention of transmembrane proteins, including nectins, Tspan33, and the copper pump ATP7A, at cell-cell junctions and lateral membranes. However, the structural basis for the distinct subcellular localizations of PLEKHA5, PLEKHA6, and PLEKHA7 is not clear. Here we expressed mutant and chimeric proteins of WW-PLEKHAs in cultured cells to clarify the role of their structural domains in their localization. We found that the WW-mediated interaction between PLEKHA5 and PDZD11 is required for their respective association with cytoplasmic microtubules. The PH domain of PLEKHA5 is required for its localization along the lateral plasma membrane and promotes the lateral localization of PLEKHA7 in a chimeric molecule. Although the PH domain of PLEKHA7 is not required for its localization at the adherens junctions (AJ), it promotes a AJ localization of chimeric proteins. The C-terminal region of PLEKHA6 and PLEKHA7 and the coiled-coil region of PLEKHA7 promote their localization at AJ of epithelial cells. These observations indicate that the localizations of WW-PLEKHAs at specific subcellular sites, where they recruit PDZD11, are the result of multiple cooperative protein-lipid and protein-protein interactions and provide a rational basis for the identification of additional proteins involved in trafficking and sorting of WW-PLEKHAs.

Highlights

IntroductionThe PLEKHA (Pleckstrin Homology domain containing family A) family of proteins is characterized by the presence of a pleckstrin homology (PH) domain and comprises PLEKHA1 and PLEKHA2 (TAPP1 and TAPP2) (Kimber et al, 2002; Marshall et al, 2002), PLEKHA3 and PLEKHA8 (FAPP1 and FAPP2) (Godi et al, 2004), PLEKHA4 (PEPP1) (Dowler et al, 2000; Shami Shah et al, 2019), PLEKHA5 and PLEKHA6 (PEPP2 and PEPP3) (Dowler et al, 2000; Zou and Cox, 2013; Sluysmans et al, 2021), and PLEKHA7 (Meng et al, 2008; Pulimeno et al, 2010)
The apical junctional complex (AJC) in polarized epithelial cells comprises adherens junctions (AJ, called zonulae adhaerentes in epithelia) and tight junctions (TJ)
Chimeric PLEKHA6 molecules containing the pleckstrin homology (PH) domain of either PLEKHA5 or PLEKHA7 were still localized at AJ and along lateral contacts, but chimeric molecules containing the PH domain of PLEKHA5 were detectable in the subapical cytoplasm (Supplementary Figure 1). These results suggest that in cultured polarized epithelial cells the PH domain of PLEKHA5 directs WW-PLEKHAs to lateral contacts and subapical cytoplasm, and the PH domains of PLEKHA6 and PLEKHA7 promote the accumulation of chimeric proteins along lateral membranes and junctions, they are not required for the localizations of the non-chimeric proteins at these sites

Summary

Introduction

The PLEKHA (Pleckstrin Homology domain containing family A) family of proteins is characterized by the presence of a pleckstrin homology (PH) domain and comprises PLEKHA1 and PLEKHA2 (TAPP1 and TAPP2) (Kimber et al, 2002; Marshall et al, 2002), PLEKHA3 and PLEKHA8 (FAPP1 and FAPP2) (Godi et al, 2004), PLEKHA4 (PEPP1) (Dowler et al, 2000; Shami Shah et al, 2019), PLEKHA5 and PLEKHA6 (PEPP2 and PEPP3) (Dowler et al, 2000; Zou and Cox, 2013; Sluysmans et al, 2021), and PLEKHA7 (Meng et al, 2008; Pulimeno et al, 2010). PLEKHA7 together with PDZD11 controls the localization of nectins, and of the Tspan33-ADAM10 complex at adherens junctions (AJ) (Guerrera et al, 2016; Shah et al, 2018). PLEKHA5 colocalizes with microtubules in the cytoplasm of epithelial, endothelial and myeloblastic-derived cells and when exogenously expressed recruits PDZD11 to this localization (Sluysmans et al, 2021). We explore the role of PH, WW, and coiledcoil domains of each of the three WW-PLEKHAs in determining their subcellular localizations, by expressing mutant and chimeric proteins in epithelial cells. The results indicate specific roles for WW, PH and coiled-coil domains in orchestrating the subcellular localizations of distinct WW-PLEKHAs

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