PLEKHA5, PLEKHA6 and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

Abstract

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown. Here we identify PLEKHA5 and PLEKHA6 as new interactors of PDZD11, which similarly to the junctional protein PLEKHA7 bind to PDZD11 N-terminus through their WW domains. Using CRISPR-KO kidney epithelial cells, we show by immunofluorescence that WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) recruit PDZD11 to distinct plasma membrane localizations, and that they are required for the efficient anterograde targeting of ATP7A to the cell periphery in elevated copper. Pulldown experiments show that WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. However, WW-PLEKHAs and PDZD11 are not necessary for ATP7A Golgi localization in basal copper, ATP7A copper-induced exit from the Golgi, and ATP7A retrograde trafficking to the Golgi. Finally, measuring bioavailable copper with the labile copper probe CF4 shows that WW-PLEKHAs and PDZD11 are required to maintain low intracellular copper levels when cells are exposed to elevated copper. These data indicate that WW-PLEKHAs-PDZD11 complexes regulate the localization and function of ATP7A to modulate cellular copper homeostasis.

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

“…Culture conditions for mouse cortical collecting duct cells (mCCD Tet-on) (Vasileva et al, 2017), Madin-Darby canine kidney cells (MDCKII Tet-off) (Vasileva et al, 2017) and haploid human cells (Hap1) (Popov et al, 2015) were described previously. PLEKHA6-KO, PLEKHA7-KO, PLEKHA6/7-KO, and PDZD11-KO mCCD, PLEKHA5-KO and PLEKHA6-KO MDCK, as well as PLEKHA5-KO, PLEKHA7-KO, and PLEKHA5/7-KO Hap1 were obtained by CRISPR/Cas9 genomic editing (Popov et al, 2015;Guerrera et al, 2016;Shah et al, 2018;Sluysmans et al, 2021). MDCK cysts were grown in Matrigel as described previously (Sluysmans et al, 2021).…”

“…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). We denoted as WW-PLEKHAs a subset of the PLEKHA family of proteins, i.e., PLEKHA5, PLEKHA6, and PLEKHA7, which include isoforms that also contain Trp-Trp (WW) domains, in addition to the characteristic PH domain (Sluysmans et al, 2021). All three WW-PLEKHA proteins also contain proline-rich and coiled-coil domains of unknown function (Figures 1A-C).…”

“…WW-PLEKHAs together with PDZD11 control the targeting of the Menkes copper pump ATP7A to the cell periphery under elevated copper conditions (Sluysmans et al, 2021). Furthermore, 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).…”

“…Furthermore, 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). PLEKHA6 is localized at AJ and along lateral membranes of polarized epithelial cells, and PLEKHA7 exclusively at AJ of epithelial and non-epithelial cells (Pulimeno et al, 2010;Shah et al, 2018;Sluysmans et al, 2021).…”

“…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). PLEKHA6 is localized at AJ and along lateral membranes of polarized epithelial cells, and PLEKHA7 exclusively at AJ of epithelial and non-epithelial cells (Pulimeno et al, 2010;Shah et al, 2018;Sluysmans et al, 2021). However, the structural basis for the specific subcellular localizations of WW-PLEKHAs is unclear.…”