Electrostatic Plasma Membrane Targeting Contributes to Dlg Function in Cell Polarity and Tumorigenesis

Abstract

SUMMARYDiscs large (Dlg) is an essential polarity protein and a tumor suppressor originally characterized in Drosophila but is also well conserved in vertebrates. Like the majority of polarity proteins, plasma membrane (PM)/cortical localization of Dlg is required for its function in regulating apical-basal polarity and tumorigenesis, but the exact mechanisms targeting Dlg to PM remain to be unclear. Here we show that, similar to recently discovered polybasic polarity proteins such as Lgl and aPKC, Dlg also contains a positively charged polybasic domain that electrostatically binds the PM phosphoinositides PI4P and PI(4,5)P2. Electrostatic targeting by the polybasic domain acts as the primary mechanism localizing Dlg to the PM in follicular and early embryonic epithelial cells, and is crucial for Dlg to regulate both polarity and tumorigenesis. The electrostatic PM targeting of Dlg is controlled by a potential phosphorylation-dependent allosteric regulation of its polybasic domain, and is specifically enhanced by interactions between Dlg and another basolateral polarity protein and tumor suppressor Scrib. Our studies highlight an increasingly significant role of electrostatic PM targeting of polarity proteins in regulating cell polarity.

Evidence for a Nuclear Role forDrosophilaDlg as a Regulator of the NURF Complex

“…We were unable to predict an NES, although such sequences have been detected in vertebrate ZO-1 (Islas et al, 2002), ZO-2 (Islas et al, 2002;Jaramillo et al, 2004;González-Mariscal et al, 2006), and Nagie Oko (Bit-Avragim et al, 2008). However, various transgenically-expressed truncations of Dlg localize strongly to nuclei (Hough et al, 1997;Thomas et al, 2000;Bachmann et al, 2004;Lu et al, 2021), including one that deletes the E-F region itself. Additionally, coding exons that are alternatively spliced in a neural-specific isoform of Dlg are sufficient to drive nuclear localization; these protein sequences are conserved with hDlg1 (Bachmann et al, 2004).…”

“…The predicted NLS in the Dlg E-F/HOOK region is conserved in hDlg1. The SH3-HOOK-GUK region undergoes an intramolecular interaction that regulates exposure of sequences that can mediate several Dlg functions (Nix et al, 2000;McGee et al, 2001;Qian and Prehoda, 2006;Marcette et al, 2009;Newman and Prehoda, 2009;Zeng et al, 2017;Rademacher et al, 2019;Lu et al, 2021); the increased nuclear localization of transgenic Dlg or hDlg1 deleted for the GUK domain raises the possibility that nuclear entry could be one of these (Thomas et al, 2000;Kohu et al, 2002;Lu et al, 2021). The predicted NLS in Dlg PDZ1 is conserved not only in hDlg1 but also in the MAGUKs ZO-1, ZO-2 and their Drosophila homolog Polychaetoid.…”

“…Quantitation of membrane localization in follicle cells showed that, while Dlg 2XNLS>A was enriched at the membrane (PM Index >1), its localization was reduced compared to WT Dlg (Supplemental Figure 4A-D,G). We considered whether this might be due to disruption of electrostatic membrane binding, since the mutated residues in NLS2 of Dlg 2XNLS>A partially overlap with a recently described membrane binding polybasic region (Figure 6A) (Lu et al, 2021). However, localization was further reduced in scrib-depleted cells (Supplemental Figure 4E,F), while Dlg constructs that lack electrostatic membrane binding are insensitive to scrib depletion (Lu et al, 2021).…”

“…Both are conserved in the human homolog hDlg1, and for the latter, experimental evidence consistent with an NLS has been demonstrated. Transgenic constructs have previously deleted either the E-F or the entire HOOK domain; each results in mutant Dlg proteins that show strongly increased nuclear localization visible by immunohistochemistry (Hough et al, 1997;Lu et al, 2021). Since these data indicate that the E-F region cannot constitute the sole NLS of Dlg, we mutated basic residues within the predicted NLSs in both the E-F region as well as PDZ1 to alanine (Dlg 2XNLS>A , Figure 6A).…”

“…We considered whether this might be due to disruption of electrostatic membrane binding, since the mutated residues in NLS2 of Dlg 2XNLS>A partially overlap with a recently described membrane binding polybasic region (Figure 6A) (Lu et al, 2021). However, localization was further reduced in scrib-depleted cells (Supplemental Figure 4E,F), while Dlg constructs that lack electrostatic membrane binding are insensitive to scrib depletion (Lu et al, 2021). Most importantly, to determine if mutation of both predicted NLS altered Dlg's ability to enter the nucleus, we carried out subcellular fractionation studies on protein extracted from imaginal discs.…”