Scribble associates with two polarity proteins, lgl2 and vangl2, via distinct molecular domains

Abstract

Scribble (Scrib) is a large multi-domain cytoplasmic protein that was first identified through its requirement for the establishment of epithelial polarity. We tested the hypotheses that Scrib asssociates with the basolateral membrane via multiple domains, binds specific protein partners, and is part of a multimeric complex. We generated a series of EGFP-tagged Scrib fusion proteins and examined their membrane localizations in two types of polarized mammalian epithelial cells using biochemical and morphological approaches. We found that Scrib's Leucine-rich-repeat (LRR) and PDS-95/Discs Large/ZO-1 (PDZ) domains independently associate with the plasma membrane in both cell types. We identified multiple large Scrib complexes, demonstrated that Scrib and the cytoplasmic protein Lethal giant larvae2 (Lgl2) co-IP and that this association occurs via Scrib's LRR domain. Further, this report demonstrates that the membrane protein Vangl2 binds selectively to specific PDZ domains in Scrib. Our identification of Scrib's associations highlights its function in multiple biologic pathways and sets the stage for future identification of more proteins that must interact with Scrib's remaining domains. J. Cell. Biochem. 99: 647-664, 2006. (c) 2006 Wiley-Liss, Inc.

Vang-like protein 2 and Rac1 interact to regulate adherens junctions

“…However, the exact mode of interaction between Vangl2 and Rac1 remains to be elucidated. Other molecules might be involved, for example Scrib, which can bind Vangl2 via its PDZ-binding domain (Kallay et al, 2006).…”

“…To test whether the PDZ (post synaptic density protein, Drosophila disc large tumor suppressor and zonula occludens-1 protein)-binding domain was necessary for the ability of Vangl2 to affect the actin cytoskeleton, we transfected MDCK cells with Vangl24-EGFP, a construct lacking the four amino acids in the PDZ-binding domain (Kallay et al, 2006). Cells expressing Vangl24-EGFP did not display any aberrant phenotype, indicating that interaction with other proteins via the PDZ-binding domain is essential for Vangl2 to cause the phenotype (Fig.…”

“…It was then inserted into the XhoI and/or HindIII site of the pcDNA3-HA expression vector or the NotI site of the human nestin (hnestin) 1852 vector (Lothian and Lendahl, 1997;Shariatmadari et al, 2005). The (human) Vangl2-EGFP and Vangl2D4-EGFP constructs were kind gifts from Lelita T. Braiterman (Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD) (Kallay et al, 2006).…”

JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells

“…On day 7 or 8 after FACS, whole cell extracts from the short-term cultures were prepared to evaluate JAM-A levels (see below and Ref. 27). Frozen stocks of virus-infected cells were prepared after several rounds of propagation and then thawed, cultured, and FACS sorted on postinfection days 40 (KO1) and 29 (KO2).…”

Scrib, un gène suppresseur de tumeurs impliqué dans la polarité cellulaire

“…Ce domaine permet, entre autres, son interaction avec Lgl [24], l'orthologue de Lethal giant larvae, un autre suppresseur de tumeurs génétiquement lié à Scribble chez la drosophile. Scrib possède aussi quatre domaines PDZ (post synaptic disc-large zona ou zonula occludens) 1 [16,25].…”