DLG1 is an anchor for the E3 ligase MARCH2 at sites of cell–cell contact

Abstract

PDZ domain containing molecular scaffolds play a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination.

Viral and cellular MARCH ubiquitin ligases and cancer

“…However, the RING-CH domains in the N termini of MARCH2 and MARCH3 and their PDZ-binding motifs in their C termini have been shown to be essential for their effects on intracellular location of syntax6 and TGN38/46 [94,95]. More recently, MARCH2 was found to interact with DLG1, a PDZ domain containing molecular scaffold involved in determining cell polarity [99]. MARCH2 co-localizes with DLG1 at sites of cell-cell contact and promotes DLG1 ubiquitination.…”

DLG1 (discs, large homolog 1 (Drosophila))

“…MARCH 2, which is part of the MARCH family ubiquitin ligases and is implicated in the endosomal trafficking interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, MARCH2 colocalized with DLG1 at sites of cell-cell contact (Cao et al, 2008). SAP97 is a binding partner of the cytoplasmic domain of TACE, which is the Tumour necrosis factor alpha converting enzyme and is the metalloproteasedisintegrin responsible for the ectodomain shedding of several proteins, including tumour necrosis factor alpha.…”

DLG1 (discs, large homolog 1 (Drosophila))

“…MARCH 2, which is part of the MARCH family ubiquitin ligases and is implicated in the endosomal trafficking interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, MARCH2 co-localized with DLG1 at sites of cell-cell contact (Cao et al, 2008). SAP97 is a binding partner of the cytoplasmic domain of TACE, which is the Tumour necrosis factor alpha converting enzyme and is the metalloprotease-disintegrin responsible for the ectodomain shedding of several proteins, including tumour necrosis factor alpha.…”