Sorting Nexin 27 Protein Regulates Trafficking of a p21-activated Kinase (PAK) Interacting Exchange Factor (β-Pix)-G Protein-coupled Receptor Kinase Interacting Protein (GIT) Complex via a PDZ Domain Interaction

Julie L Valdes,Jingrong Tang,Mark I Mcdermott,Jean-Cheng Kuo,Seth P Zimmerman,Stephen M Wincovitch,Clare M Waterman,Sharon L Milgram,Martin P Playford

doi:10.1074/jbc.m111.260802

Julie L Valdes, Jingrong Tang + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m111.260802

Copy DOI

Abstract

Sorting nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27-PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells using a GST-SNX27 fusion construct as bait. We found that β-Pix (p21-activated kinase-interactive exchange factor), a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted with SNX27. The association of β-Pix and SNX27 is specific for β-Pix isoforms terminating in the type-1 PDZ binding motif (ETNL). In the same screen we also identified Git1/2 as a potential SNX27 interacting protein. The interaction between SNX27 and Git1/2 is indirect and mediated by β-Pix. Furthermore, we show recruitment of the β-Pix·Git complex to endosomal sites in a SNX27-dependent manner. Finally, migration assays revealed that depletion of SNX27 from HeLa and mouse principal kidney cortical collecting duct cells significantly decreases cell motility. We propose a model by which SNX27 regulates trafficking of β-Pix to focal adhesions and thereby influences cell motility.

Highlights

Sorting Nexin 27 regulates intracellular trafficking of proteins through the endosomal system
Cells lacking Sorting nexin 27 (SNX27) have decreased cell motility, which we propose to be due to ␤-Pix intracellular trafficking defects
We found that ␤-Pix (p21-activated kinase-interactive exchange factor), a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted with SNX27

Summary

Background

Sorting Nexin 27 regulates intracellular trafficking of proteins through the endosomal system. Results: An interaction between SNX27 and the guanine nucleotide exchange factor ␤-Pix in complex with Git family proteins is identified. Sorting nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. We propose a model by which SNX27 regulates trafficking of ␤-Pix to focal adhesions and thereby influences cell motility. The relatively large number of proteins in the sorting nexin family along with the diverse array of protein interaction domains present in each suggests multiple roles for this family during endosomal trafficking [3]. Regulation of cell motility by ␤-Pix might involve the Git family of proteins [14]. The involvement of ␤-Pix-Git in cell migration is complex and involves phosphorylation events, transient protein-protein interactions, and intracellular trafficking. We show that SNX27 is responsible for recruitment of the ␤-Pix1⁄7Git complex to endosomal sites and propose that, during cell motility, the ␤-Pix1⁄7Git complex recycles between the endocytotic system and focal contact sites

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION