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.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Adaptor Proteins, Signal Transducing / genetics
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Adaptor Proteins, Signal Transducing / metabolism*
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Amino Acid Motifs
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Animals
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Movement / physiology
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Focal Adhesions / genetics
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Focal Adhesions / metabolism
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GTPase-Activating Proteins / genetics
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GTPase-Activating Proteins / metabolism*
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Guanine Nucleotide Exchange Factors / genetics
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Guanine Nucleotide Exchange Factors / metabolism*
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HeLa Cells
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Humans
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Intercellular Signaling Peptides and Proteins
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Kidney Tubules, Collecting / cytology
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Kidney Tubules, Collecting / metabolism*
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Mice
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Multiprotein Complexes / genetics
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Multiprotein Complexes / metabolism
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NIH 3T3 Cells
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PDZ Domains
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Phosphoproteins / genetics
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Phosphoproteins / metabolism*
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Protein Transport
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Rho Guanine Nucleotide Exchange Factors
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Sorting Nexins / genetics
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Sorting Nexins / metabolism*
Substances
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ARHGEF7 protein, human
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Adaptor Proteins, Signal Transducing
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Cell Cycle Proteins
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GIT1 protein, human
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GIT2 protein, human
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GTPase-Activating Proteins
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Git1 protein, mouse
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Git2 protein, mouse
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Guanine Nucleotide Exchange Factors
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Intercellular Signaling Peptides and Proteins
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Multiprotein Complexes
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Phosphoproteins
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Rho Guanine Nucleotide Exchange Factors
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SNX27 protein, human
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Snx27 protein, mouse
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Sorting Nexins