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First published online 3 August 2004
doi: 10.1242/jcs.01233

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Sorting nexin 16 regulates EGF receptor trafficking by phosphatidylinositol-3-phosphate interaction with the Phox domain

¹ Division of Molecular and Life Science, Pohang University of Science and Technology, San 31, Hojadong, Pohang, Kyungbuk 790-784, Republic of Korea
² Department of Physiology, College of Medicine, Pusan National University, Pusan 602-739, Republic of Korea

View larger version (42K): [in a new window]   Fig. 1. Identification and cellular distribution of SNX16. (A) Domain structure of SNX16. (B) Multiple sequence alignment of sorting nexins (SNXs) 1-4 (GenBank accession nos. U53225, NP0030901, NP003786 and NP003785, respectively) and SNX16 (GenBank accession no. AF305780) was performed using the ClustalW program, and the aligned sequences are shown. (C) SNX16 and its mutant forms are depicted schematically with black boxes representing the PX domain. Amino acid positions within each construct are indicated by the numbers. (D) COS-7 cells were transiently transfected with FLAG-tagged SNX16 and its mutants followed 24-30 hours later by preparation of total cell lysates (Total), cytosolic fractions (cytosol), and membrane particulates (membrane), as described in the Materials and Methods. The distribution of FLAG-SNX16 and its mutants were determined in each fraction by immunoblotting with anti-FLAG antibody. (E) GFP-tagged SNX16 and its point mutant form in PX domain (green) were expressed in COS-7 cells. Cells were stained for endogenous EEA1 and transferrin receptor (TfR) with anti-EEA1 and anti-TfR primary antibody and visualized with Rhodamine Red-conjugated anti-rabbit IgG secondary antibody (red).

View larger version (46K): [in a new window]   Fig. 2. PX domain-dependent direct interaction between SNX16 and PtdIns(3)P. (A) The ability of GST-fused SNX16WT and SNX16Y145A to bind a variety of phosphoinositides (PI) was analyzed by protein-lipid overlay assay. 100 pmols of the relevant PI was spotted on to a nitrocellulose membrane, which was then incubated with the purified proteins. The membranes were washed and proteins bound to the membrane by lipid interaction were detected with anti-GST antibody. Results representative of at least three separate experiments are shown. (B) COS-7 cells were transfected with GFP-tagged SNX16 and fixed. Cells were processed to detect SNX16 (green) and TfR (red) simultaneously by confocal microscopy. Like TfR, SNX16 was redistributed from endosomal membranes to the cytosol after treatment with wortmannin. Bars, 10 µm. (C) COS-7 cells were transfected with FLAG-tagged SNX16WT and SNX16Y145A, cultured in the absence of serum for 3 hours and then treated with or without wortmannin (100 nM) for 15 min. Cells were lysed and lysates were immunoreacted with anti-TfR antibody. Complexes associated with TfR were analyzed with anti-FLAG and anti-TfR antibodies. Total cell lysates (40 µg) were immunoblotted with anti-FLAG antibody.

View larger version (55K): [in a new window]   Fig. 3. SNX16 associates with EGFR. (A) COS-7 cells were transfected with FLAG-tagged SNX16 and treated with EGF for the indicated times. Cell lysates were immunoprecipitated with anti-FLAG antibody and then immunoblotted with anti-EGFR antibody. Total cell lysates (40 µg) were then immunoblotted with anti-EGFR and anti-FLAG antibody. (B) COS-7 cells were transfected with GFP-tagged SNX16 and treated with EGF for the indicated times. Fixed cells were processed to simultaneously detect SNX16 (green) and EGFR (red) by confocal microscopy. Bars, 10 µm.

View larger version (59K): [in a new window]   Fig. 4. Effect of SNX16 on the EGF-induced degradation of EGFR. (A) COS-7 cells expressing FLAG-tagged SNX16WT and SNX16Y145A were treated with EGF for the indicated times and then immunoblotted with antibodies against EGFR, FLAG or actin. The activation of MAPK (phospho-ERK1/2) was also assessed by immunoblotting with anti-phospho ERK1/2 antibody. Anti-ERK1/2 antibody was used to detect levels of ERK1/2 as a loading control. (B) COS-7 cells were transfected with GFP-tagged SNX16 and incubated with Rhodamine-conjugated LysoTracker. Cells were treated with EGF for the indicated times. Fixed cells were processed to detect SNX16 (green) and LysoTracker (red) simultaneously by confocal microscopy. Bars, 10 µm. (C) COS-7 cells were treated with EGF at the indicated times. Cell surface proteins were biotinylated as described in Materials and Methods. Biotinylated proteins were recovered using Streptavidin and the amount of EGFR recovered was assessed by western blotting using anti-EGFR antibody.

View larger version (19K): [in a new window]   Fig. 5. Role of SNX16 in the regulation of EGF-induced gene transcription. COS-7 cells were transfected with the reporter genes SRE-Luc (10 ng) and lacZ (20 ng) and with FLAG-tagged SNX16WT and SNX16Y145A (10 ng). After EGF treatment for the indicated times, EGF-induced increase in luciferase activity was quantified and expressed as mean±s.d.

View larger version (41K): [in a new window]   Fig. 6. The PX domain is required for the homo-oligomerization of SNX16. Various HA or FLAG-tagged SNX mutants were coexpressed in COS-7 cells. FLAG-tagged proteins were immunoprecipitated and analyzed by immunoblotting using anti-HA antibody. Total cell lysates (40 µg) were then immunoblotted with anti-HA and anti-FLAG antibody.

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