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The tails of apical scaffolding proteins EBP50 and E3KARP regulate their localization and dynamics.

Garbett D, Sauvanet C, Viswanatha R, Bretscher A - Mol. Biol. Cell (2013)

Bottom Line: Proteomic analysis of the effects of EBP50 dynamics on binding-partner preferences identified a novel PDZ1 binding partner, the I-BAR protein insulin receptor substrate p53 (IRSp53).Additionally, the tails promote different microvillar localizations for EBP50 and E3KARP, which localized along the full length and to the base of microvilli, respectively.Thus the tails define the localization and dynamics of these scaffolding proteins, and the high dynamics of EBP50 is regulated by the occupancy of its PDZ domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853.

ABSTRACT
The closely related apical scaffolding proteins ERM-binding phosphoprotein of 50 kDa (EBP50) and NHE3 kinase A regulatory protein (E3KARP) both consist of two postsynaptic density 95/disks large/zona occludens-1 (PDZ) domains and a tail ending in an ezrin-binding domain. Scaffolding proteins are thought to provide stable linkages between components of multiprotein complexes, yet in several types of epithelial cells, EBP50, but not E3KARP, shows rapid exchange from microvilli compared with its binding partners. The difference in dynamics is determined by the proteins' tail regions. Exchange rates of EBP50 and E3KARP correlated strongly with their abilities to precipitate ezrin in vivo. The EBP50 tail alone is highly dynamic, but in the context of the full-length protein, the dynamics is lost when the PDZ domains are unable to bind ligand. Proteomic analysis of the effects of EBP50 dynamics on binding-partner preferences identified a novel PDZ1 binding partner, the I-BAR protein insulin receptor substrate p53 (IRSp53). Additionally, the tails promote different microvillar localizations for EBP50 and E3KARP, which localized along the full length and to the base of microvilli, respectively. Thus the tails define the localization and dynamics of these scaffolding proteins, and the high dynamics of EBP50 is regulated by the occupancy of its PDZ domains.

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IRSp53-T binds to EBP50 and localizes to microvilli. (A) Experimental schematic of SILAC mass spectrometry to compare binding-partner differences of EBP50 and EBP50-E3tail. Briefly, JEG-3 cells stably expressing 3xFLAG-tagged versions of EBP50 or EBP50-E3tail were labeled with either light or heavy arginine and lysine, respectively. After being immunoprecipitated, samples were trypsin digested and analyzed by mass spectrometry. The ratio of light:heavy peptide can be compared to yield highly sensitive and quantitative differences in protein abundance. (B) Table of SILAC mass spectrometry results. The number of peptides and overall ratio of binding to EBP50/EBP50-E3tail is indicated. The last four C-terminal amino acids are also listed for reference. (C) Sequence of alignment of human IRSp53-S and IRSp53-T isoforms. Conserved regions are highlighted in blue; the PDZ binding motif of IRSp53-S and potential PDZ binding site in IRSp53-T are in red. (D) 3xFLAG-tagged vector alone or EBP50 were immunoprecipitated from transiently transfected cells also expressing either GFP-IRSp53-S or IRSp53-T and blotted for FLAG and GFP. (E) Maximum projection image of JEG-3 cells expressing TagRFPT-IRSp53-T (red) and ezrin (green). Scale bar: 5 μm.
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Figure 5: IRSp53-T binds to EBP50 and localizes to microvilli. (A) Experimental schematic of SILAC mass spectrometry to compare binding-partner differences of EBP50 and EBP50-E3tail. Briefly, JEG-3 cells stably expressing 3xFLAG-tagged versions of EBP50 or EBP50-E3tail were labeled with either light or heavy arginine and lysine, respectively. After being immunoprecipitated, samples were trypsin digested and analyzed by mass spectrometry. The ratio of light:heavy peptide can be compared to yield highly sensitive and quantitative differences in protein abundance. (B) Table of SILAC mass spectrometry results. The number of peptides and overall ratio of binding to EBP50/EBP50-E3tail is indicated. The last four C-terminal amino acids are also listed for reference. (C) Sequence of alignment of human IRSp53-S and IRSp53-T isoforms. Conserved regions are highlighted in blue; the PDZ binding motif of IRSp53-S and potential PDZ binding site in IRSp53-T are in red. (D) 3xFLAG-tagged vector alone or EBP50 were immunoprecipitated from transiently transfected cells also expressing either GFP-IRSp53-S or IRSp53-T and blotted for FLAG and GFP. (E) Maximum projection image of JEG-3 cells expressing TagRFPT-IRSp53-T (red) and ezrin (green). Scale bar: 5 μm.

Mentions: The difference in dynamics specified by the tail domains of EBP50 and E3KARP could be a direct result of a regulator interacting with the E3KARP tail to stabilize its interaction with ezrin, or a regulator binding to the EBP50 tail to enhance its release from ezrin. To search for such proteins, we used an unbiased proteomic approach. First, we generated stable JEG-3 cell lines expressing either 3xFLAG-tagged wild-type EBP50 or the chimeric fusion EBP50-E3tail. Next the cells were subjected to stable isotope labeling of amino acids in cell culture (SILAC), with the EBP50-expressing cells grown in “light” medium, and the EBP50-E3tail expressing cells grown in “heavy” medium to allow for uniform labeling. The 3xFLAG-EBP50 and 3xFLAG-EBP50-E3tail were separately immunoprecipitated, mixed, and subjected to trypsin digestion followed by mass spectrometry (Ong et al., 2002; Figure 5A). Consistent with our earlier results by Western blot (Figure 3D), the EBP50-E3tail chimera bound almost twice as much ezrin and three times as much radixin compared with EBP50 (Figure 5B). Interactions with known PDZ binding partners, such as EBP50 PDZ interactor of 64 kDa (EPI64), nadrin, and yes-associated protein-1, were unchanged in the EBP50-E3tail chimera (Mohler et al., 1999; Reczek and Bretscher, 2001). We also found a significant number of peptides for the N-terminal Inverse-Bin-Amphiphysin-RVS (I-BAR) domain containing protein insulin receptor substrate p53 (IRSp53) isoform–T, which preferentially bound ∼50% more to EBP50 than EBP50-E3tail (Figure 5B). Endogenous IRSp53-T runs at the same size as EBP50, so we were unable to confirm this interaction preference because its immunoblot signal was masked by the large quantity of 3xFLAG-EBP50 precipitated (unpublished data). However, due to the highly quantitative and sensitive nature of SILAC mass spectrometry, it is likely that IRSp53-T has a modest binding preference for EBP50 over EBP50-E3tail.


The tails of apical scaffolding proteins EBP50 and E3KARP regulate their localization and dynamics.

Garbett D, Sauvanet C, Viswanatha R, Bretscher A - Mol. Biol. Cell (2013)

IRSp53-T binds to EBP50 and localizes to microvilli. (A) Experimental schematic of SILAC mass spectrometry to compare binding-partner differences of EBP50 and EBP50-E3tail. Briefly, JEG-3 cells stably expressing 3xFLAG-tagged versions of EBP50 or EBP50-E3tail were labeled with either light or heavy arginine and lysine, respectively. After being immunoprecipitated, samples were trypsin digested and analyzed by mass spectrometry. The ratio of light:heavy peptide can be compared to yield highly sensitive and quantitative differences in protein abundance. (B) Table of SILAC mass spectrometry results. The number of peptides and overall ratio of binding to EBP50/EBP50-E3tail is indicated. The last four C-terminal amino acids are also listed for reference. (C) Sequence of alignment of human IRSp53-S and IRSp53-T isoforms. Conserved regions are highlighted in blue; the PDZ binding motif of IRSp53-S and potential PDZ binding site in IRSp53-T are in red. (D) 3xFLAG-tagged vector alone or EBP50 were immunoprecipitated from transiently transfected cells also expressing either GFP-IRSp53-S or IRSp53-T and blotted for FLAG and GFP. (E) Maximum projection image of JEG-3 cells expressing TagRFPT-IRSp53-T (red) and ezrin (green). Scale bar: 5 μm.
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Figure 5: IRSp53-T binds to EBP50 and localizes to microvilli. (A) Experimental schematic of SILAC mass spectrometry to compare binding-partner differences of EBP50 and EBP50-E3tail. Briefly, JEG-3 cells stably expressing 3xFLAG-tagged versions of EBP50 or EBP50-E3tail were labeled with either light or heavy arginine and lysine, respectively. After being immunoprecipitated, samples were trypsin digested and analyzed by mass spectrometry. The ratio of light:heavy peptide can be compared to yield highly sensitive and quantitative differences in protein abundance. (B) Table of SILAC mass spectrometry results. The number of peptides and overall ratio of binding to EBP50/EBP50-E3tail is indicated. The last four C-terminal amino acids are also listed for reference. (C) Sequence of alignment of human IRSp53-S and IRSp53-T isoforms. Conserved regions are highlighted in blue; the PDZ binding motif of IRSp53-S and potential PDZ binding site in IRSp53-T are in red. (D) 3xFLAG-tagged vector alone or EBP50 were immunoprecipitated from transiently transfected cells also expressing either GFP-IRSp53-S or IRSp53-T and blotted for FLAG and GFP. (E) Maximum projection image of JEG-3 cells expressing TagRFPT-IRSp53-T (red) and ezrin (green). Scale bar: 5 μm.
Mentions: The difference in dynamics specified by the tail domains of EBP50 and E3KARP could be a direct result of a regulator interacting with the E3KARP tail to stabilize its interaction with ezrin, or a regulator binding to the EBP50 tail to enhance its release from ezrin. To search for such proteins, we used an unbiased proteomic approach. First, we generated stable JEG-3 cell lines expressing either 3xFLAG-tagged wild-type EBP50 or the chimeric fusion EBP50-E3tail. Next the cells were subjected to stable isotope labeling of amino acids in cell culture (SILAC), with the EBP50-expressing cells grown in “light” medium, and the EBP50-E3tail expressing cells grown in “heavy” medium to allow for uniform labeling. The 3xFLAG-EBP50 and 3xFLAG-EBP50-E3tail were separately immunoprecipitated, mixed, and subjected to trypsin digestion followed by mass spectrometry (Ong et al., 2002; Figure 5A). Consistent with our earlier results by Western blot (Figure 3D), the EBP50-E3tail chimera bound almost twice as much ezrin and three times as much radixin compared with EBP50 (Figure 5B). Interactions with known PDZ binding partners, such as EBP50 PDZ interactor of 64 kDa (EPI64), nadrin, and yes-associated protein-1, were unchanged in the EBP50-E3tail chimera (Mohler et al., 1999; Reczek and Bretscher, 2001). We also found a significant number of peptides for the N-terminal Inverse-Bin-Amphiphysin-RVS (I-BAR) domain containing protein insulin receptor substrate p53 (IRSp53) isoform–T, which preferentially bound ∼50% more to EBP50 than EBP50-E3tail (Figure 5B). Endogenous IRSp53-T runs at the same size as EBP50, so we were unable to confirm this interaction preference because its immunoblot signal was masked by the large quantity of 3xFLAG-EBP50 precipitated (unpublished data). However, due to the highly quantitative and sensitive nature of SILAC mass spectrometry, it is likely that IRSp53-T has a modest binding preference for EBP50 over EBP50-E3tail.

Bottom Line: Proteomic analysis of the effects of EBP50 dynamics on binding-partner preferences identified a novel PDZ1 binding partner, the I-BAR protein insulin receptor substrate p53 (IRSp53).Additionally, the tails promote different microvillar localizations for EBP50 and E3KARP, which localized along the full length and to the base of microvilli, respectively.Thus the tails define the localization and dynamics of these scaffolding proteins, and the high dynamics of EBP50 is regulated by the occupancy of its PDZ domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853.

ABSTRACT
The closely related apical scaffolding proteins ERM-binding phosphoprotein of 50 kDa (EBP50) and NHE3 kinase A regulatory protein (E3KARP) both consist of two postsynaptic density 95/disks large/zona occludens-1 (PDZ) domains and a tail ending in an ezrin-binding domain. Scaffolding proteins are thought to provide stable linkages between components of multiprotein complexes, yet in several types of epithelial cells, EBP50, but not E3KARP, shows rapid exchange from microvilli compared with its binding partners. The difference in dynamics is determined by the proteins' tail regions. Exchange rates of EBP50 and E3KARP correlated strongly with their abilities to precipitate ezrin in vivo. The EBP50 tail alone is highly dynamic, but in the context of the full-length protein, the dynamics is lost when the PDZ domains are unable to bind ligand. Proteomic analysis of the effects of EBP50 dynamics on binding-partner preferences identified a novel PDZ1 binding partner, the I-BAR protein insulin receptor substrate p53 (IRSp53). Additionally, the tails promote different microvillar localizations for EBP50 and E3KARP, which localized along the full length and to the base of microvilli, respectively. Thus the tails define the localization and dynamics of these scaffolding proteins, and the high dynamics of EBP50 is regulated by the occupancy of its PDZ domains.

Show MeSH
Related in: MedlinePlus