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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 the PDZ1 domain of EBP50 and shows slow exchange from microvilli. (A) 3xFLAG-tagged vector alone, EBP50 wild-type, or a PDZ1 mutant were immunoprecipitated from transiently transfected cells also expressing GFP-IRSp53-T and blotted for FLAG, GFP, and endogenous EPI64. (B) GST-IRSp53-T tail or a mutant with an additional alanine added to the C-terminus (Amut) were used to precipitate purified full-length EBP50 wild-type or PDZ1, PDZ2, and PDZ1&2 mutants. Gel was stained for total protein with IRDye. (C) Photobleaching recovery curves of GFP-tagged EBP50, ezrin, and IRSp53-T. Error bars show SD; n ≥ 10 for all experiments. (D) Photobleaching recovery curves of GFP-tagged EBP50 alone or in cells also expressing TagRFPT-IRSp53-T. Error bars show SD; n ≥ 10 for all experiments. (E) Representative time points from photobleaching experiments of cells expressing GFP-EBP50 alone or with TagRFPT-IRSp53-T. Photobleached regions are indicated by green boxes. Scale bars: 2 μm.
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Figure 6: IRSp53-T binds to the PDZ1 domain of EBP50 and shows slow exchange from microvilli. (A) 3xFLAG-tagged vector alone, EBP50 wild-type, or a PDZ1 mutant were immunoprecipitated from transiently transfected cells also expressing GFP-IRSp53-T and blotted for FLAG, GFP, and endogenous EPI64. (B) GST-IRSp53-T tail or a mutant with an additional alanine added to the C-terminus (Amut) were used to precipitate purified full-length EBP50 wild-type or PDZ1, PDZ2, and PDZ1&2 mutants. Gel was stained for total protein with IRDye. (C) Photobleaching recovery curves of GFP-tagged EBP50, ezrin, and IRSp53-T. Error bars show SD; n ≥ 10 for all experiments. (D) Photobleaching recovery curves of GFP-tagged EBP50 alone or in cells also expressing TagRFPT-IRSp53-T. Error bars show SD; n ≥ 10 for all experiments. (E) Representative time points from photobleaching experiments of cells expressing GFP-EBP50 alone or with TagRFPT-IRSp53-T. Photobleached regions are indicated by green boxes. Scale bars: 2 μm.

Mentions: To further investigate how IRSp53-T interacts with EBP50, we performed immunoprecipitations from JEG-3 cells transiently transfected with GFP-IRSp53-T and 3xFLAG-tagged versions of either EBP50 wild-type, a PDZ1 mutant, or vector control (Figure 6A). Both GFP-IRSp53-T and endogenous EPI64, a known PDZ1 binding partner, bound very strongly to wild-type EBP50 but not to the PDZ1 mutant or vector control. To determine whether the interaction between IRSp53-T and EBP50 occurs directly through the potential PDZ binding motif in the tail of IRSP53-T and the PDZ1 domain of EBP50, we created glutathione S-transferase (GST) fusions of the IRSp53-T wild-type tail and a mutant with an alanine added (IRSp53-T-Amut), which blocks PDZ interactions, and performed GST pull downs against purified recombinant untagged full-length EBP50 (Figure 6B). The GST-IRSp53-T-Amut tail did not bind to any of the EBP50 constructs used. GST-IRSp53-T wild-type tail efficiently precipitated wild-type EBP50 and the PDZ2 mutant to a lesser degree, but did not bind to the PDZ1 or PDZ1&2 mutants. Altogether, this demonstrates that IRSp53-T directly interacts with the PDZ1 domain of EBP50 via its C-terminal PDZ binding motif.


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 the PDZ1 domain of EBP50 and shows slow exchange from microvilli. (A) 3xFLAG-tagged vector alone, EBP50 wild-type, or a PDZ1 mutant were immunoprecipitated from transiently transfected cells also expressing GFP-IRSp53-T and blotted for FLAG, GFP, and endogenous EPI64. (B) GST-IRSp53-T tail or a mutant with an additional alanine added to the C-terminus (Amut) were used to precipitate purified full-length EBP50 wild-type or PDZ1, PDZ2, and PDZ1&2 mutants. Gel was stained for total protein with IRDye. (C) Photobleaching recovery curves of GFP-tagged EBP50, ezrin, and IRSp53-T. Error bars show SD; n ≥ 10 for all experiments. (D) Photobleaching recovery curves of GFP-tagged EBP50 alone or in cells also expressing TagRFPT-IRSp53-T. Error bars show SD; n ≥ 10 for all experiments. (E) Representative time points from photobleaching experiments of cells expressing GFP-EBP50 alone or with TagRFPT-IRSp53-T. Photobleached regions are indicated by green boxes. Scale bars: 2 μm.
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Related In: Results  -  Collection

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Figure 6: IRSp53-T binds to the PDZ1 domain of EBP50 and shows slow exchange from microvilli. (A) 3xFLAG-tagged vector alone, EBP50 wild-type, or a PDZ1 mutant were immunoprecipitated from transiently transfected cells also expressing GFP-IRSp53-T and blotted for FLAG, GFP, and endogenous EPI64. (B) GST-IRSp53-T tail or a mutant with an additional alanine added to the C-terminus (Amut) were used to precipitate purified full-length EBP50 wild-type or PDZ1, PDZ2, and PDZ1&2 mutants. Gel was stained for total protein with IRDye. (C) Photobleaching recovery curves of GFP-tagged EBP50, ezrin, and IRSp53-T. Error bars show SD; n ≥ 10 for all experiments. (D) Photobleaching recovery curves of GFP-tagged EBP50 alone or in cells also expressing TagRFPT-IRSp53-T. Error bars show SD; n ≥ 10 for all experiments. (E) Representative time points from photobleaching experiments of cells expressing GFP-EBP50 alone or with TagRFPT-IRSp53-T. Photobleached regions are indicated by green boxes. Scale bars: 2 μm.
Mentions: To further investigate how IRSp53-T interacts with EBP50, we performed immunoprecipitations from JEG-3 cells transiently transfected with GFP-IRSp53-T and 3xFLAG-tagged versions of either EBP50 wild-type, a PDZ1 mutant, or vector control (Figure 6A). Both GFP-IRSp53-T and endogenous EPI64, a known PDZ1 binding partner, bound very strongly to wild-type EBP50 but not to the PDZ1 mutant or vector control. To determine whether the interaction between IRSp53-T and EBP50 occurs directly through the potential PDZ binding motif in the tail of IRSP53-T and the PDZ1 domain of EBP50, we created glutathione S-transferase (GST) fusions of the IRSp53-T wild-type tail and a mutant with an alanine added (IRSp53-T-Amut), which blocks PDZ interactions, and performed GST pull downs against purified recombinant untagged full-length EBP50 (Figure 6B). The GST-IRSp53-T-Amut tail did not bind to any of the EBP50 constructs used. GST-IRSp53-T wild-type tail efficiently precipitated wild-type EBP50 and the PDZ2 mutant to a lesser degree, but did not bind to the PDZ1 or PDZ1&2 mutants. Altogether, this demonstrates that IRSp53-T directly interacts with the PDZ1 domain of EBP50 via its C-terminal PDZ binding motif.

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