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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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The tail of EBP50 contains a unique dynamic property that is absent in E3KARP. (A) Sequence alignment of human EBP50 and E3KARP. Conserved residues are highlighted in blue; the site where the tails of EBP50 and E3KARP were swapped is marked in red. (B) Photobleaching recovery curves of GFP-tagged EBP50, EBP50-E3tail, E3KARP, and E3KARP-50tail. Error bars show SD; n ≥ 9 for all experiments. (C) Representative time points from photobleaching experiments of GFP-tagged EBP50, E3KARP, EBP50-E3tail, and E3KARP-50tail. Photobleached areas are indicated by green boxes. Scale bars: 2 μm. (D) 3xFLAG-tagged EBP50 and E3KARP constructs expressed in JEG-3 cells were immunoprecipitated (IP) and blotted for FLAG, endogenous ezrin, and E-cadherin as a control.
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Figure 3: The tail of EBP50 contains a unique dynamic property that is absent in E3KARP. (A) Sequence alignment of human EBP50 and E3KARP. Conserved residues are highlighted in blue; the site where the tails of EBP50 and E3KARP were swapped is marked in red. (B) Photobleaching recovery curves of GFP-tagged EBP50, EBP50-E3tail, E3KARP, and E3KARP-50tail. Error bars show SD; n ≥ 9 for all experiments. (C) Representative time points from photobleaching experiments of GFP-tagged EBP50, E3KARP, EBP50-E3tail, and E3KARP-50tail. Photobleached areas are indicated by green boxes. Scale bars: 2 μm. (D) 3xFLAG-tagged EBP50 and E3KARP constructs expressed in JEG-3 cells were immunoprecipitated (IP) and blotted for FLAG, endogenous ezrin, and E-cadherin as a control.

Mentions: To determine whether other regions in the tail of EBP50 also contribute to its higher exchange from microvilli, we swapped the entire tail regions of EBP50 (residues 238–358) and E3KARP (residues 235–337) (Figure 3A) and again examined their exchange from microvilli by FRAP and ability to coprecipitate ezrin from JEG-3 lysates. Surprisingly, fusion of the tail of E3KARP to the EBP50 PDZ domains (EBP50-E3tail) showed greatly reduced dynamics compared with EBP50 and was indistinguishable from E3KARP (Figure 3, B and C). Additionally, fusion of the EBP50 tail to the E3KARP PDZ domains (E3KARP-50tail) greatly enhanced dynamics compared with E3KARP and was very similar to that of EBP50 (Figure 3, B and C). This suggests that the tail of EBP50 possesses a unique property that can fully account for EBP50’s highly dynamic nature, as it is able to transfer this ability to the E3KARP-50tail chimera.


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)

The tail of EBP50 contains a unique dynamic property that is absent in E3KARP. (A) Sequence alignment of human EBP50 and E3KARP. Conserved residues are highlighted in blue; the site where the tails of EBP50 and E3KARP were swapped is marked in red. (B) Photobleaching recovery curves of GFP-tagged EBP50, EBP50-E3tail, E3KARP, and E3KARP-50tail. Error bars show SD; n ≥ 9 for all experiments. (C) Representative time points from photobleaching experiments of GFP-tagged EBP50, E3KARP, EBP50-E3tail, and E3KARP-50tail. Photobleached areas are indicated by green boxes. Scale bars: 2 μm. (D) 3xFLAG-tagged EBP50 and E3KARP constructs expressed in JEG-3 cells were immunoprecipitated (IP) and blotted for FLAG, endogenous ezrin, and E-cadherin as a control.
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Related In: Results  -  Collection

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Figure 3: The tail of EBP50 contains a unique dynamic property that is absent in E3KARP. (A) Sequence alignment of human EBP50 and E3KARP. Conserved residues are highlighted in blue; the site where the tails of EBP50 and E3KARP were swapped is marked in red. (B) Photobleaching recovery curves of GFP-tagged EBP50, EBP50-E3tail, E3KARP, and E3KARP-50tail. Error bars show SD; n ≥ 9 for all experiments. (C) Representative time points from photobleaching experiments of GFP-tagged EBP50, E3KARP, EBP50-E3tail, and E3KARP-50tail. Photobleached areas are indicated by green boxes. Scale bars: 2 μm. (D) 3xFLAG-tagged EBP50 and E3KARP constructs expressed in JEG-3 cells were immunoprecipitated (IP) and blotted for FLAG, endogenous ezrin, and E-cadherin as a control.
Mentions: To determine whether other regions in the tail of EBP50 also contribute to its higher exchange from microvilli, we swapped the entire tail regions of EBP50 (residues 238–358) and E3KARP (residues 235–337) (Figure 3A) and again examined their exchange from microvilli by FRAP and ability to coprecipitate ezrin from JEG-3 lysates. Surprisingly, fusion of the tail of E3KARP to the EBP50 PDZ domains (EBP50-E3tail) showed greatly reduced dynamics compared with EBP50 and was indistinguishable from E3KARP (Figure 3, B and C). Additionally, fusion of the EBP50 tail to the E3KARP PDZ domains (E3KARP-50tail) greatly enhanced dynamics compared with E3KARP and was very similar to that of EBP50 (Figure 3, B and C). This suggests that the tail of EBP50 possesses a unique property that can fully account for EBP50’s highly dynamic nature, as it is able to transfer this ability to the E3KARP-50tail chimera.

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