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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 last ∼50 residues in tail of EBP50 are more dynamic than those in E3KARP both in vivo and in vitro. (A) Sequence alignment of human EBP50 and E3KARP. Conserved residues are highlighted in blue, the site where the tail and ebd+ regions of EBP50 and E3KARP were swapped is marked in red. (B) Photobleaching recovery curves of GFP-tagged EBP50, EBP50-E3tail, and EBP50-E3ebd+. Error bars show SD; n ≥ 10 for all experiments. (C) Photobleaching recovery curves of GFP-tagged E3KARP, E3KARP-50tail, and E3KARP-50ebd+. Error bars show SD. n ≥ 9 for all experiments. (D) Photobleaching recovery curves of GFP-tagged wild-type EBP50, EBP50(242-358), and EBP50-PDZ1&2mut. Error bars show SD; n ≥ 10 for all experiments. (E) Photobleaching recovery curves of GFP-tagged wild-type E3KARP, E3KARP(239-337), and E3KARP-PDZ1&2mut. Error bars show SD; n ≥ 11 for all experiments. (F) Graph of the normalized intensity of SUMO-tagged EBP50 or E3KARP that remained bound to ezrin FERM beads after incubation with untagged EBP50 or E3KARP for the times indicated. The data from three independent experiments were fitted to a single exponential decay curve shown as a solid line. Error bars indicate SD; dotted lines indicate the 95% confidence interval of the fit. (G) A representative experiment from (F). Limiting amounts of SUMO-EBP50 or E3KARP were prebound to ezrin FERM beads and competed off with untagged EBP50 and E3KARP for the times indicated. Gel was stained for total protein with IRDye.
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Figure 4: The last ∼50 residues in tail of EBP50 are more dynamic than those in E3KARP both in vivo and in vitro. (A) Sequence alignment of human EBP50 and E3KARP. Conserved residues are highlighted in blue, the site where the tail and ebd+ regions of EBP50 and E3KARP were swapped is marked in red. (B) Photobleaching recovery curves of GFP-tagged EBP50, EBP50-E3tail, and EBP50-E3ebd+. Error bars show SD; n ≥ 10 for all experiments. (C) Photobleaching recovery curves of GFP-tagged E3KARP, E3KARP-50tail, and E3KARP-50ebd+. Error bars show SD. n ≥ 9 for all experiments. (D) Photobleaching recovery curves of GFP-tagged wild-type EBP50, EBP50(242-358), and EBP50-PDZ1&2mut. Error bars show SD; n ≥ 10 for all experiments. (E) Photobleaching recovery curves of GFP-tagged wild-type E3KARP, E3KARP(239-337), and E3KARP-PDZ1&2mut. Error bars show SD; n ≥ 11 for all experiments. (F) Graph of the normalized intensity of SUMO-tagged EBP50 or E3KARP that remained bound to ezrin FERM beads after incubation with untagged EBP50 or E3KARP for the times indicated. The data from three independent experiments were fitted to a single exponential decay curve shown as a solid line. Error bars indicate SD; dotted lines indicate the 95% confidence interval of the fit. (G) A representative experiment from (F). Limiting amounts of SUMO-EBP50 or E3KARP were prebound to ezrin FERM beads and competed off with untagged EBP50 and E3KARP for the times indicated. Gel was stained for total protein with IRDye.

Mentions: To determine whether a smaller region of the tail is capable of conferring this dynamic property, we next generated chimeras in which we swapped tail residues 294–337 of E3KARP onto residues 1–307 of EBP50 (EBP50-E3ebd+) and EBP50 tail residues 308–358 onto residues 1–293 of E3KARP (E3KARP-50ebd+) (Figure 4A). FRAP of GFP-tagged EBP50-E3ebd+ revealed that its exchange from microvilli was indistinguishable from EBP50-E3tail (Figure 4B). Importantly, the dynamics of GFP-E3KARP-50ebd+ was also similar to that of E3KARP-50tail (p value of 0.44) and different from E3KARP wild type (p value < 0.005) (Figure 4C). Taken together, this indicates that the last 50 residues of EBP50 contain this dynamic property, which is transferrable to E3KARP.


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 last ∼50 residues in tail of EBP50 are more dynamic than those in E3KARP both in vivo and in vitro. (A) Sequence alignment of human EBP50 and E3KARP. Conserved residues are highlighted in blue, the site where the tail and ebd+ regions of EBP50 and E3KARP were swapped is marked in red. (B) Photobleaching recovery curves of GFP-tagged EBP50, EBP50-E3tail, and EBP50-E3ebd+. Error bars show SD; n ≥ 10 for all experiments. (C) Photobleaching recovery curves of GFP-tagged E3KARP, E3KARP-50tail, and E3KARP-50ebd+. Error bars show SD. n ≥ 9 for all experiments. (D) Photobleaching recovery curves of GFP-tagged wild-type EBP50, EBP50(242-358), and EBP50-PDZ1&2mut. Error bars show SD; n ≥ 10 for all experiments. (E) Photobleaching recovery curves of GFP-tagged wild-type E3KARP, E3KARP(239-337), and E3KARP-PDZ1&2mut. Error bars show SD; n ≥ 11 for all experiments. (F) Graph of the normalized intensity of SUMO-tagged EBP50 or E3KARP that remained bound to ezrin FERM beads after incubation with untagged EBP50 or E3KARP for the times indicated. The data from three independent experiments were fitted to a single exponential decay curve shown as a solid line. Error bars indicate SD; dotted lines indicate the 95% confidence interval of the fit. (G) A representative experiment from (F). Limiting amounts of SUMO-EBP50 or E3KARP were prebound to ezrin FERM beads and competed off with untagged EBP50 and E3KARP for the times indicated. Gel was stained for total protein with IRDye.
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Figure 4: The last ∼50 residues in tail of EBP50 are more dynamic than those in E3KARP both in vivo and in vitro. (A) Sequence alignment of human EBP50 and E3KARP. Conserved residues are highlighted in blue, the site where the tail and ebd+ regions of EBP50 and E3KARP were swapped is marked in red. (B) Photobleaching recovery curves of GFP-tagged EBP50, EBP50-E3tail, and EBP50-E3ebd+. Error bars show SD; n ≥ 10 for all experiments. (C) Photobleaching recovery curves of GFP-tagged E3KARP, E3KARP-50tail, and E3KARP-50ebd+. Error bars show SD. n ≥ 9 for all experiments. (D) Photobleaching recovery curves of GFP-tagged wild-type EBP50, EBP50(242-358), and EBP50-PDZ1&2mut. Error bars show SD; n ≥ 10 for all experiments. (E) Photobleaching recovery curves of GFP-tagged wild-type E3KARP, E3KARP(239-337), and E3KARP-PDZ1&2mut. Error bars show SD; n ≥ 11 for all experiments. (F) Graph of the normalized intensity of SUMO-tagged EBP50 or E3KARP that remained bound to ezrin FERM beads after incubation with untagged EBP50 or E3KARP for the times indicated. The data from three independent experiments were fitted to a single exponential decay curve shown as a solid line. Error bars indicate SD; dotted lines indicate the 95% confidence interval of the fit. (G) A representative experiment from (F). Limiting amounts of SUMO-EBP50 or E3KARP were prebound to ezrin FERM beads and competed off with untagged EBP50 and E3KARP for the times indicated. Gel was stained for total protein with IRDye.
Mentions: To determine whether a smaller region of the tail is capable of conferring this dynamic property, we next generated chimeras in which we swapped tail residues 294–337 of E3KARP onto residues 1–307 of EBP50 (EBP50-E3ebd+) and EBP50 tail residues 308–358 onto residues 1–293 of E3KARP (E3KARP-50ebd+) (Figure 4A). FRAP of GFP-tagged EBP50-E3ebd+ revealed that its exchange from microvilli was indistinguishable from EBP50-E3tail (Figure 4B). Importantly, the dynamics of GFP-E3KARP-50ebd+ was also similar to that of E3KARP-50tail (p value of 0.44) and different from E3KARP wild type (p value < 0.005) (Figure 4C). Taken together, this indicates that the last 50 residues of EBP50 contain this dynamic property, which is transferrable to E3KARP.

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