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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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EBP50, E3KARP, and ezrin show similar dynamic profiles in several polarized cell lines. Left, photobleaching recovery curves of GFP-tagged EBP50, E3KARP, and ezrin in JEG-3 (A), Caco-2 (B), and MDCK (C) cells. Error bars show SD; n ≥ 7 for all experiments. Right, representative time points for the recovery curves of the respective cells types. Scale bars: 2 μm. (D) 3xFLAG-tagged EBP50 and E3KARP expressed in JEG-3, Caco-2, and MDCK cells were immunoprecipitated (IP) and blotted for FLAG and endogenous ezrin.
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Figure 1: EBP50, E3KARP, and ezrin show similar dynamic profiles in several polarized cell lines. Left, photobleaching recovery curves of GFP-tagged EBP50, E3KARP, and ezrin in JEG-3 (A), Caco-2 (B), and MDCK (C) cells. Error bars show SD; n ≥ 7 for all experiments. Right, representative time points for the recovery curves of the respective cells types. Scale bars: 2 μm. (D) 3xFLAG-tagged EBP50 and E3KARP expressed in JEG-3, Caco-2, and MDCK cells were immunoprecipitated (IP) and blotted for FLAG and endogenous ezrin.

Mentions: We have recently analyzed the dynamics of green fluorescent protein (GFP)-EBP50 and ezrin-GFP in the choriocarcinoma-derived JEG-3 cell line and have shown that EBP50 has remarkably rapid dynamics in microvilli (recovery in ∼5 s) compared with its binding partner ezrin (recovery in ∼30 s). Interestingly, EBP50’s most closely related family member, E3KARP, has slow dynamics (recovery in ∼20 s; Garbett and Bretscher, 2012). To see whether these effects extend to other epithelial cell lines, we used fluorescence recovery after photobleaching (FRAP) on Caco-2 and MDCK cells expressing low levels of GFP-EBP50, GFP-E3KARP, and ezrin-GFP (Figure 1, A–C). Despite polarized Caco-2 and MDCK cells having much shorter microvilli than JEG-3 cells, they show nearly identical exchange rates for EBP50, E3KARP, and ezrin Previously we have shown that mutations to EBP50 that reduce its dynamics also increase its ability to precipitate ezrin from JEG-3 cell lysate (Garbett and Bretscher, 2012). To see whether the slower dynamics of E3KARP was also representative of a stronger interaction with ezrin, we transiently expressed 3xFLAG-tagged EBP50 or E3KARP in JEG-3, Caco-2, and MDCK cells, performed FLAG immunoprecipitations, and probed for endogenous ezrin (Figure 1D). In all three cell lines, EBP50 precipitated less endogenous ezrin than seen with E3KARP, indicating the reduced dynamics of E3KARP was likely due to a more persistent interaction with ezrin. We continued our study using JEG-3 cells, which are easier to manipulate in culture and have microvilli that are more readily observed by confocal microscopy.


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)

EBP50, E3KARP, and ezrin show similar dynamic profiles in several polarized cell lines. Left, photobleaching recovery curves of GFP-tagged EBP50, E3KARP, and ezrin in JEG-3 (A), Caco-2 (B), and MDCK (C) cells. Error bars show SD; n ≥ 7 for all experiments. Right, representative time points for the recovery curves of the respective cells types. Scale bars: 2 μm. (D) 3xFLAG-tagged EBP50 and E3KARP expressed in JEG-3, Caco-2, and MDCK cells were immunoprecipitated (IP) and blotted for FLAG and endogenous ezrin.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814156&req=5

Figure 1: EBP50, E3KARP, and ezrin show similar dynamic profiles in several polarized cell lines. Left, photobleaching recovery curves of GFP-tagged EBP50, E3KARP, and ezrin in JEG-3 (A), Caco-2 (B), and MDCK (C) cells. Error bars show SD; n ≥ 7 for all experiments. Right, representative time points for the recovery curves of the respective cells types. Scale bars: 2 μm. (D) 3xFLAG-tagged EBP50 and E3KARP expressed in JEG-3, Caco-2, and MDCK cells were immunoprecipitated (IP) and blotted for FLAG and endogenous ezrin.
Mentions: We have recently analyzed the dynamics of green fluorescent protein (GFP)-EBP50 and ezrin-GFP in the choriocarcinoma-derived JEG-3 cell line and have shown that EBP50 has remarkably rapid dynamics in microvilli (recovery in ∼5 s) compared with its binding partner ezrin (recovery in ∼30 s). Interestingly, EBP50’s most closely related family member, E3KARP, has slow dynamics (recovery in ∼20 s; Garbett and Bretscher, 2012). To see whether these effects extend to other epithelial cell lines, we used fluorescence recovery after photobleaching (FRAP) on Caco-2 and MDCK cells expressing low levels of GFP-EBP50, GFP-E3KARP, and ezrin-GFP (Figure 1, A–C). Despite polarized Caco-2 and MDCK cells having much shorter microvilli than JEG-3 cells, they show nearly identical exchange rates for EBP50, E3KARP, and ezrin Previously we have shown that mutations to EBP50 that reduce its dynamics also increase its ability to precipitate ezrin from JEG-3 cell lysate (Garbett and Bretscher, 2012). To see whether the slower dynamics of E3KARP was also representative of a stronger interaction with ezrin, we transiently expressed 3xFLAG-tagged EBP50 or E3KARP in JEG-3, Caco-2, and MDCK cells, performed FLAG immunoprecipitations, and probed for endogenous ezrin (Figure 1D). In all three cell lines, EBP50 precipitated less endogenous ezrin than seen with E3KARP, indicating the reduced dynamics of E3KARP was likely due to a more persistent interaction with ezrin. We continued our study using JEG-3 cells, which are easier to manipulate in culture and have microvilli that are more readily observed by confocal microscopy.

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