The scaffolding protein EBP50 regulates microvillar assembly in a phosphorylation-dependent manner

Garbett, Damien; LaLonde, David P.; Bretscher, Anthony

doi:10.1083/jcb.201004115

Cited by 67 publications

(114 citation statements)

References 40 publications

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“…These observations suggest that simple NHERF1 binary interactions or dimerization do not play an essential role in assembling a functional ternary complex and are unlikely to account for defective regulation of P i transport by the three NHERF1 mutants. Such a conclusion is compatible also with recent evidence that NHERF1 remains monomeric in cells (55). Further evidence that the described NHERF1 mutations do not act as dominant negative proteins comes from the absence of an inhibitory action when coexpressed with wild-type NHERF1.…”

Section: Discussionsupporting

confidence: 77%

Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

Wang

Means

Yang

et al. 2012

Journal of Biological Chemistry

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Background: Some inherited defects in NHERF1 are associated with high phosphate (P i ) excretion and skeletal abnormalities. Results: NHERF1 forms a multiprotein complex with Npt2a and ezrin. Mutants fail to assemble this ternary complex. Conclusion:The NHERF1 ternary complex is required for PTH-sensitive P i transport. Significance: NHERF1 mutations may cause disease processes through structural changes that prevent assembly of multiprotein complexes.

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Section: Discussionsupporting

confidence: 77%

Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

Wang

Means

Yang

et al. 2012

Journal of Biological Chemistry

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“…Both basal and MRC-5 conditioned medium-dependent Rac1 activation decreased upon b-PIX knockdown, confirming the involvement of b-PIX in the Rac1 activation, using MRC-5 conditioned medium (Figure 5b). Because the accessibility of EBP50 PDZ domains for binding partners can be modulated by phosphorylation at the ezrin-binding domain of EBP50, 14,20 we hypothesized that phosphorylation of ser347 and 348 residues in EBP50 might affect the binding between EBP50 and b-PIX. Using glutathione S-transferase (GST) fusion protein pull-downs, we found that the C-terminal of b-PIX interacts with ezrin only in the presence of EBP50 and not in the EBP50 knockdown cells (Figure 5c).…”

Section: Resultsmentioning

confidence: 99%

“…23 In addition, protein kinase C (PKC) phosphorylates human EBP50 at serine 162, 339 and 340, which regulates the affinity between the PDZ domains of EBP50 and the PDZ ligands, and promotes EBP50 oligomerization. 14,20,24,25 Recently, it has been known that microvilli assembly and the activity of cystic fibrosis transmembrane conductance regulator are modulated by EBP50 phosphorylation. 14,25 Although the accumulating evidence supports the functional importance of EBP50 phosphorylation, the underlying regulatory mechanisms involved remain poorly understood.…”

Section: Ebp50mentioning

confidence: 99%

“…10 EBP50 participates in the modulation of ion transport, organization of apical microvilli, cancer development, and the trafficking and stabilization of membrane proteins. 9,[12][13][14] Understanding the mechanisms that regulate the function of EBP50 is, therefore, a critical problem.…”

mentioning

confidence: 99%

“…3 The phosphorylation state of EBP50 regulates the accessibility of its PDZ domains for cognate ligands and the formation of EBP50 oligomers, both of which are required for EBP50 to act as a scaffold protein. 14,20 Several kinases phosphorylate EBP50. G protein-coupled receptor kinase 6A (GRK6A) constitutively phosphorylates rabbit EBP50 at serine 289, which may facilitate EBP50 oligomerization.…”

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confidence: 99%

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Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

Chen

Jou

2012

Cell Death Differ

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We demonstrated a protein kinase C (PKC)-dependent phosphorylation of canine ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) at serine 347/348 by site-directed mutagenesis and a phospho-specific antibody. Cell fractionation and confocal imaging revealed the relocation of EBP50 from the plasma membrane to cytosol that accompanied this phosphorylation event. Increased phosphorylation at these serine residues led to the dissociation of EBP50 from ezrin and b-PIX, which are two upstream regulators of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to hepatocyte growth factor-induced cell spreading and scattering, which is normally mediated by Rac1 activation. Detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation did not undergo apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function. Scaffold proteins have many modular domains that mediate protein-protein interactions involved in orchestrating different signaling cascades. As a result, scaffold proteins are thought to act as stable structural proteins that facilitate many cell signaling pathways. However, this traditional role of scaffold proteins has been challenged, 1 and the dynamic role of scaffold as both an activator and suppressor of cellular signaling remains poorly characterized. Furthermore, the factors that modulate such a bifunctional role of scaffold proteins remain largely unknown.Ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) is a scaffold protein with two tandem PDZ domains and a C-terminal ERM-binding domain. 2,3 EBP50 binds proteins that contain a PDZ-binding motif including ion exchangers, 4,5 ion channels, 6 G protein-coupled receptors, 7-9 growth factor tyrosine kinase receptors 10,11 and PTEN. 10 EBP50 participates in the modulation of ion transport, organization of apical microvilli, cancer development, and the trafficking and stabilization of membrane proteins. 9,12-14 Understanding the mechanisms that regulate the function of EBP50 is, therefore, a critical problem.Ras family proteins are locked in a GDP-bound, inactivated state by binding GDI in the GTP-rich cellular environment. 15,16 Dissociation of small GTPases from GDI is required, but is not sufficient for their activation. GEFs drive the exchange of GTP for GDP, leading to the activation of small GTPases. 17 EBP50 can activate Rho-family small GTPases by recruiting ezrin, which is thought to cause the dissociation of GDI from the small GTPase, and b-PIX, a Rac1 GEF that contains a PDZ-binding motif. 18,19 EBP50 is a serine/threonine-phosphorylated protein. 3 The phosphorylation state of EBP50 regulates the acc...

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Regulation of cell proliferation and adhesion by means of a novel region of drosophila merlin interacting with Sip1

Abeysundara

Leung

Primrose

et al. 2014

Developmental Dynamics

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Background: The tumor suppressor protein merlin is thought to regulate cell proliferation and cell adhesion through interaction with protein partners. Loss of merlin is associated with Neurofibromatosis Type 2 (NF2) tumors. NHERF1 or EBP50 is a scaffolding protein that functions in apical organization of polarized cells. Merlin and NHERF1 have been shown to interact in vitro in vertebrates. We investigate how the Drosophila NHERF1 orthologue, Sip1, and Merlin function to regulate cell proliferation and adhesion. Results: We identify two conserved arginine residues (R325 and R335) in Merlin which, in addition to the FERM domain, are required for interaction with Sip1. Mutation of the arginine residues result in reduced Sip1 binding to Merlin and loss of Merlin growth suppressor function. Over-expression of Merlin R325A and/or Merlin R335L in Drosophila wings result in increased proliferation in the adult wing (increase in size), which is rescued by co-over-expression of constitutively active Merlin protein. Reduced Sip1 binding to Merlin also produces defects in adhesion in follicle epithelial cells. Conclusions: Sip1 facilitates the activation of Merlin as a tumor suppressor protein. Thus, our work provides insight into how Merlin functions as a tumor suppressor and in adhesion and this provides insight into the mechanism of NF2 pathogenesis. Developmental Dynamics 243:1554-1570,

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The scaffolding protein EBP50 regulates microvillar assembly in a phosphorylation-dependent manner

Abstract: Both Cdc2 and PKC-mediated phosphorylation of EBP50 alter accessibility of its first PDZ domain when its second PDZ domain is occupied to regulate microvilli formation on epithelial cells.

Cited by 67 publications

References 40 publications

Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

Regulation of cell proliferation and adhesion by means of a novel region of drosophila merlin interacting with Sip1

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