G Protein-coupled Receptor Kinase 6A Phosphorylates the Na+/H+ Exchanger Regulatory Factor via a PDZ Domain-mediated Interaction

Hall, Randy A.; Spurney, Robert F.; Premont, Richard T.; Rahman, Nadeem U.; Blitzer, Jeremy T.; Pitcher, Julie A.; Lefkowitz, Robert J.

doi:10.1074/jbc.274.34.24328

Cited by 136 publications

(161 citation statements)

References 32 publications

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“…G protein-coupled receptor kinase 6A (GRK6A) constitutively phosphorylates rabbit EBP50 at serine 289, which may facilitate EBP50 oligomerization. 21,22 During mitosis, rabbit EBP50 is phosphorylated by cyclin-dependent kinase Cdc2 at serine 279 and 301, which inhibits EBP50 oligomerization. 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.…”

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confidence: 99%

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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confidence: 99%

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

Chen

Jou

2012

Cell Death Differ

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“…Interestingly, the sequence identity between GRK4, GRK5, and GRK6 diverges immediately after the predicted C-terminal amphipathic helices, and this difference is coincident with exonexon boundaries (12). After the amphipathic helix, GRK4 and GRK6A, one of three GRK6 splice variants (27,28), contain sites for palmitoylation (Fig. 1A), whereas GRK5 contains numerous serine sites of phosphorylation.…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, the two additional splice variants of GRK6, GRK6B and GRK6C (Fig. 1A), lack cysteine sites of palmitoylation in their C termini (27) yet retain the predicted amphipathic helix motif. One would predict that palmitoylation combined with a membrane binding helix, as is found in GRK4 and GRK6A, would result in tighter membrane binding compared with GRK5, GRK6B, and GRK6C, which all appear to lack lipid modification, and possibly would promote localization to different membrane microdomains.…”

Section: Discussionmentioning

confidence: 99%

A Predicted Amphipathic Helix Mediates Plasma Membrane Localization of GRK5

Thiyagarajan

Stracquatanio

Pronin³

et al. 2004

Journal of Biological Chemistry

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G protein-coupled receptor kinases (GRKs) specifically phosphorylate agonist-occupied G protein-coupled receptors at the inner surface of the plasma membrane (PM), leading to receptor desensitization. GRKs utilize a variety of mechanisms to bind tightly, and sometimes reversibly, to cellular membranes. Previous studies demonstrated the presence of a membrane binding domain in the C terminus of GRK5. Here we define a mechanism by which this short C-terminal stretch of amino acids of GRK5 mediates PM localization. Secondary structure predictions suggest that a region contained within amino acids 546 -565 of GRK5 forms an amphipathic helix, with the key features of the predicted helix being a hydrophobic patch of amino acids on one face of the helix, hydrophilic amino acids on the opposite face, and a number of basic amino acids surrounding the hydrophobic patch. We show that amino acids 546 -565 of GRK5 are sufficient to target the cytoplasmic green fluorescent protein (GFP) to the PM, and the hydrophobic amino acids are necessary for PM targeting of GFP-546 -565. Moreover, full-length GRK5-GFP is localized to the PM, but mutation of the hydrophobic patch or the surrounding basic amino acids prevents PM localization of GRK5-GFP. Last, we show that mutation of the hydrophobic residues severely diminishes phospholipid-dependent autophosphorylation of GRK5 and phosphorylation of membrane-bound rhodopsin by GRK5. The findings in this report thus suggest the presence of a membrane binding motif in GRK5 and define the importance of a group of hydrophobic amino acids within this motif in mediating its PM localization.Cell surface localized G protein-coupled receptors (GPCRs) 1 detect a large variety of extracellular stimuli and initiate numerous intracellular signaling pathways. Proper regulation of GPCR signaling is maintained in part by a process known as desensitization (1), which refers to the turning off of a GPCRinitiated signaling event in the continuous presence of agonist. A key mechanism of GPCR desensitization is phosphorylation of agonist-occupied receptor by the G protein-coupled receptor kinases (GRKs) (2, 3). This phosphorylation promotes binding of arrestin proteins to the GPCR and results in subsequent uncoupling of the GPCR from G protein.To function properly GRKs need to be localized to the cytoplasmic face of the plasma membrane (PM) where their GPCR substrates are located. Different members of the GRK family utilize a variety of mechanisms to bind to cellular membranes (2, 3). GRK2 and GRK3 contain well characterized C-terminal pleckstrin homology domains (4) that mediate translocation of the kinases from the cytoplasm to the PM in response to GPCR activation (5, 6). The pleckstrin homology domains allow PM binding by interacting with both phospholipids and free G protein ␤␥ subunits. In contrast to GRK2/3, other GRK family members exhibit a more constitutive association with cellular membranes. GRK1 and GRK7 contain a C-terminal CAAX motif, which directs covalent modification by a farnesyl or gerany...

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“…Mammalian expression constructs, containing the HA-tagged NHERF, D1, and D2 constructs were kindly provided by Drs. Robert Lefkowitz (Howard Hughes Medical Institute, Duke University, Durham, NC) and Randy Hall (Emory University, Atlanta, GA) and have been described (18,38).…”

Section: Methodsmentioning

confidence: 99%

The Role of the C Terminus and Na+/H+ Exchanger Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia

Benharouga

Sharma

et al. 2003

Journal of Biological Chemistry

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The conserved C-terminal peptide motif ( 1476 DTRL) of the cystic fibrosis transmembrane conductance regulator (CFTR) ensures high affinity binding to different PSD-95/Disc-large/zonula occludens-1 (PDZ) domaincontaining molecules, including the Na ؉ /H ؉ exchanger regulatory factor (NHERF)/ezrin-radixin-moesin-binding phosphoprotein of 50 kDa. The physiological relevance of NHERF binding to CFTR is not fully understood. Individuals with mutations resulting in premature termination of CFTR (S1455X or ⌬26 CFTR) have moderately elevated sweat Cl ؊ concentration, without an obvious lung and pancreatic phenotype, implying that the CFTR function is largely preserved. Surprisingly, when expressed heterologously, the ⌬26 mutation was reported to abrogate channel activity by destabilizing the protein at the apical domain and inducing its accumulation at the basolateral membrane

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G Protein-coupled Receptor Kinase 6A Phosphorylates the Na+/H+ Exchanger Regulatory Factor via a PDZ Domain-mediated Interaction

Cited by 136 publications

References 32 publications

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

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

A Predicted Amphipathic Helix Mediates Plasma Membrane Localization of GRK5

The Role of the C Terminus and Na+/H+ Exchanger Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia

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