Protein kinase C regulates the phosphorylation and oligomerization of ERM binding phosphoprotein 50

doi:10.1016/j.yexcr.2005.02.011

Experimental Cell Research

Volume 306, Issue 1, 15 May 2005, Pages 264-273

https://doi.org/10.1016/j.yexcr.2005.02.011 Get rights and content

Abstract

Ezrin–Radixin–Moesin (ERM) binding phosphoprotein 50 (EBP50, a.k.a. NHERF-1) is a scaffold protein essential for the localization and coordinated activity of apical transporters, enzymes and receptors in epithelial cells. EBP50 acts via multiple protein binding interactions, including oligomerization through interactions of its PSD95–Dlg–ZO1 (PDZ) domains. EBP50 can be phosphorylated on multiple sites and phosphorylation of specific sites modulates the extent of oligomerization. The aim of the present study was to test the capacity of protein kinase C (PKC) to phosphorylate EBP50 and to regulate its oligomerization. In vitro experiments showed that the catalytic subunit of PKC directly phosphorylates EBP50. In HEK-293 cells transfected with rat EBP50 cDNA, a treatment with 12 myristate 13-acetate (PMA) induced a translocation of PKCα and β isoforms to the membrane and increased ³²P incorporation into EBP50. In co-transfection/co-precipitation studies, PMA treatment stimulated EBP50 oligomerization. Mass spectrometry analysis of full-length EBP50 and phosphorylation analyses of specific domains, and of mutated or truncated forms of EBP50, indicated that PKC-induced phosphorylation of EBP50 occurred on the Ser³³⁷/Ser³³⁸ residue within the carboxyl-tail domain of the protein. Truncation of Ser³³⁷/Ser³³⁸ also diminished PKC-induced oligomerization of EBP50. These results suggest the PKC signaling pathway can impact EBP50-dependent cellular functions by regulating EBP50 oligomerization.

Introduction

Ezrin–Radixin–Moesin (ERM) binding phosphoprotein 50 (EBP50; a.k.a. NHERF-1) is a membrane-cytoskeleton linking protein localized in the apical region of epithelial cells [1], [2], [3]. Through protein–protein interactions in the apical region, EBP50 coordinates the activities of transporters, enzymes and receptors [3], [4], [5], [6], [7]. EBP50 is tethered to the actin cytoskeleton via a binding interaction of its carboxyl end with ezrin [8], while its amino portion consists of two PSD95–Dlg–ZO1 (PDZ) domains. PDZ domains consist of conserved sequences of ∼90 amino acids. They bind the COOH-tail of proteins that terminate with a PDZ ligand motif (-[T/S]-X-[Hydrophobic]) [9], [10]. Proteins with PDZ domains typically serve to sequester and coordinate the activities of integrated proteins within membrane microdomains [11]. To diversify and amplify their binding interactions, PDZ proteins either contain multiple PDZ domains, contain additional protein binding motifs or oligomerize. Oligomerization of these proteins may be mediated by PDZ domain-independent [12] or PDZ domain-dependent mechanisms [13], [14]. EBP50 oligomerization is mediated by PDZ–PDZ interactions [14] and while being oligomerized, EBP50 retains the ability to concurrently bind integral membrane proteins to its PDZ domains [15], [16].

EBP50 oligomerization is both positively and negatively regulated through site-specific phosphorylation [16], [17], [18]. Ser²⁸⁹ in rabbit EBP50 is a putative site of G protein-coupled receptor kinase 6a (GRK6a)-dependent phosphorylation [19]. A S289D mutation, which mimics constitutive phosphorylation at this site, enhances EBP50 oligomerization [16]. Rabbit EBP50 may also be phosphorylated on Ser²⁷⁷ and Ser³⁰¹ by the cyclin-dependent kinase cdc2 during mitosis [18] and, in contrast with phosphorylation at Ser²⁸⁹, phosphorylation at these sites inhibits EBP50 oligomerization. The aim of the present study was to investigate the capacity of protein kinase C (PKC) to phosphorylate EBP50 and to regulate EBP50 oligomerization. Several lines of evidence suggest that PKC might contribute to the regulation of EBP50 functions. During activation, PKC isoforms translocate from the cytosol to the plasma membrane, a site where EBP50 resides to interpose between membrane proteins and the underlying cytoskeleton. Protein–protein interactions of other PDZ domain-containing proteins are regulated by PKC phosphorylation. For example, PKC isoforms associate with EBP50 via the protein receptor, receptor for activated C kinase 1 (RACK1) and PKC activity regulates the net activity of NHE3 and CFTR, two EBP50-bound proteins [20], [21], [22], [23], [24]. Furthermore, it was shown in a recent study that PKC phosphorylates the PDZ2 domain of human EBP50 with a consequent modulation of associated CFTR activity [25]. The results in the present study demonstrate that PKC directly phosphorylates EBP50 on a previously uncharacterized phosphorylation site, Ser³³⁷/Ser³³⁸ within the C-tail domain, and that PKC-mediated phosphorylation of EBP50 enhances EBP50 oligomerization.

Section snippets

Preparation of EBP50 mutants

EBP50 point mutants were generated by overlap polymerase chain reaction from wild type Flag-EBP50 [14] using mismatched complementary primers to incorporate the appropriate mutation and wild-type primers encompassing the start codon and the termination codon (Table 1). Truncated EBP50, Δ336 and Δ346 were generated from wild type Flag-EBP50 using specific primers (Table 1). Products were digested with NotI, an internal restriction site within EBP50, and BamHI, and subsequently ligated into the

In vitro phosphorylation of EBP50 by PKC catalytic subunit

The capacity of PKC to directly phosphorylate EBP50 was assessed by incubating the catalytic subunit of PKC with isolated Flag-EBP50 in the presence of ³²P-ATP (Fig. 1). In the absence of the PKC catalytic subunit, no phosphorylation of EBP50 was observed. The addition of the PKC catalytic subunit resulted in marked EBP50 phosphorylation. Subsequent incubation in alkaline phosphatase reduced the levels of incorporated ³²P, confirming that the ³²P signal arose from phosphorylation of the

Discussion

EBP50 (a.k.a. NHERF-1) was first identified as a phosphorylated factor required for cAMP-inhibition of NHE3 activity in epithelial cells of the proximal tubule [1], [2]. It was later determined that the regulation of NHE3 by EBP50 did not require EBP50 phosphorylation [32]. Subsequently, EBP50 was shown to oligomerize through PDZ–PDZ interactions [14] and the phosphorylation status of EBP50 was found to moderate the oligomerization process [16], [18], [19]. Furthermore, there is both a positive

Acknowledgments

This work was supported by a postdoctoral fellowship from the “ Association pour la Recherche sur le Cancer ”, France (to L.F.), by National Institutes of Health Grants DK57729 and DK34039 (to R.B.D.) and GM42629 (to K.E.H.) and by a grant from the association “ Vaincre la Mucoviscidose ”, Paris (to C.H.).

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Na⁺-H⁺ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium–phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser⁴⁶, Ser¹⁶², Ser¹⁸¹, Ser²⁶⁹, Ser²⁸⁰, Ser²⁹¹, Thr²⁹³, Ser²⁹⁹, and Ser³⁰² did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser²⁹⁰ was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser²⁹⁰. Mutating ²⁵⁷VPF²⁵⁹ eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser²⁹⁰ dephosphorylation of recombinant NHERF1. Hydrogen–deuterium exchange MS revealed that β-sheets in NHERF1’s PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser²⁹⁰ rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein–coupled receptor kinase 6A controls the Ser²⁹⁰ phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.
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Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) is a scaffold protein, with two tandem PDZ domains and a carboxyl-terminal ezrin-binding (EB) region. This particular sticky structure is responsible for its interaction with different molecules to form multi-complexes that have a pivotal role in a lot of diseases. In particular, its involvement during carcinogenesis and cancer progression has been deeply analyzed in different tumors. The role of NHERF1 is not unique in cancer; its activity is connected to its subcellular localization. The literature data suggest that NHERF1 could be a new prognostic/predictive biomarker from breast cancer to hematological cancers. Furthermore, the high potential of this molecule as therapeutical target in different carcinomas is a new challenge for precision medicine. These evidences are part of a future view to improving patient clinical management, which should allow different tumor phenotypes to be treated with tailored therapies. This article reviews the biology of NHERF1, its engagement in different signal pathways and its involvement in different cancers, with a specific focus on breast cancer. It also considers NHERF1 potential role during inflammation related to most human cancers, designating new perspectives in the study of this “Janus-like” protein.
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The phosphorylation of specific residues in a flexible disordered activation loop yields precise control of signal transduction. One paradigm is the phosphorylation of S339/S340 in the intrinsically disordered tail of the multi-domain scaffolding protein NHERF1, which affects the intracellular localization and trafficking of NHERF1 assembled signaling complexes. Using neutron spin echo spectroscopy (NSE), we show salt-concentration-dependent excitation of nanoscale motion at the tip of the C-terminal tail in the phosphomimic S339D/S340D mutant. The “tip of the whip” that is unleashed is near the S339/S340 phosphorylation site and flanks the hydrophobic Ezrin-binding motif. The kinetic association rate constant of the binding of the S339D/S340D mutant to the FERM domain of Ezrin is sensitive to buffer salt concentration, correlating with the excited nanoscale dynamics. The results suggest that electrostatics modulates the activation of nanoscale dynamics of an intrinsically disordered protein, controlling the binding kinetics of signaling partners. NSE can pinpoint the nanoscale dynamics changes in a highly specific manner.
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It is possible that the preference of EGFR for a specific PDZ domain depends on the cellular context. In particular, it is now clear that phosphorylation of EBP50 by a number of kinases (including GRK6a, PKC and PKA) profoundly affects its interactions [40–43]. Also, the C-terminal sequence of EBP50 is a PDZ ligand itself and evidence supports the interaction between this PDZ-binding motif and the PDZ2 domain, resulting in a “closed” conformation [44–46].
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Arrestin scaffolds NHERF1 to the P2Y12 receptor to regulate receptor internalization
2012, Journal of Biological Chemistry
Citation Excerpt :
Interestingly, NHERF1 also underwent a small molecular mass increase of ∼3–4 kDa upon agonist treatment. This molecular weight shift may be a consequence of NHERF1 phosphorylation following receptor activation that is postulated to increase receptor affinity for NHERF1 (18, 19). We subsequently attempted to co-immunoprecipitate endogenous P2Y12R with NHERF1 from human platelets.
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Significance: NHERF1 mutations may cause disease processes through structural changes that prevent assembly of multiprotein complexes.

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Protein kinase C regulates the phosphorylation and oligomerization of ERM binding phosphoprotein 50

Abstract

Introduction

Section snippets

Preparation of EBP50 mutants

In vitro phosphorylation of EBP50 by PKC catalytic subunit

Discussion

Acknowledgments

Hepatology

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Neuron

Cell

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Osteoarthr. Cartil.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.