Protein kinase C regulates the phosphorylation and oligomerization of ERM binding phosphoprotein 50
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.).
References (39)
- et al.
Ezrin–radixin–moesin-binding phosphoprotein 50 is expressed at the apical membrane of rat liver epithelia
Hepatology
(2001) - et al.
Epithelial inducible nitric-oxide synthase is an apical EBP50-binding protein that directs vectorial nitric oxide output
J. Biol. Chem.
(2002) - et al.
Ezrin–radixin–moesin–binding phosphoprotein–50/Na+/H+ exchanger regulatory factor (EBP50/NHERF) blocks U50,488H-induced down-regulation of the human kappa opioid receptor by enhancing its recycling rate
J. Biol. Chem.
(2002) - et al.
Regulation of GTP-binding protein alpha q (Galpha q) signaling by the ezrin–radixin–moesin-binding phosphoprotein-50 (EBP50)
J. Biol. Chem.
(2002) - et al.
PDZ domains: structural modules for protein complex assembly
J. Biol. Chem.
(2002) - et al.
Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin
Neuron
(1999) - et al.
Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains
Cell
(1996) - et al.
Evidence for ezrin–radixin–moesin-binding phosphoprotein 50 (EBP50) self-association through PDZ–PDZ interactions
J. Biol. Chem.
(2000) - et al.
N-terminal PDZ domain is required for NHERF dimerization
FEBS Lett.
(2001) - et al.
Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase
J. Biol. Chem.
(2001)
G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domain-mediated interaction
J. Biol. Chem.
Binding specificity for RACK1 resides in the V5 region of beta II protein kinase C
J. Biol. Chem.
Protein kinase C epsilon-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor
J. Biol. Chem.
Activation of integrin-RACK1/PKCalpha signalling in human articular chondrocyte mechanotransduction
Osteoarthr. Cartil.
Separate C-terminal domains of the epithelial specific brush border Na+/H+ exchanger isoform NHE3 are involved in stimulation and inhibition by protein kinases/growth factors
J. Biol. Chem.
Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A
J. Biol. Chem.
Crystal structure of the PDZ1 domain of human Na(+)/H(+) exchanger regulatory factor provides insights into the mechanism of carboxyl-terminal leucine recognition by class I PDZ domains
J. Mol. Biol.
cAMP-induced phosphorylation and inhibition of Na(+)/H(+) exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor
J. Biol. Chem.
Specific interaction of the PDZ domain protein PICK1 with the COOH terminus of protein kinase C-alpha
J. Biol. Chem.
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