Protein Kinase C Phosphorylation Disrupts Na+/H+ Exchanger Regulatory Factor 1 Autoinhibition and Promotes Cystic Fibrosis Transmembrane Conductance Regulator Macromolecular Assembly

Zhongping Dai,David J.E Callaway,Joseph R Testa,Poulikos I Poulikakos,Zimei Bu,Jianquan Li

doi:10.1074/jbc.m702019200

Zhongping Dai, David J.E Callaway + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m702019200

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Abstract

An emerging theme in cell signaling is that membrane-bound channels and receptors are organized into supramolecular signaling complexes for optimum function and cross-talk. In this study, we determined how protein kinase C (PKC) phosphorylation influences the scaffolding protein Na(+)/H(+) exchanger regulatory factor 1 (NHERF) to assemble protein complexes of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel that controls fluid and electrolyte transport across cell membranes. NHERF directs polarized expression of receptors and ion transport proteins in epithelial cells, as well as organizes the homo- and hetero-association of these cell surface proteins. NHERF contains two modular PDZ domains that are modular protein-protein interaction motifs, and a C-terminal domain. Previous studies have shown that NHERF is a phosphoprotein, but how phosphorylation affects NHERF to assemble macromolecular complexes is unknown. We show that PKC phosphorylates two amino acid residues Ser-339 and Ser-340 in the C-terminal domain of NHERF, but a serine 162 of PDZ2 is specifically protected from being phosphorylated by the intact C-terminal domain. PKC phosphorylation-mimicking mutant S339D/S340D of NHERF has increased affinity and stoichiometry when binding to C-CFTR. Moreover, solution small angle x-ray scattering indicates that the PDZ2 and C-terminal domains contact each other in NHERF, but such intramolecular domain-domain interactions are released in the PKC phosphorylation-mimicking mutant indicating that PKC phosphorylation disrupts the autoinhibition interactions in NHERF. The results demonstrate that the C-terminal domain of NHERF functions as an intramolecular switch that regulates the binding capability of PDZ2, and thus controls the stoichiometry of NHERF to assemble protein complexes.

Highlights

PDZ2 and the C-terminal domain of NHERF, the hydrogen bond cluster formed among Ser-170, Gln-177, and His-212 in PDZ2 may impede PDZ2 from binding to target peptides, which contributes to the weak binding capabilities of
NHERF (PDB code 2OZF) shows that Ser-162 is located in the indicate that the truncated PDZ2 and the PDZ2 domain in the loop formed between ␤1 and ␤2 strands and is exposed to sol- PDZ2-CT construct have different conformational states and vent and PKC phosphorylation
The findings presented in this study implicate that PKC may influence CFTR functions indirectly by phosphorylating scaffolding proteins and changing the affinity and stoichiometry of these scaffolding proteins to assemble CFTR complexes

Summary

MATERIALS AND METHODS

Rylation reaction was performed by adding 25 milliunits of PKC to a 20-␮l solution containing 1.0 ␮g of purified protein, 0.020 mM ATP, 20 mM HEPES, pH 7.4, 10 mM MgCl2, 1.0 mM dithiothreitol, and 1.7 mM CaCl2. The SPR response curves were obtained by subtracting the background signal generated by injecting the analyte over a control cell without ligand coating to remove the bulk refractive index effects. The three flow cells of a sensor chip were coated with C-CFTR at three different densities by injecting the C-CFTR solution twice to each flow cell at 3 ␮l of 3 ␮g/ml, 1 ␮l of 10 ␮g/ml, and 3 ␮l of 20 ␮g/ml. Light scattering experiments show that, at these protein concentrations, NHERF, NHERF(S339D/S340D), PDZ2-CT, and PDZ2CT(S339D/S340D) are monomeric, and the inter-molecular interference effects are negligible. The three-dimensional molecular envelopes reconstructed from SAXS were generated using the program package Situs, developed by Wriggers and Birmanns (61)

RESULTS

73 Ϯ 16 64 Ϯ 10 5300 Ϯ 200 1600 Ϯ 100

DISCUSSION