C-terminal phosphorylation of MRP2 modulates its interaction with PDZ proteins q Tam as Heged€ us, a Tam as Sessler, a Robert Scott, b William Thelin, b  Eva Bakos, c Andr as V aradi, c Katalin Szab o, c L aszl  o Homolya, a Sharon L. Milgram, b and Bal azs Sarkadi a, * a Department of Molecular Cell Biology, Membrane Research Group of the Hungarian Academy of Sciences, National Medical Center, Di oszegi u. 64, 1113 Budapest, Hungary b Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, USA c Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary Received 31 January 2003 Abstract MRP2, a member of the ABC protein superfamily, functions as an ATP-dependent export pump for anionic conjugates in the apical membranes of epithelial cells. It has been reported that the trafficking of MRP2 is modulated by PKC. Adjacent to the C- terminal PDZ binding motif, which may be involved in the targeting of MRP2, we found a potential PKC phosphorylation site (Ser 1542 ). Therefore, we examined the interaction of MRP2 and its phosphorylation-mimicking mutants with different PDZ proteins (EBP50, E3KARP, PDZK1, IKEPP, b2-syntrophin, and SAP-97). The binding of these PDZ proteins to CFTR and ABCA1, other ABC proteins, possessing PDZ binding motif, was also studied. We observed a strong binding of apically localized PDZ proteins to both MRP2 and CFTR, whereas b2-syntrophin exhibited binding only to ABCA1. The phosphorylation-mimicking MRP2 mutant and a phosphorylated C-terminal MRP2 peptide showed significantly increased binding to IKEPP, EBP50, and both individual PDZ domains of EBP50. Our results suggest that phosphorylation of the MRP2 PDZ binding motif has a profound effect on the PDZ binding of MRP2. Ó 2003 Published by Elsevier Science (USA). Keywords: MRP2; PDZ; Protein–protein interaction; Phosphorylation PDZ (PSD-95/Dlh/ZO1) proteins have been de- scribed to take part in scaffolding multiprotein com- plexes, targeting, and regulation of membrane proteins [1,2]. PDZ proteins contain 80–90 amino acid long PDZ domains, in some cases in multiple copies, which may anchor different binding partners. Some PDZ proteins also contain other protein–protein or protein–lipid in- teraction domains. These include the ERM domain which facilitates binding to cytoskeletal elements, the SH3, or the PH domains providing additional protein– protein and protein–lipid interactions (Fig. 1A) [3]. PDZ proteins interact with proteins containing characteristic PDZ binding motifs. The classical PDZ binding motifs are localized at the C termini of proteins and their most generally recognized consensus pattern is composed of three amino acids, [T,S]-X-[F,V,A,I,L,M], or [F,Y]-B-[F,V,A,I,L,M], where X represents any, and B represents a bulky amino acid [1,4,5]. It has been proposed that additional residues upstream (e.g., posi- tions )3, )5, and )8 from the C-terminus) are also Biochemical and Biophysical Research Communications 302 (2003) 454–461 www.elsevier.com/locate/ybbrc BBRC q Abbreviations: ABC, ATP binding cassette; CFTR, cystic fibrosis transmembrane conductance regulator; E3KARP, NHE3 kinase A regulatory protein; EBP50, ERM (Ezrin/Radixin/Moesin) binding phosphoprotein 50; GRK5, G protein-coupled receptor kinase 5; GST, glutathione S-transferase; IKEPP, intestinal and kidney enriched PDZ protein; Kir2.3, inwardly rectifying K þ channel 2.3; MAGUK, membrane associated guanylate kinase; MRP2, multidrug resistance protein2; NHERF, Na þ =H þ exchanger regulatory factor; PDZ domain, Psd95/Dlg/ZO-1 domain; PH domain, pleckstrin homology domain; PKA, protein kinase A; PKC, protein kinase C; PSD-95, postsynaptic density protein 95; PVDF, polyvinylidene difluoride; SAP-97, synapsis associated protein 97; Sf9 cells, Spodoptera fru- giperda ovarian cells; TBS, Tris buffered saline. * Corresponding author. Fax: +36-1-372-4353. E-mail address: sarkadi@biomembrane.hu (B. Sarkadi). 0006-291X/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0006-291X(03)00196-7