Department of Genetics, University of Melbourne, Parkville, Victoria 3010, Australia
Submitted 6 April 2004 ; accepted in final form 13 July 2004
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ABSTRACT |
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Menkes disease; PDZ; copper; trafficking
In epithelial cells, transport proteins are specifically targeted to particular membranes to effect vectorial transport of fluid, solutes, and electrolytes (29). Targeting from the TGN and endocytic compartments to either apical (AP) or basolateral (BL) membrane domains is regulated by sorting and/or retention mechanisms (10). Basolateral targeting signals are almost exclusively found in the cytoplasmic domains of transmembrane proteins and usually contain either tyrosine or dihydrophobic motifs (2). Studies have shown these motifs to interact with adaptor proteins AP1 (44) and AP2 (45, 46), which are involved in clathrin assembly at the TGN and the PM, respectively. Furthermore, basolateral sorting determinants often overlap internalization signals (21, 22). Because the hydrophobic dileucine L1487L1488 of wild-type MNK is required for internalization of MNK from the PM to the TGN, we investigated whether it also acts as a basolateral targeting motif.
PDZ domain proteins interact with PDZ target sequences usually located at the carboxyl termini of membrane-associated proteins (20). These interactions have been implicated in stabilization/retention of membrane proteins at specific membranes in polarized cells (34). The COOH terminus of MNK has the amino acid sequence DTAL, which fits the consensus target motif for class 1 PDZ proteins (20).
Defects or alterations in trafficking of membrane proteins have been linked with disease (25, 27, 30). Furthermore, a known mutation in a Menkes disease patient has been shown to inhibit MNK trafficking in cultured fibroblasts (1, 26). In the current study our aims were to characterize the normal localization of MNK in polarized cells under varying copper conditions and to identify signals in the COOH-terminal domain of MNK that regulate its trafficking to discrete membrane locations in polarized Madin-Darby canine kidney (MDCK) cells.
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MATERIALS AND METHODS |
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Antibodies. Polyclonal anti-rabbit MNK antibodies were described previously (5). Antibodies against Na-K-ATPase were a gift from Dr. Judy Callaghan (Dept. of Pathology and Immunology, Monash Medical School, Prahran, Australia). Anti-Golgi marker to protein 58K was purchased from Sigma-Aldrich (St. Louis, MO). The antibody anti-OKT9, a mouse monoclonal antibody used for the detection of the apical marker NPP3/TfR, was a gift from Prof. J. W. Goding. Anti-uvomorulin (E-cadherin) was purchased from Sigma-Aldrich and used as a marker for tight cell-cell contacts in polarized cells. Monoclonal mouse anti-ZO-1, a widely used antibody for detection of tight junctions associated with polarized epithelial cells, was purchased from Zymed Laboratories (South San Francisco, CA). For detection of primary antibodies, the Alexa Fluor range of IgG-fluorophore conjugates (Molecular Probes, Eugene, OR) was used.
Confocal microscopy. Confocal imaging was performed with an Optiscan F900e Personal Confocal System with a krypton-argon laser (Optiscan, Melbourne, Australia). An Olympus BX60 microscope with a x60 PlanApo oil-immersion lens was used for visualization of samples. F900e control software was used for image capture and analysis. At least 20 polarized cells, expressing varying levels of the transfected MNK constructs, were analyzed per sample, and representative images are shown in Figs. 2, 3, 5, and 6. Cell thickness was between 10 and 15 µm, scanned in the X-Y axis in 1-µm increments to create three-dimensional (3D) image stacks. Generally, 20 scans per image were required to bring out detail in membrane structures. Maximum-brightness images were obtained by flattening the 3D stack into a single image. Images in the X-Z axis were obtained from cross sections of the X-Y scans. Images were taken at normal, x1.25, or x1.5 software zoom and saved as RGB TIFFs to maintain acceptable image quality. Images were then opened in Photopaint 10 (Corel) for channel separation and brightness/contrast correction.
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RESULTS |
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MNK undergoes copper-induced relocalization to BL membrane. MDCK cells have a low detectable level of endogenous MNK that makes them ideal for studying the effects of mutations in transfected mutant MNK cDNAs. To investigate the role of MNK trafficking in polarized epithelia, MDCK cells were transfected with wild-type MNK and various MNK cDNA constructs containing mutations in distinct regions of the protein (see Fig. 1). Wild-type MNK localized at the Golgi of cells in basal medium (Fig. 3, A and C) was found to relocalize to the BL membrane after elevated copper treatment (Fig. 3, D and F). The restoration of basal copper levels to these copper-treated cells resulted in the return of MNK to the Golgi (Fig. 3, G and I). Uniform expression of ZO-1 under all copper conditions confirmed that these cells were polarized (Fig. 3, B, E, and H).
Surface biotinylation. To independently confirm the apparent basolateral localization of wild-type MNK we examined the levels of MNK in pools of proteins isolated from BL or AP membranes. AP or BL surface proteins were biotinylated with sulfo-NHS-SS-biotin, precipitated with streptavidin-agarose, separated by SDS-PAGE, and then detected with anti-MNK antibodies (36). In basal medium, the relative abundance of MNK in BL membranes was greater than that in AP membranes (Fig. 4A), suggesting that in the absence of elevated copper MNK is targeted to the BL membrane. When the copper concentration in the AP chamber was increased to 315 µM, there was a 2.3-fold increase in MNK at the BL surface, whereas levels of MNK at the AP surface were not altered by this treatment (Fig. 4B). When media containing 315 µM copper were added to the BL chamber, AP levels of MNK were unchanged but there was a fourfold increase in MNK at the BL surface (Fig. 4C). Adding 315 µM copper to both AP and BL chambers resulted in a 3.8-fold increase in MNK at the BL surface (Fig. 4D). These findings independently confirmed our immunofluorescence analyses and suggested that elevated copper stimulates the relocalization of MNK to the BL membrane.
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Identification of trafficking signals in COOH-terminal domain of MNK. To further characterize the copper-induced relocalization of MNK to the BL membrane, we used site-directed mutagenesis to identify candidate motifs critical for this targeting process. The dileucine internalization motif L1487-L1488 was of particular interest in this study because it was previously shown to be important for MNK recycling from the PM to the TGN in nonpolar cells (14, 38). In polarized MDCK cells the dileucine mutant L1487A-L1488A was mislocalized in basal medium, with only residual levels apparent at the Golgi (Fig. 6A). Interestingly, in elevated-copper conditions the weak labeling of L1487A-L1488A in the Golgi was no longer observed and there was an increased level of MNK above the level of E-cadherin staining, consistent with an apical or subapical localization (Fig. 6B). The L1487A-L1488A MNK failed to relocalize to the Golgi when cells were returned to basal medium (Fig. 6C). These data suggest that the dileucine motif L1487-L1488 is essential for basolateral targeting of MNK in elevated copper and endocytic retrieval from the PM after a restoration of copper homeostasis. Examination of the regions downstream of Leu1488 revealed a putative PDZ target motif, 1497DTAL1500. We investigated the role of this putative PDZ target motif in basolateral localization of MNK in MDCK cells by truncation of the 1497DTAL1500 sequence. This truncated form of MNK was localized to the Golgi in basal medium (Fig. 6D), dispersed to a region consistent with the AP membrane and intracellularly after elevated-copper treatment (Fig. 6E), and recycled to the Golgi when basal medium copper levels were restored after elevated-copper treatment (Fig. 6F). The localization of MNK at the AP membrane and intracellularly in elevated copper, along with its ability to recycle back to the Golgi in basal medium, suggested that the COOH-terminal PDZ binding domain is important for retention of MNK at the BL membrane when copper levels are elevated. To confirm the apparent AP membrane localization of mutants described above, MDCK cells stably expressing wtMNK were transfected with the chimeric protein NPP3 TM/cyto-HTFR EC, which resides primarily at the AP membrane of MDCK cells (4, 31). When cells were incubated in basal medium, transferred to elevated-copper medium, and returned to basal medium after copper treatment (Fig. 6, G, H, and I, respectively), the AP membrane was very clearly labeled with NPP3 TM/cyto-HTFR EC, similar to the localization of the L1487A-L1488A mutant MNK (as in Fig. 6, AC) as well as the 1497DTAL1500 truncated MNK in elevated copper (Fig. 6E).
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DISCUSSION |
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The mechanisms for sorting and targeting proteins to specific membranes in polarized epithelial cells are only partially understood (4). However, several recent studies have elucidated key domains of the cytoplasmic region of many membrane proteins. Basolateral targeting determinants may be complex in that they are often composed of several motifs with overlapping signals. For example, dileucine motifs may be read differently at specific subcellular sites according to their surrounding structural context (48). In the case of MNK it is known that a dileucine motif in the COOH-terminal cytoplasmic domain, L1487-L1488, is required for endocytic retrieval from the PM (14, 37, 38). We have identified an additional role for this particular dileucine motif to include BL membrane targeting in polarized epithelial cells. This dual role for dileucine motifs is similar to the dual endocytic and polarized targeting roles for a dileucine motif in the macrophage IgG receptor FcRII-2B in MDCK cells (21). Other studies have shown dileucine residues to be important BL membrane signaling motifs, but not as endocytic motifs (4, 13), and further studies have shown that dileucine motifs only target proteins to endosomal/lysosomal compartments (47). It is unclear why removal of the BL targeting dileucine motif of MNK results in AP membrane targeting (Fig. 6, AC). It is possible that the default trafficking pathway of MNK, without BL sorting via the dileucine motif, is to the AP membrane. Alternatively, mutation of the L1487-L1488 motif may have exposed a "cryptic" apical sorting signal as has been suggested in some other systems (35).
PDZ target motifs are mostly localized at the COOH terminus of membrane proteins. The mislocalization of DTAL in copper-supplemented medium (Fig. 6E) suggests that the putative PDZ target motif is important in sorting or BL membrane retention of MNK. In light of data from studies on other membrane proteins (34), an interaction of the PDZ target motif with PDZ binding proteins may be important for selective stabilization/retention of MNK at the BL membrane under conditions of elevated copper. This is consistent with our recent observation (36) from surface biotinylation studies of a rapid recycling pool of MNK proximal to the PM at elevated copper levels. A possible role of PDZ interaction in sorting of MNK cannot be excluded, and elucidation will require kinetic analysis (36), which was not possible in the current study because of relatively low transfection frequencies and difficulties in isolating stable cell lines (unpublished data).
Functional diversity of signaling mechanisms of membrane-associated proteins, including dileucine and PDZ motifs, has been highlighted by many recent studies in polarized cells. Furthermore, overlapping signals and the importance of amino acids surrounding previously characterized motifs suggest that secondary and tertiary protein structure is critical in determining intracellular localization via specific protein-protein interactions (4). Given our recent findings suggesting that there is a copper-regulated kinase phosphorylation of MNK (51), it is possible that signaling via phosphorylation mediates the "reading" of sorting signals as observed in some other systems (12). In the current studies, the identification of two signals, a dileucine motif and a PDZ target motif, has provided insight into the mechanisms of MNK trafficking to the BL membrane in polarized epithelial cells, where absorption/reabsorption is vital for maintaining systemic copper homeostasis. The copper-regulated trafficking of MNK to specific membrane domains in polarized cells provides an exciting paradigm for understanding the molecular interactions involved in regulating the function of a transporter through changes in its subcellular localization.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present addresses: I. Voskoboinik, Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne, Victoria 8006, Australia; M. J. Petris, Depts. of Biochemistry and Nutritional Sciences, University of Missouri, Columbia, MO 65211.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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