Report |
Address correspondence to Elizabeth Smythe, Dept. of Biomedical Sciences, University of Sheffield, Sheffield S10 2TN, England, UK. Tel.: 44-114-222-4635. Fax: 44-114-222-2788. email: e.smythe{at}sheffield.ac.uk
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Abstract |
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Key Words: regulation; endocytosis; sorting; coated vesicles; phosphorylation
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Introduction |
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Recent reports have demonstrated that µ2 phosphorylation regulates the interaction of AP2 with cargo. In permeabilized cells, it was shown that µ2 phosphorylation was required for the incorporation of transferrin bound to its receptor into newly formed coated pits (Olusanya et al., 2001). Furthermore, transfection of HeLa cells with mutant forms of µ2, which could not be phosphorylated, resulted in an inhibition of transferrin endocytosis, indicating the importance of µ2 phosphorylation in vivo (Olusanya et al., 2001). Other complementary analyses, measuring affinities of AP2 for peptides containing tyrosine-based internalization motifs, demonstrated that µ2 phosphorylation significantly enhances the association of AP2 with these motifs (Ricotta et al., 2002). The x-ray structure of the AP2 complex (lacking the hinge and ear domains of - and ß2-adaptin) has revealed that the binding site for cargo on µ2 is partially blocked by ß2-adaptin in the unphosphorylated complex (Collins et al., 2002). This suggests that a conformational change is required to reveal the binding site, and such a change could be effected by phosphorylation. In particular, the phosphorylation site on µ2, Thr156 (Pauloin and Thurieau, 1993), is located on an exposed loop of µ2 and is thus ideally situated to elicit such a change (Collins et al., 2002). Interestingly, the paradigm of phosphorylation of µ subunits regulating cargo interactions has been further supported by the demonstration that phosphorylation of µ1, the medium subunit of the AP1 adaptor complex found in clathrin-coated vesicles budding from the TGN, also enhances its association with cargo (Ghosh and Kornfeld, 2003).
These data support the premise that reversible cycles of phosphorylation are important in the control of the coated vesicle cycle. However, they raise the question of how µ2 phosphorylation is itself regulated so that AP2 adaptors are activated only in the correct functional context. Previous reports have shown that AP2 within clathrin coats has a higher affinity for sorting signals, suggesting that the processes of coated vesicle formation might be coupled to cargo sorting (Rapoport et al., 1997). Here, we provide a mechanistic explanation for how this occurs by showing that clathrin can directly regulate the levels of phosphorylated µ2 in vivo and in vitro via activation of the µ2 kinase.
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Results and discussion |
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To pursue the role of phosphorylated µ2 in coated pit formation, we generated anti-peptide antibodies to a phosphopeptide that corresponds to the Thr156 phosphorylation site on µ2. In addition, we purified AP2 from bovine brain, under conditions whereby µ2 kinase remains associated with the AP2 complex (Campbell et al., 1984). Untreated AP2, or AP2 that had been incubated with Mg2+ATP, were immunoblotted either with an antibody that recognizes the brain-specific insert of -adaptin or with the anti-phospho-Thr156 µ2 antibodies (Fig. 2 a). Although blotting with the
-adaptin antibodies showed that equal amounts of AP2 were present in each lane, immunoreactivity with anti-phospho-Thr156 µ2 antibodies was maximal where AP2 had been incubated with Mg2+ATP (Fig. 2 a, lane 2). Treatment of the phosphorylated complex with alkaline phosphatase resulted in a loss of immunoreactivity (Fig. 2 a, lane 3). Reactivity against phosphorylated µ2 was also lost when the antibody was incubated in the presence of a 100-fold molar excess of phosphopeptide (Fig. 2 a, lane 4), but not when incubated with nonphosphorylated peptide (Fig. 2 a, lane 5). Together, these data confirm that the antibody recognizes phospho-Thr156 µ2 only.
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When permeabilized cell membranes were incubated with APs prelabeled with [32P]ATP, there was a time-dependent decline in radioactivity that was prevented by microcystin (Fig. 2 e, bottom). This suggests that phosphate turnover was occurring on the µ2 subunit. When this experiment was repeated with APs prelabeled with cold ATP, there was an initial decline in phospho-µ2 immunoreactivity followed by an increase (Fig. 2 e, compare 0 to 5 min). However, the level of phospho-µ2 immunoreactivity after 30 min in the absence of microcystin was less than in its presence for the same time period (Fig. 2 e, middle). This strongly suggests that the level of phospho-µ2 is determined by the net activities of both kinase and phosphatases.
To investigate the connection between clathrin and µ2 phosphorylation in vivo, we examined the level of phospho-Thr156 µ2 in the chicken DT40 B cell line DKO-R. In this cell line, both endogenous alleles of clathrin heavy chain have been inactivated by homologous recombination and replaced with clathrin under the control of a tetracycline-responsive repressor (Wettey et al., 2002). From EST database searches (http://chick.umist.ac.uk), we confirmed that the amino acid sequence of chicken µ2 is 98% identical to the mammalian protein, and shows perfect conservation in the region surrounding Thr156 (unpublished data). We analyzed the distribution of phosphorylated µ2 in assembled and unassembled pools of clathrin prepared from DKO-R cells under clathrin-expressing conditions. Most of the clathrin was found in the assembled fraction (Fig. 3 a), consistent with the high endocytic capacity of the DKO-R cell line (Wettey et al., 2002). Immunoblotting of µ2 showed that the AP2 complex distributed more equally between the assembled and unassembled fractions. However, almost all the phospho-Thr156 µ2 was found in the assembled fraction, indicating that µ2 phosphorylation occurs primarily when AP2 is associated with the assembled pool of clathrin. In DKO-R cells treated with doxycycline for 96 h, clathrin expression was undetectable by immunoblotting (Fig. 3 b). Using a more sensitive clathrin triskelion ELISA (Cheetham et al., 1996), residual expression corresponding to 0.5% of the unrepressed level was detected under these conditions (unpublished data). As judged by immunoblotting, clathrin depletion did not affect the level of µ2 (Fig. 3 b) or, in separate experiments,
- and ß-adaptin (unpublished data). However, the level of µ2 phosphorylation was strikingly reduced by clathrin removal (Fig. 3 b).
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Materials and methods |
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Clathrin, AP2, and µ2 kinase
Coat proteins were prepared by differential centrifugation followed by gel filtration as described previously (Campbell et al., 1984). This allowed separation of clathrin from the APs. Further preparation of AP2 from the µ2 kinase was performed on hydroxyapatite as described previously (Pauloin and Thurieau, 1993). Some batches of AP2 and kinase were prepared by Linda Adams (University of Dundee, Dundee, UK) and Sophia Semerdjieva (University of Sheffield, Sheffield, UK). Clathrin heavy and light chains were separated as described previously (Winkler and Stanley, 1983).
Kinase assays
Kinase assays (20-µl final volume) contained µ2 kinase, AP2, 1 mM MgCl2, and 100 µM [32P]ATP (specific activity of 0.5 µCi/nmol) in 10 mM Tris, pH 7.5. Incubations were performed for 10 min at 30°C, reactions were stopped by addition of 5x Laemmli sample buffer, and the samples were heated at 60°C for 5 min before electrophoresis. Gels were fixed, dried, and radioactivity was quantified using a PhosphorImager (Fuji) followed by analysis using Fuji Science lab. Peptide kinase assays were performed using the peptide (EEQSQITSQVTCQIGWRRR) as substrate. Incubations were as described above, except that ATP was used at a specific activity of 2 µCi/nmol; incubations were processed as described previously (Cross and Smythe, 1998).
Permeabilized cell assay
The permeabilized cell assays were performed as described previously (Smythe et al., 1994; McLauchlan et al., 1998) with the following modifications: assay mixes were pretreated with 1 µM microcystin at 4°C for 10 min before allowing the reaction to proceed.
Growth and preparation of DKO-R cells
Maintenance of DKO-R cells and doxycycline treatment were as described previously (Wettey et al., 2002). 5 x 106 cells were lysed in PBS containing 0.1% Triton X-100, 0.2 mM PMSF, and 50 mM NaF, and were centrifuged at 1,000 g for 5 min. Cytosol was prepared as described previously (Wettey et al., 2002). Clathrin was detected with mAb TD1 (Wettey et al., 2002), µ2 with an mAb from Transduction Laboratories. To separate assembled from unassembled clathrin, cells were lysed in 40 mM Hepes, pH 7.4, 75 mM KCl, 4.5 mM MgCl2, 1.5 mM CaCl2, 0.5% NP-40, 0.01% PMSF, and 50 mM NaF, and were centrifuged at 1,000 g. After centrifugation at 100,000 g for 20 min, the supernatant (unassembled clathrin) was removed and the pellet (assembled clathrin) was resuspended in the same volume of lysis buffer as the supernatant (Cheetham et al., 1996). Both fractions were subjected to immunoblotting as above.
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Acknowledgments |
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This work was supported by grants from the Medical Research Council (G117/267), the Biotechnology and Sciences Research Council (94/C15666), and the British Heart Foundation (FS/2001053) to E. Smythe. F. Wettey received support from the Marie Curie Foundation, and L. Hufton from funds provided by Cambridge University.
Submitted: 14 April 2003
Accepted: 2 September 2003
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