Article |
Address correspondence to Stuart Kornfeld, Washington University School of Medicine, Department of Internal Medicine, 660 S. Euclid Ave., Box 8125, St. Louis, MO 63110. Tel: (314)-362-8803. Fax: (314)-362-8826. E-mail: skornfel{at}im.wustl.edu
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Abstract |
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Key Words: adaptor protein-1; phosphoregulation; protein phosphatase 2A; clathrin-coated vesicle; uncoating
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Introduction |
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AP-1 is a heterotetrameric complex composed of two 100-kD subunits (termed
and ß1), an
47-kD subunit (µ1), and an
20-kD subunit (
1). The hinge regions of the ß1 and
subunits bind clathrin via clathrin box motifs (Shih et al., 1995; Doray and Kornfeld, 2001), and the appendage of the
subunit binds a number of accessory proteins, including
synergin (Page et al., 1999); Rabaptin-5 (Shiba et al., 2002); Golgi-localized,
earcontaining, adenosine 5' diphosphate-ribosylation factorbinding proteins (GGAs) (Doray et al., 2002b); and enthoprotin/clint (Kalthoff et al., 2002; Wasiak et al., 2002). The µ1 subunit interacts with tyrosine-based sorting motifs present on the cytoplasmic tails of the MPRs and other cargo molecules (Bremnes et al., 1998; Ohno et al., 1998; Owen and Evans, 1998), and either it or the ß1 subunit binds dileucine-based sorting motifs (Bremnes et al., 1998; Rapoport et al., 1998). These various proteinprotein interactions must be highly coordinated if the assembly/disassembly of AP-1containing CCVs is to proceed efficiently. Several reports have indicated that this process may be regulated by phosphorylation/dephosphorylation events. To date, clathrin (Hill et al., 1988), ß1 and µ1 subunits of AP-1(Wilde and Brodsky, 1996), GGAs 1 and 3 (Doray et al., 2002a), and the cytoplasmic tails of MPRs (Meresse et al., 1990) have been shown to be phosphorylated. Further, it has been established that phosphorylation of the ß1 hinge impairs clathrin binding and assembly (Wilde and Brodsky, 1996). Although the effect of µ1 phosphorylation on ligand binding has not been examined, it has been found that phosphorylation of the µ2 subunit of AP-2 greatly enhances the binding of sorting signals at the plasma membrane (Fingerhut et al., 2001; Ricotta et al., 2002). Finally, the phosphorylation of casein kinase-2 (CK2) sites in the cytoplasmic tails of the MPRs enhances binding to AP-1 (Le Borgne et al., 1993; Mauxion et al., 1996; Dittie et al., 1997).
We now report that phosphorylation of µ1 and ß1 is differentially regulated and that phosphorylation of µ1 strongly enhances ligand binding, whereas dephosphorylation of this subunit by protein phosphatase 2A (PP2A) is an essential step in the CCV uncoating process. These data, together with the previous findings of others, are incorporated into a model whereby cycles of AP-1 phosphorylation/dephosphorylation regulate CCV assembly and disassembly.
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Results |
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Taken together, these findings demonstrate that phosphorylation of the µ1 subunit enhances binding to sorting signals in the cytoplasmic tails of the MPRs, especially to the CK2 acidic cluster sites. This effect is most striking in the case of the CD-MPR; perhaps because it has a tyrosine-based sorting motif that is poorly recognized by AP-1 (Honing et al., 1997), and the other sorting motifs assume greater importance. One possible explanation for the enhanced binding to the CK2 acidic sorting motifs is that phosphorylation of µ1 induces a conformational change that exposes basic surfaces that are buried in the nonphosphorylated state. It is also apparent that µ1 phosphorylation is not the only factor that regulates the interaction of AP-1 with the MPR cytoplasmic tails, as phosphorylation of the CK2 sites enhances binding as well.
We next tested whether dephosphorylation of CCV-derived AP-1 impaired ligand binding. In a preliminary experiment, we showed that in vitrophosphorylated µ1 (and µ2) was dephosphorylated by low nanogram amounts of PP2A, and this dephosphorylation was completely prevented by okadaic acid (Fig. 3 A). As seen in Fig. 3 B, the PP2A treatment decreased the ability of the CCV-derived AP-1 to bind to the CD-MPR tail. Because µ1 is the only subunit of the CCV-derived AP-1 that is phosphorylated, this finding confirms that µ1 phosphorylation enhances ligand binding.
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Discussion |
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It has recently been demonstrated that the MPRs also bind to the GGA family of proteins (Puertollano et al., 2001; Takatsu et al., 2001; Zhu et al., 2001) and that this interaction is required for packaging of the MPRs into AP-1 CCVs (Doray et al., 2002b). We found that the GGAs with their bound MPRs interact with AP-1 and that the CK2 associated with AP-1 phosphorylates GGAs 1 and 3 (Doray et al., 2002b). This results in autoinhibition of cargo binding, providing a mechanism for the transfer of the MPRs from these GGAs to AP-1. The CK2 would also be expected to phosphorylate the CK2 sites in the cytoplasmic tails of the MPRs, thereby enhancing their binding to AP-1.
The membrane-associated AP-1 also binds clathrin and facilitates clathrin assembly, which proceeds to the formation of a CCV that eventually buds off from the membrane. The vesicle carries within itself a dormant CK2 (associated with AP-1) and an active GAK (Korolchuk and Banting, 2002). After budding, clathrin disassembly occurs, most likely triggered by GAK and Hsc-70 (Braell et al., 1984; Schlossman et al., 1984; Prasad et al., 1993; Barouch et al., 1994; Ungewickell et al., 1995; Umeda et al., 2000; Morgan et al., 2001). During this process, the ß1 subunit of AP-1 becomes phosphorylated, but the kinase that mediates this event is currently unknown. However, it is tempting to speculate that the phosphorylation of the ß1 hinge could additionally contribute to clathrin dissociation.
After clathrin release, the adaptors remain on the membrane, possibly to mediate "postbudding" roles (Huang et al., 2001), such as the interaction with motor kinesins like KIF13A (Nakagawa et al., 2000) or with Rabaptin-5 (Shiba et al., 2002). At some point, presumably before vesicle fusion with the acceptor compartment, the adaptors are released back to the cytosol. This event, in our model, is mediated by Hsc-70 in concert with PP2A, which is recruited via its interaction with Hsc-70 (Figs. 57). PP2A-mediated dephosphorylation would reverse the conformation change in the µ1 subunit that was induced by its phosphorylation. This would be expected to decrease the avidity for cargo sorting signals, thereby facilitating dissociation from the membrane. AP-1 dissociation may be further stimulated by PP2A-mediated dephosphorylation of the CK2 sites in the cytoplasmic tails of the cargo molecules (Molloy et al., 1998). In addition, dephosphorylation of µ1 could decrease surface charge interactions between the µ1 subunit and the lipid bilayer of the membrane, as speculated for µ2 (Collins et al., 2002). The released AP-1 will have a phosphorylated ß1 subunit and a dephosphorylated µ1 subunit, as found in the cytosol of mouse L cells (Fig. 1). A similar mechanism appears to account for AP-2 release from CCVs (Fig. 5 B). This requirement for µ2 dephosphorylation in CCV recycling could explain why maintaining µ2 in a phosphorylated state eventually results in inhibition of CCV-mediated transferrin uptake (Conner and Schmid, 2002).
This model explains the critical role of cycles of phosphorylation and dephosphorylation in regulating clathrin-mediated cargo transport at the TGN. In future studies, it will be necessary to identify the PP2A family member associated with the TGN and the kinase that phosphorylates the ß1 subunit. In addition, it will be important to gain an understanding of how the kinases and phosphatases are themselves regulated so that they act at the correct time to coordinate this complex series of events.
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Materials and methods |
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Buffers.
The following buffers were used: buffer A (10 mM [NH4]2 SO4, 20 mM Hepes, pH 7.0, 2 mM magnesium acetate, 25 mM KCl, 1 mM PMSF); buffer B (20 mM Hepes, pH 7.2, 5 mM magnesium acetate, 125 mM potassium acetate, 0.1% Triton X-100, 1 mM DTT); buffer C (1.0 M Tris-HCl, pH 7.0, 1 mM EDTA, 1 mM PMSF); buffer D (0.1 M Tris, pH 8.0, 0.1 M NaCl, 0.5% sodium deoxycholate, 0.2% SDS, 1% Triton X-100, 10 nM okadaic acid); and buffer E (25 mM Tris-Cl, pH 7.5, 125 mM potassium acetate, 5 mM magnesium acetate, 1 mM DTT). All chemicals were reagent grade and obtained from Sigma-Aldrich.
Antibodies.
Rat antiHsc-70 monoclonal antibody was obtained from Calbiochem. Rabbit antiPP2A-A/ß was from Santa Cruz Biotechnology, Inc. AP-1 was probed with antiAP-1
-hinge monoclonal 100/3 antibody and anti-ß1/2 monoclonal 100/1 antibody; AP-2 with antiAP-2
monoclonal 100/2 antibody from Sigma-Aldrich. Rabbit anti-µ1 (RY/1) was prepared as described earlier (Traub et al., 1995). Anti-clathrin monoclonal antibody TD.1 was from BAbCo. AntiAP-1
monoclonal antibody from Transduction Laboratories was used for immunoprecipitation of AP-1. Species-specific HRP-conjugated secondary antibodies used for Western blotting were from Amersham Biosciences.
Methods
Bovine liver was harvested immediately after slaughter and was used for CCV preparation as previously described (Meresse et al., 1990). Sucrose gradientpurified CCVs were stored at -80°C until usage. Bovine brain cytosol was prepared as before (Drake et al., 2000), and bovine liver cytosol was the post70,000 g centrifugation byproduct during the CCV preparation. Tris extraction of the bovine liver CCVs was performed at 4°C overnight in buffer C. After centrifugation at 100,000 g for 1 h, the supernatant containing the released AP-1 was collected and dialyzed against PBS. AP-1 was immunopurified from the cytosol and Tris extracts of CCVs using affinity chromatography and peptide elution as described earlier (Zhu et al., 1998).
Coat release assay.
5 µg of bovine liver CCVs was incubated with or without 0.5 µg of Hsc-70/10 ng PP2A/50 µg bovine brain cytosol, as indicated in the figure legends for Figs. 5 and 6, in the presence of an ATP-regenerating system (containing 800 µM ATP, creatine phosphokinase, and 5 mM creatine phosphate) in a final volume 50 µl of buffer A. The reactions were performed in thick-walled ultracentrifuge Eppendorf tubes at 25°C. At the indicated times, the reactions were centrifuged in a Beckman TLA 100.3 rotor at 100,000 g for 10 min at 4°C. 40 µl of the supernatant was removed without disturbing the pellet and analyzed by SDS-PAGE and Western blotting for the presence of released coat components.
Pull-down assays.
GSTCI-MPR and CD-MPR peptides, wild-type and mutants, were expressed and purified as before (Doray et al., 2002a). GSTPP2A-A plasmid was a generous gift from Marc C. Mumby (University of Texas Southwestern Medical Center, Dallas, TX). The reactions contained 100 µg of purified GST fusion ligand that had been prebound to glutathione-Sepharose beads at room temperature for 2 h and 4 µg of immunopurified AP-1 from either bovine liver cytosol or Tris extracts of bovine liver CCVs in a final volume of 350 µl of buffer B. The reactions were incubated at 4°C for 4 h with constant tumbling. The beads were then collected by centrifugation at 3,000 rpm and given four washes, each with 1 ml of buffer B. The pellet was boiled in SDS sample buffer for Western blotting.
Western blotting.
Samples boiled in SDS sample buffer were subjected to SDS-PAGE on a 10% gel, transferred onto nitrocellulose, and immunoblotted with the antibodies mentioned before. The blot was developed using an ECL detection kit from Amersham Biosciences and filmed with KODAK X-OMAT K (Eastman Kodak Co.).
Immunodepletion of cytosolic PP2A.
An affinity column was prepared by coupling 200 µg of antiPP2A-A/ß to 250 mg of CNBr-activated Sepharose beads (Sigma-Aldrich). Bovine brain cytosol was subjected to repeated passes through this column until the PP2A was depleted, as determined by immunoblotting. The beads were regenerated by washing with glycine, pH 2.5.
In vivo phosphorylation, membranecytosol fractionation, and immunoprecipitation of AP-1.
Mouse L cells (LSS) were grown in -MEM (supplemented with 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin) at 37°C and 5% CO2. Four confluent P100 dishes were labeled with 1 mCi/ml [32P]orthophosphoric acid/phosphate-free
-MEM/10% FBS/20 mM Hepes, pH 7.4, with or without 10 nM okadaic acid for 4 h at 37°C and 5% CO2. The medium was removed and the dishes washed with ice cold PBS. The cells were scraped in 1 ml of homogenization buffer (100 mM MES, pH 6.5, 0.25% sucrose, 1 mM EGTA, 10 mM okadaic acid, and protease inhibitor cocktail). The cell suspension was sonicated three times, 10 s each, at intensity 3.5 using a Fisher Scientific sonic dismembrator (model no. 550), followed by centrifugation at 650 g for 15 min to remove intact cells and nuclei. The postnuclear supernatant was collected and subjected to centrifugation at 100,000 g in a Beckman TLA 100.3 rotor for 60 min. The supernatant was collected (cytosol) and the pellet was resuspended in buffer B (supplemented with 10 mM okadaic acid and 0.4% Triton X-100), sonicated, and centrifuged at 14,000 g for an additional 15 min to remove insoluble membrane proteins. The two supernatant fractions were incubated with 10 µl of antiAP-1
antibody overnight with tumbling. The next morning, BSA-blocked protein Gagarose beads were added to the tubes and incubated for an additional hour. The beads were then pelleted and washed repeatedly with buffer D until no further radioactivity was released. The washed pellets were boiled in sample buffer and subjected to SDS-PAGE using a 10% gel. The gel was dried and filmed with KODAK X-OMAT MR. Duplicate sets were subjected to Western blotting to confirm the specificity of the immunoprecipitation.
Dephosphorylation assays.
Dephosphorylation assays were performed on phosphorylated AP-1 prepared both in vivo and in vitro. AP-1 was phosphorylated in vivo in the presence of okadaic acid and immunoprecipitated from whole cell lysates as above. The washed beads were then subjected to dephosphorylation in buffer A with either commercially obtained bovine kidney PP2A or Golgi-enriched membranes. The duration of incubations and the amount of PP2A and Golgi membrane used are indicated in the figure legends. CCVs were used to phosphorylate the µ1 subunit of AP-1 in vitro because they contain AP-1 as well as GAK kinase, which is known to act on µ1 (Umeda et al., 2000). 25 µg of CCVs was incubated for 4 h at 30°C in buffer A supplemented with 1.5 mM ATP 32P (ICN Biomedicals; specific activity 12.5 µCi/mmol ATP) in a final volume of 40 µl. After incubation, PP2A was added, as indicated, for an additional 15 min, and the entire mixture was subjected to SDS-PAGE on a 12% gel and autoradiography after drying. The phosphorylated 50-kD and 47-kD bands were the only detectable products of the reaction. The use of a 12% gel was required to resolve the µ1 and µ2 subunits (Loeb et al., 1989), which otherwise run together (Korolchuk and Banting, 2002).
Limited proteolysis of purified AP-1.
Aliqouts (2.5 µg) of purified AP-1, of either cytosolic or CCV source, were incubated with various amounts of bovine pancreatic trypsin (Sigma-Aldrich), as indicated in the figure legend for Fig. 4, in a final volume of 100 µl of buffer E at 37°C for 8 min. The tubes were then placed on ice, and the reactions were arrested by the addition of an excess of soybean trypsin inhibitor (Sigma-Aldrich). Aliquots of the reactions were subjected to SDS-PAGE and immunoblotting. The bands were quantitated using an Eastman Kodak Co. densitometer and plotted using KaleidaGraph.
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Footnotes |
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Acknowledgments |
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This work was supported by National Institutes of Health grant RO1 CA-08759 to S. Kornfeld.
Submitted: 19 November 2002
Revised: 9 January 2003
Accepted: 10 January 2003
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References |
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