AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation

Abstract

The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its beta1 and mu1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its beta1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated beta1 compared with phosphorylated mu1. Once on the membrane, the mu1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of mu1 (and mu2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70-mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.

Figure 1.

In vivo phosphorylation of AP-1 subunits. Confluent P100 dishes of mouse L cells (LSS) were labeled with 1 mCi/ml of [³²P]orthophosphoric acid for 4 h in the presence or absence of 10 nM okadaic acid in the labeling medium. Membrane–cytosol fractionation was performed after harvesting the cells, and each fraction was immunoprecipitated with 2 μg of anti–AP-1 γ monoclonal antibody. The immunoprecipitated proteins were eluted in sample buffer and subjected to SDS-PAGE for subsequent autoradiography (A) and Western blotting (B). The band corresponding to the AP-1 γ subunit in the Western blots was quantitated using a densitometer and expressed as a ratio with the first lane as the point of reference. M, membrane; C, cytosol.

Figure 2.

AP-1 from cytosolic versus CCV source display differential ligand binding. A schematic representation of the GST-fused MPR cytoplasmic tail constructs that were expressed and purified from E. coli is shown in the top panel. The CK2 sites are represented symbolically. (A and B) 100 μg of GST-fused CD-MPR or CI-MPR cytoplasmic tail constructs was incubated at 37°C for 3 h in the presence or absence of 500 U of recombinant human CK2 (Calbiochem) in buffer A supplemented with 1.5 mM ATP and 2 μM polylysine. Efficacy of phosphorylation achieved by this method was 75–80%. The CK2-treated ligands were then incubated with GSH-beads at room temperature for 2 h in 350 μl of buffer B. The beads were sedimented and washed with 1 ml of buffer B to remove unbound ligand and CK2. The glutathione-Sepharose beads with the various bound ligands were used in pull-down assays to assess their binding avidity toward AP-1 immunopurified from cytosol or CCVs. 10% of the input and 25% of the pellets (except as noted) were loaded onto a 10% SDS-PAGE gel and transferred onto nitrocellulose for immunoblotting with anti–AP-1γ (100/3) monoclonal antibody. The figures represent one of two to four independent experiments. fl, full length.

Figure 3.

Dephosphorylation of the μ1 subunit of AP-1 decreases ligand binding avidity. (A, top) 25 μg of purified CCVs was incubated at 30°C for 4 h in the presence of ATP (γ³²P) to phosphorylate μ1 (and μ2) in vitro using endogenous GAK as the kinase, as described in the Materials and methods. The CCVs were then incubated with purified bovine kidney–derived PP2A at the indicated amounts for an additional 15 min. The complete reaction was subjected to SDS-PAGE on a 12% gel, dried, and filmed. Another identical set was subjected to Western blotting with an antibody toward μ1. (A, bottom) An autorad of a similar in vitro dephosphorylation reaction that was performed in the presence or absence of 10 mM okadaic acid. (B) 50-μg aliquots of immunopurified AP-1 derived from bovine liver CCVs were incubated with or without 50 ng of PP2A in buffer A at 37°C for 1 h. Aliquots were then analyzed for binding to phosphorylated GST-fused MPR ligands as in Fig. 2. 10% of the input and 25% of the pellets were subjected to SDS-PAGE on a 10% gel and immunoblotted with anti–AP-1γ (100/3) monoclonal antibody.

Figure 4.

Phosphorylated AP-1 μ1 has increased sensitivity to trypsin. 2.5 μg of immunopurified AP-1 from bovine liver cytosol (A) or CCVs (B) in 100 μl buffer E was incubated with varying amounts of trypsin as indicated. The reaction was performed at 37°C for 8 min and immediately terminated by the addition of an excess of soybean trypsin inhibitor at 4°C. 25% of the reaction was subjected to SDS-PAGE and Western blotting for the γ, β1, and μ1 subunits of AP-1 with their respective antibodies, as mentioned under the Materials and methods. The bands were quantitated using a densitometer. The amount of uncleaved (trypsin resistant) subunits expressed as a percentage of the starting amount was plotted against trypsin concentration using KaleidaGraph. The inset shows the blots of the three subunits, γ, β1, and μ1.

Figure 5.

PP2A mediates adaptor release in the presence of Hsc-70 and an ATP-regenerating system. (A and B) 5 μg of purified bovine liver CCVs was incubated with varying combinations of bovine brain cytosol, Hsc-70, PP2A, and ATP-regenerating system at 25°C for 3 min (A and B) or 5 min (A) in a final volume of 50 μl. At the indicated time, the reaction was centrifuged at 100,000 g in a Beckman Coulter ultracentrifuge. 40 μl of the supernatant was collected and subjected to SDS-PAGE, followed by immunoblotting for released AP-1 (A) and AP-2 (B). (C) 5 μg of CCVs and 0.5 μg Hsc-70 were incubated with varying concentrations of PP2A in the presence of the ATP-regenerating system for 5 min and worked up as above. The bands corresponding to the released AP-1 were quantitated using a densitometer and expressed as a percentage of the input. The figures represent 1 of 5–10 independent experiments.

Figure 6.

PP2A is the cytosolic cofactor that mediates AP-1 uncoating. (A) Coat release assays were performed as described in the Materials and methods and in Fig. 5 in the presence or absence of 5 mM okadaic acid. The duration of incubation was 4 min, and the released coat components were subjected to electrophoresis followed by immunoblotting as before. (B) PP2A was immunodepleted from bovine brain cytosol by repeated passage through an anti-PP2A affinity column as described in the Materials and methods. The depletion was confirmed by immunoblotting the runthrough fraction after each passage through the affinity column. (C) Whole cytosol and depleted cytosol were used in the coat release assay as described before. 10 ng of bovine kidney PP2A (Calbiochem) was added back to the depleted cytosol in the “addback” lane.

Figure 7.

PP2A interacts with Hsc-70 via its regulatory A subunit. (A) 100 μg of GST or GST fusion peptide corresponding to the PP2A A subunit protein was bound to glutathione-Sepharose beads and used to pull down Hsc-70 from 2 mg of bovine liver cytosol. 20% of the pellet was subjected to SDS-PAGE and subsequent immunoblotting with anti–Hsc-70 mAb. The “smile” in the bound Hsc-70 is caused by the GST–PP2A-A fusion protein, which runs at a similar molecular weight as Hsc-70, i.e., ∼70 kD. (B) Aliquots (2.5 μg) of recombinant bovine Hsc-70 were incubated at 25°C for 15 min in the presence or absence of 800 μM ATP and 5 mM magnesium acetate in 25 μl of buffer A. The reactions were then used for binding to 100 μg of GST or GST–PP2A-A fusion proteins prebound to glutathione-Sepharose beads at 4°C for 4 h. 20% of the pellet was subjected to SDS-PAGE and subsequent immunoblotting with anti–Hsc-70 mAb. (C) Bovine liver cytosol (2.5 mg) was subjected to immunoprecipitation with either rabbit preimmune sera or rabbit polyclonal antibody toward PP2A-Aα/β. The immune complexes were captured onto BSA-blocked protein A–agarose beads (Sigma-Aldrich). After four washes with PBS, the bound proteins were eluted by boiling in SDS sample buffer and subjected to SDS-PAGE. Immunoblotting was performed using mAb against Hsc-70.

Figure 8.

AP-1 released from CCVs by PP2A and Hsc-70 displays low-avidity ligand binding. AP-1 was extracted from CCVs either with 1.0 M Tris at 4°C or by incubation at 25°C in the presence of PP2A, Hsc-70, and ATP. The supernatants from these reactions were collected as sources of AP-1. GST-fused CK-phosphorylated MPR tails bound to glutathione-Sepharose beads were used as ligands for binding to AP-1 from the two sources. The beads were washed, eluted by boiling in SDS sample buffer, and subjected to SDS-PAGE and immunoblotting with mAb100/3 to detect bound AP-1.

Figure 9.

Golgi-associated PP2A has a preference for phosphorylated β1 versus μ1. (A and B) Confluent P60 dishes of LSS cells were labeled with 1 mCi/ml of [³²P]orthophosphoric acid for 4 h in the presence of 10 nM okadaic acid in the labeling media. The cells were harvested and whole cell lysates were subjected to immunoprecipitation with 2 μg of anti–AP-1 γ monoclonal antibody. The immune complexes were captured on BSA-blocked protein G–agarose beads, washed, and thereafter treated with or without 10 ng of PP2A (A) or 50 μg of rat liver Golgi-enriched membranes in buffer B (B) for the times indicated in the figures. The complete reaction was subjected to SDS-PAGE followed by autoradiography.

Figure 10.

Working model for phosphoregulation of AP-1. (1) Cytosolic AP-1 has a phosphorylated β1 hinge that prevents its association with clathrin and a dephosphorylated μ1 subunit, which presumably is in its inactive/ “closed” conformation. AP-1 is recruited onto the Golgi membranes via ARF-GTP (gray circle) where the β1 hinge gets dephosphorylated by a Golgi-associated isoform of PP2A. (2) Dephosphorylation of the β1 hinge allows AP-1 to initiate clathrin assembly. Concurrently, μ1 is phosphorylated, most likely by GAK (auxilin 2) (Umeda et al., 2000). This results in a conformational change that exposes ligand binding sites on μ1, thereby enhancing interactions with cargo sorting signals at the TGN. (3) During the maturation of the clathrin-coated bud, the β1 is maintained in a dephosphorylated state and μ1 in a phosphorylated state, presumably by GAK, which is incorporated into the forming CCV as an active enzyme (Korolchuk and Banting, 2002). (4) After budding, clathrin dissociation is mediated by GAK and Hsc-70. β1 phosphorylation could occur at this point, contributing to clathrin dissociation. The AP-1 remains on the vesicle membrane, possibly performing postbudding roles. (5) At some point before vesicle fusion with the acceptor compartment, AP-1 is released from the vesicle membrane. Hsc-70 may recruit PP2A from the cytosol and, in concert, mediate AP-1 release. This is achieved by dephosphorylation of μ1, which results in decreased avidity for cargo sorting signals due to reversion to its inactive conformation. (6) Released AP-1 returns to the cytosolic pool with phosphorylated β1 subunit and dephosphorylated μ1 subunit, both in their functionally inactive states.