Phosphorylation-induced Conformational Changes Regulate GGAs 1 and 3 Function at the Trans-Golgi Network*

Pradipta Ghosh and Stuart KornfeldDagger

From the Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, December 9, 2002, and in revised form, February 6, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The GGAs (Golgi-localizing, gamma -adaptin ear homology domain, ARF-binding) are a family of multidomain proteins implicated in protein trafficking between the Golgi and the endosomes. All three GGAs (1, 2, and 3) bind to the mannose 6-phosphate receptor tail via their VHS domains, as well as to the adaptor protein complex-1 via their hinge domains. The latter interaction has been proposed to be important for cooperative packaging of cargo into forming clathrin-coated carriers at the trans-Golgi network. Here we present evidence that GGA1 function is highly regulated by cycles of phosphorylation and dephosphorylation. Cell fractionation showed that the phosphorylated pool of GGA1 resided predominantly in the cytosol and that recruitment onto membranes was associated with dephosphorylation. Okadaic acid inhibition studies and in vitro dephosphorylation assays indicated that dephosphorylation is mediated by a protein phosphatase 2A-like phosphatase. Dephosphorylation of GGA1 induced a change in the conformation to an "open" form as measured by gel filtration and sucrose gradient analyses. This was associated with enhanced binding to ligands because of release of autoinhibition and increased binding to the adaptor protein complex-1 gamma -appendage. A model is proposed for the regulation of GGA1 function at the trans-Golgi network.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The family of multidomain proteins named Golgi-localized, gamma -ear-containing, ARF-binding molecules (or GGAs)1 has been shown to facilitate protein trafficking from the trans-Golgi network (TGN) to lysosomes in mammalian cells and to the vacuole in yeast (1-4). The three members, named GGA 1, 2, and 3 have an NH2-terminal VHS (Vps, Hrs, and STAM) domain, followed by a coiled coil GAT (GGA and Tom) domain, a variable hinge region, and a COOH-terminal appendage that is homologous to the adaptor protein-1 (AP-1) gamma -appendage (1, 2, 5-7). All three GGAs bind to an acidic cluster dileucine (AC-LL) sorting motif on the cytoplasmic tails of the two mannose 6-phosphate receptors (MPRs) via their VHS domains (8-10). This interaction appears to be essential for MPR sorting at the TGN (8, 9). However, the GGAs differ from each other in certain aspects. GGAs 1 and 3, but not GGA 2, are subject to autoinhibition mediated by binding of internal AC-LL motifs to the ligand binding site on the VHS domain (11). This autoinhibition requires phosphorylation of a casein kinase 2 (CK2) site just upstream of the internal AC-LL (11). The mechanism whereby the autoinhibition is relieved to enable GGAs 1 and 3 to function in cargo binding has not been examined.

Recent evidence indicates that the GGAs cooperate with AP-1 in the sorting and packaging of the MPR cargo molecules into clathrin-coated carriers. Immunoelectron microscopic studies showed that the GGAs and AP-1 co-localize within clathrin-coated buds and vesicles at the TGN (12, 13) and live cell fluorescent imaging has revealed that the GGAs and AP-1 exit the TGN in the same clathrin-coated tubules (13). In addition to these morphologic studies, in vitro binding assays have provided evidence for a direct interaction between the hinge domains of GGAs and the gamma -appendage of AP-1 (12). Expression of a truncated form of GGA, lacking the hinge domain that mediates AP-1 interaction, caused accumulation of CD-MPR at the TGN (8). Taken together, these observations indicate that the GGAs mediate sorting of MPRs at the TGN and facilitate their entry into forming AP-1-containing clathrin-coated vesicles (CCVs). In support of this, a mutant MPR that failed to bind GGAs, but did bind AP-1, was poorly incorporated into AP-1-positive clathrin-coated carriers forming at the TGN (12).

AP-1 was also shown to possess an associated CK2, which was capable of phosphorylating GGAs 1 and 3 (12). The resulting phosphorylation-induced autoinhibition of the ligand binding site on the VHS domain was postulated to cause a directed transfer of the MPR cargo from GGAs 1 and 3 to AP-1. To explain the lack of detectable GGAs in isolated CCVs (2), it was proposed that the phosphorylated GGAs 1 and 3 return to the cytosol after transferring their cargo to AP-1 (12).

In the present study we have examined the regulation of GGAs 1 and 3 phosphorylation. Using in vivo metabolic labeling, we established that membrane-associated GGA1 is predominantly dephosphorylated, whereas the cytosolic pool is phosphorylated. Dephosphorylation was associated with a marked change in conformation that favored increased ligand binding as well as binding to the gamma -appendage of AP-1. A model is proposed whereby GGAs 1 and 3 function at the TGN is regulated by cycles of phosphorylation and dephosphorylation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The anti-c-myc (clone 9E10) monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) whereas the anti-HA monoclonal was from Covance (Berkeley, CA). Affinity purified rabbit polyclonal anti-GGA1 antibody was a generous gift from Margaret Robinson, University of Cambridge, Cambridge, UK. All chemicals used were of reagent grade and purchased from Sigma. Superose-6 resin, the gel filtration column, and FPLC apparatus were from Amersham Biosciences. Gel filtration standards were obtained from Bio-Rad. Bovine kidney heterotrimeric PP2A1 and recombinant human casein kinase 2 were from Calbiochem, La Jolla, CA. [32P]Orthophosphoric acid for in vivo labeling was from Amersham and [gamma -32P]ATP was obtained from ICN radiochemicals. The c-myc epitope peptide (clone 9E10) was prepared by the protein and nucleic acid chemistry laboratory (PNACL) at the Washington University School of Medicine.

Buffers-- The following buffers were used: A, 25 mM Hepes-KOH, pH 7.2, 125 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM DTT, and 0.4% Triton X-100; B, 25 mM HEPES-KOH, pH 7.4, 250 mM sucrose, 1 mM EDTA; C, 25 mM Hepes-KOH, pH 7.2, 125 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM DTT, and 0.1% Triton X-100; D, 100 mM MES, pH 6.5, 0.25% sucrose, 1 mM EGTA, 10 mM okadaic acid and protease inhibitors; E, 0.1 M Tris-Cl, pH 8.0, 0.1 M NaCl, 0.5% Na deoxycholate, 0.2% SDS, 1% Triton X-100, 1 mM okadaic acid; F, 25 mM Hepes-OH, pH 7.2, 125 mM KCl, 5 mM MgCl2, 20 mM MnCl2, 2 µM polylysine; G, 25 mM Tris-Cl, pH 7.0, 0.2 mM MnCl2, 1 mM DTT.

Plasmids and Protein Expression-- Myc-tagged GGA1 wild type and mutant plasmids were obtained as described earlier (11, 14). Bacmid DNAs were transfected into Sf9 insect cells to produce recombinant baculoviruses that were amplified and used to express the various GGAs in the insect cells as before (11). Insect cells expressing the GGA proteins were routinely harvested 48 h post-infection, lysed into cold buffer A containing protease inhibitor mixture (Roche Molecular Biochemicals) by sonication, and centrifuged at 20,000 × g for 10 min. The supernatant containing the GGA protein was stored at -80 °C before use in various experiments. Bovine adrenal cytosol was prepared from fresh adrenal glands obtained from a local slaughterhouse. The glands were thawed on ice and transferred to chilled homogenization buffer B (supplemented with 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 0.1 unit of trypsin inhibitory unit/ml aprotinin). Homogenization was performed with the use of a Potter-Elvehjem homogenizer with a 2:1 (w/w) ratio of buffer to tissue. All subsequent steps were carried out at 4 °C. The crude homogenate was centrifuged sequentially at 3000 × g for 10 min, 10,000 × g for 20 min, and 100,000 × g for 60 min. The final supernatant, i.e. the cytosolic fraction, served as the source of endogenous GGA1 in our experiments.

Plasmids encoding GST-CI-MPR (18AA), GST-GGA1 hinge peptide (residues 342-367), and GST-AP-1gamma ear (residues 703-822) fusion proteins were used to express these proteins in Escherichia coli strain BL21(RIL) (Stratagene) (11). Pelleted bacteria from 1 liter of culture were lysed into 20 ml of cell lytic B reagent (Sigma), sonicated, and centrifuged at 27,000 × g at 4 °C for 15 min to remove insoluble material. The clarified lysate was then mixed by tumbling at 4 °C for 4 h with glutathione-Sepharose 4B (Amersham Biosciences) pre-equilibrated with 20 mM Tris-Cl, pH 7.5, containing 0.1% Triton X-100. Following 4 washes with the same buffer and a single wash with detergent-free 50 mM Tris-Cl, pH 8.0, the GST fusion proteins were competitively eluted with 10 mM reduced glutathione in 50 mM Tris-Cl, pH 8.0, and dialyzed against phosphate-buffered saline before use.

Immunopurification of GGA1-- His-myc-GGA1 (12) was expressed in Sf9 cells and purified on a nickel-nitrilotriacetic acid column (Qiagen) as instructed by the manufacturer. GGA1 was also purified from Sf9 cells expressing myc-tagged GGA1 using affinity chromatography followed by peptide elution. For this purpose, an anti-c-myc affinity column was prepared by coupling 200 µg of anti-c-myc (clone 9E10) monoclonal antibody to 1 g of activated CNBr-Sepharose beads (Sigma). The beads were washed with 100 ml of 100 mM sodium bicarbonate, pH 8.3, 0.5 M sodium chloride, and 100 mM sodium acetate, pH 4.0, 0.5 M sodium chloride alternating for four cycles and equilibrated with TBS buffer (100 mM Tris-HCl, pH 7.5, 150 mM sodium chloride) before incubating with a Sf9 cell lysate containing myc-tagged GGA1 overnight at 4 °C with constant tumbling. The following morning the beads were pelletted and washed repeatedly in buffer C (with 0.5% Triton X-100) until there was no detectable protein being released. Thereafter, the beads were incubated for 15 min at room temperature with 5 mg/ml c-myc epitope peptide in buffer C without DTT. Elutions were repeated three times and the fractions pooled, concentrated using a Centricon-10 apparatus, and frozen in aliquots at -80 °C for further use. The antibody affinity column was regenerated by washing with glycine, pH 2.5.

In Vivo Phosphorylation of GGA1 and Membrane-Cytosol Fractionation-- COS-7 cells were plated in six-well plates the night before transfection in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in the presence of penicillin (100 units/ml) and streptomycin (100 µg/ml). The following morning the cells were transfected with 4 µg of plasmid DNA encoding myc-tagged wild type GGA1 using LipofectAMINE Plus (Invitrogen) as per the manufacturer's protocol. 48 h post-transfection the cells were labeled with 1 mCi/ml [32P]orthophosphoric acid in phosphate-free Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal bovine serum, 20 mM Hepes-OH, pH 7.4, with or without 10 nM okadaic acid for 4 h at 37 °C and 5% CO2. At the end of labeling, the medium was removed and the cells were washed with 1 ml of cold phosphate-buffered saline. Cell fractionation studies were done as described earlier (5) with a few modifications. In brief, the 32P-labeled cells were scraped in 0.5 ml of buffer D and the cell suspension was sonicated for 10 s × 3 pulses of intensity 3.5 using a Fisher sonic dismembrator. This was followed by centrifugation at 650 × g for 15 min at 4 °C to remove intact cells and nuclei. Postnuclear supernatant was collected and subjected to centrifugation at 100,000 × g in a Beckman TLA 100.3 rotor for 1 h. The supernatant was collected (cytosol) and the pellet was resuspended in buffer A (+10 mM okadaic acid), sonicated, and centrifuged at 14,000 × g for an additional 15 min to remove insoluble material. The two supernatant fractions were incubated with 2 µg of anti-c-myc monoclonal antibody at 4 °C with constant tumbling. The following morning, bovine serum albumin-blocked protein G-agarose beads were added to capture the immune complexes and were incubated for an additional 1 h at 4 °C. The beads were collected by centrifugation at 750 × g for 1 min, and washed repeatedly with buffer E until no further radioactivity was released. The washed pellets were boiled in sample buffer and subjected to SDS-PAGE on a 10% gel. The gel was dried and filmed using Kodak X-Omat MR. Duplicate sets were subjected to Western blotting to confirm the specificity of the immunoprecipitation.

In Vitro Phosphorylation-Dephosphorylation Assays-- This was done as a two-step experiment. In the first step, 100 µg each of GST-CI-MPR tail (18AA) and GST-GGA1 hinge peptide (residues 342-367) were incubated with 250 units of rh-CK2 (Calbiochem) in the presence of [gamma -32P]ATP (1.5 mM, specific activity 12 mCi/mmol) in buffer F in a final volume of 100 µl at 37 °C for 1 h. The phosphopeptides were then dialyzed against 2 liters of dephosphorylation buffer, buffer G with one change overnight at 4 °C to remove the ATP. In the second step, 5 µg of each phosphopeptide were incubated with varying amounts of bovine kidney-derived PP2A1 (Calbiochem) in buffer F at 37 °C for 3 h. The reactions were terminated by boiling in SDS sample buffer and subjected to SDS-PAGE. The gel was dried and filmed using Kodak X-Omat MR.

Gel Filtration-- A Superose 6 column (1.8 × 55 cm) was connected to a FPLC machine (Amersham Biosciences). The column was washed overnight before each use with buffer C and Bio-Rad gel filtration molecular weight standards were used to calibrate the column before injecting samples. 500 µl of the sample protein (either Sf9 lysates of myc-GGA1 wild type/mutants/GGA2-HA/immunopurified myc-GGA1 or bovine adrenal cytosol as a source of endogenous GGA1) were subjected to gel filtration in buffer C at room temperature with a flow rate of 0.5 ml/min, and 1-ml fractions were collected in a fraction collector. The collected fractions were immediately set on ice. 40 µl of each fraction were boiled in SDS sample buffer and subjected to SDS-PAGE followed by Western blotting with anti-c-myc antibody to detect myc-tagged GGA1 or rabbit polyclonal anti-GGA1 antibody for endogenous GGA1.

Sucrose Gradient-- 4-20% linear sucrose gradients were poured using a gradient maker (Amersham Biosciences) into 11.5-ml Beckman SW 55Ti tubes. The freshly prepared gradients were stored at 4 °C for 30 min to 1 h before use. Protein samples and standards were gently overlaid on the surface without disturbing the gradient. The tubes were centrifuged at 42,000 rpm (300,000 × g) for 17 h at 4 °C without deceleration. Thereafter, 20 fractions (each 560 µl) were collected starting from the top of each tube. Aliquots were subjected to SDS-PAGE. The standard markers were detected by Coomassie Brilliant Blue staining and the various GGAs were detected by Western blotting.

Binding Assays-- The binding of the GST fusion proteins with the GGAs was assayed in buffer C (supplemented with 1 mM DTT) in a final volume of 300 µl. GST fusion proteins (50 µg) were immobilized at room temperature on 30 µl of packed glutathione-Sepharose beads (Amersham Biosciences). The beads with bound proteins were pelleted by centrifugation at 750 × g for 1 min, washed once with cold buffer B, and resuspended in 300 µl of buffer B containing either immunopurified GGA1 at a final concentration of 25 µg/ml or 2 mg of bovine adrenal cytosol as a source of endogenous GGA1. The binding reactions were allowed to proceed for 4 h at 4 °C with tumbling followed by centrifugation at 750 × g for 1 min. An aliquot of the supernatant was saved, and the pellets were washed four times by resuspension in 1 ml of cold buffer B followed by centrifugation at 750 × g. The washed pellets were resuspended in SDS sample buffer and heated at 100 °C for 5 min.

Electrophoresis and Immunoblotting-- Proteins were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose. Blots were blocked with TBST (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat milk for 1 h at room temperature. The blots were then probed with appropriate primary antibody followed by horseradish peroxidase-conjugated anti-mouse IgG. The immunoreactive bands were visualized on x-ray films using enhanced chemiluminescence (ECL) (Amersham Biosciences).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GGA1 Is Phosphorylated in Cytosol and Dephosphorylated upon Membrane Recruitment-- Because GGAs 1 and 3 were found to be phosphoproteins (11), we asked whether the phosphorylation was associated with the membrane or cytosolic phase of GGA cycling. To address this question, COS-7 cells were transfected with myc-tagged GGA1 and 48 h post-transfection the cultures were labeled with 32P for 4 h in the presence or absence of okadaic acid. Membrane-cytosol fractionation was then carried out, and the GGA1 in each fraction was immunoprecipitated and analyzed by SDS-PAGE. As shown in Fig. 1, when labeled in the absence of okadaic acid, the GGA1 associated with membranes was dephosphorylated whereas the cytosolic GGA1 was phosphorylated. Addition of 10 nM okadaic acid resulted in the recovery of phosphorylated GGA1 from both the membrane and cytosolic fractions. This concentration of okadaic acid is thought to be relatively selective for inhibition of PP2A-like phosphatases (15, 16). These findings indicate that GGA1 exists in a phosphorylated form in the cytosol and is dephosphorylated upon recruitment onto the membrane. This dephosphorylation is likely to be mediated by a member of the PP2A family of phosphatases based on the okadaic acid effect.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   In vivo phosphorylation of GGA1. COS7 cells were transfected in six-well plates with myc-GGA1 plasmid DNA and 48 h post-transfection the cells were labeled with 1 mCi/ml [32P]orthophosphoric acid (Amersham Biosciences) 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 the myc-GGA1 in each fraction was immunoprecipitated with 2 µg of anti-c-myc monoclonal antibody. The immunoprecipitated proteins were subjected to SDS-PAGE and autoradiography. An aliquot (5%) of the immunoprecipitates was used for Western blotting with anti-c-myc mAb. M, membrane; C, cytosol.

PP2A Dephosphorylates GGA1 in Vitro-- We next examined whether GGA1 is a substrate of purified PP2A in in vitro assays. Aliquots of a Sf9 cell lysate expressing myc-GGA1 were treated with varying amounts of bovine kidney PP2A for 3 h at 30 °C followed by SDS-PAGE and Western blotting with anti-c-myc mAb. Fig. 2A shows that myc-GGA1 underwent a downward shift in mobility when incubated with increasing amounts of PP2A, consistent with dephosphorylation. This shift in mobility was inhibited by addition of okadaic acid as seen in the first lane of the gel.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 2.   PP2A dephosphorylates GGA1 in vitro. A, aliquots (100 µg of protein) of a Sf9 cell lysate expressing myc-GGA1 were treated with the indicated amounts of bovine kidney PP2A for 3 h at 30 °C in 100 µl of buffer G. 25 µl of the reaction was boiled in SDS sample buffer and subjected to SDS-PAGE on a 5% gel followed by transfer onto nitrocellulose membrane and Western blotting with anti-c-myc mAb. B and C, 100 µg of GST-GGA1 hinge peptide (residues 342-367) was phosphorylated in vitro using 250 units of rh-CK2 (Calbiochem) in the presence of 1.5 mM [gamma -32P]ATP at 37 °C for 1 h as described under "Experimental Procedures." The phosphopeptide was dialyzed overnight to remove ATP. Aliquots (5 µg) of the dialyzed phosphopeptide were incubated with PP2A at the indicated amounts in the presence (panel C) or absence (panels B and C) of 3 mM okadaic acid at 37 °C for 3 h in 50 µl of buffer G. The reactions were boiled in SDS sample buffer and subjected to SDS-PAGE on a 12% gel followed by autoradiography using Kodak X-Omat MR. The results are representative of two independent experiments.

PP2A also dephosphorylated a GST-fused hinge peptide of GGA1 (residues 342-367) that contains the critical Ser355 within a consensus CK2 site. This GST peptide was first phosphorylated with CK2 and then incubated with varying amounts of PP2A at 37 °C for 3 h in the presence or absence of okadaic acid. The entire reaction was subjected to SDS-PAGE on a 12% gel, dried, and filmed. Fig. 2, panel B, demonstrates that the GGA1 hinge peptide was readily dephosphorylated by nanogram amounts of PP2A and panel C shows that this dephosphorylation was inhibited by okadaic acid.

Dephosphorylation of GGA1 Relieves Autoinhibition and Restores Ligand Binding-- If the ligand binding property of GGA1 is autoinhibited because of phosphorylation, it should be reversed by dephosphorylation. This hypothesis was tested in binding assays examining the effect of PP2A on myc-GGA1 binding to a GST-fused CI-MPR (18 amino acids) tail peptide encoding the AC-LL motif and neighboring residues. As shown in Fig. 3A, GGA1 binding to the GST-CI-MPR ligand was greatly enhanced by PP2A treatment, establishing that dephosphorylation relieves autoinhibition and restores ligand binding. The specificity of the binding was shown by the fact that PP2A-treated GGA1 bound poorly to the GST control (first lane). Similarly, PP2A treatment of endogenous GGA1 from bovine adrenal cytosol increased GGA1 binding to the GST-CI-MPR ligand (Fig. 3B).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3.   A and B, dephosphorylation of GGA1 enhances ligand binding. Aliquots (10 µg) of immunopurified myc-GGA1 (A) or bovine adrenal cytosol (2 mg) (B) were incubated with or without PP2A at the indicated amounts for 3 h at 30 °C in 50 µl of buffer G. The PP2A/mock treated samples were then incubated with 100 µg of GST-CI-MPR (18 amino acids) pre-bound to glutathione-Sepharose beads for 4 h at 4 °C. The beads were washed and the bound protein eluted by boiling in SDS sample buffer. 2% of the eluted protein was subjected to SDS-PAGE on a 10% gel followed by Western blotting with anti-c-myc antibody to detect bound myc-GGA1 or rabbit polyclonal anti-GGA1 antibody to detect endogenous GGA1. The results with myc-GGA1 are representative of three independent experiments.

Dephosphorylation of GGA1 Results in a Major Conformational Change-- We have previously shown that the phosphorylation-induced autoinhibition of GGAs 1 and 3 is mediated by the binding of an internal AC-LL sequence located in the hinge segment to the ligand binding site in the VHS domain (11). The transition from the inactive state to the active state would be expected to be associated with a conformation change in the molecule. Therefore we used gel filtration and sucrose gradient analyses to look for a conformational change between the two forms and to determine whether phosphorylation/dephosphorylation regulated this change. Fig. 4A shows that wild type His-myc-GGA1 from Sf9 cells eluted from the Superose 6 column at a position expected for a globular protein of ~75 kDa. The Stokes radius was estimated to be 40 Å. In contrast, GGA1 that was treated with PP2A eluted in earlier fractions that corresponded to a molecular size of ~300 kDa and a Stokes radius of 60 Å. Endogenous GGA1 present in bovine adrenal cytosol showed a similar shift in the elution profile upon treatment with PP2A (Fig. 4B).


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 4.   Dephosphorylation of GGA1 results in a major conformational change. A, aliquots (25 µg) of purified His-myc-GGA1 in 500 µl of buffer G were incubated in the presence or absence of 25 ng of PP2A for 1 h at 30 °C. The reactions were then applied to a 1.8 × 55-cm Superose 6 gel filtration column. Fractions of 1 ml were collected and an aliquot of each was subjected to SDS-PAGE on a 10% gel followed by Western blotting with anti-c-myc mAb. The elution positions of protein standards of known Stokes radii are indicated with arrows along the bottom of the panel: thyroglobulin (85 Å), ovalbumin (30.5 Å), and myoglobin (19.6 Å). B, aliquots (2 mg) of bovine adrenal cytosol in 500 µl of buffer G were incubated in the presence or absence of 50 ng of PP2A for 2 h at 30 °C prior to gel filtration. Equal aliquots of the collected fractions were subjected to SDS-PAGE and Western blotting with rabbit polyclonal anti-GGA1 antibody. C, 2 mg of Sf9 cell lysates expressing wild type myc-GGA1 or mutant myc-GGA1 D358A were subjected to gel filtration and Western blotting as above. D, 500 µg of Sf9 cell lysates expressing wild type myc-GGA1 or mutant myc-GGA1 D358A were analyzed on 4-20% sucrose gradients using a SW Ti55 rotor in a Beckman ultracentrifuge as described under "Experimental Procedures." Fractions of 560 µl were collected starting at the top of the 11.5-ml gradient. Aliquots from each fraction were subjected to SDS-PAGE followed by Western blotting with anti-c-myc mAb to detect GGA1. The gradient was calibrated with proteins of known sedimentation coefficient, whose positions are indicated at the bottom of the panel. Bovine serum albumin, 4.6 S; ovalbumin, 3.5 S; where S = sedimentation coefficient expressed in Svedberg units. Each experiment was repeated two to three times except for the experiment with the endogenous GGA1, which was done once.

We next examined a mutant GGA1 with phosphorylation at the Ser355 CK2 site disrupted by substituting the critical aspartate at position 358 with an alanine (D358A). This construct exhibits high ligand binding affinity (11), consistent with the lack of phosphorylation at Ser355. When subjected to gel filtration, the mutant GGA1 D358A eluted at the same position as the dephosphorylated GGA1 (Fig. 4C). We also analyzed seven additional GGA1 mutants with the following alterations in the internal AC-LL (355SLLDDELM362) sequence: S355D, L356A/L357A/D358A/D359A/E360A, LL-AA, DDE-AAA, LM-AA, and the GGA1 Swi (358DDELM362 right-arrow HQDLA) (11). As summarized in Table I, all the GGA1 mutants that bound to the CI-MPR ligand had a Stokes radius of 60 Å whereas the mutants that failed to bind the CI-MPR ligand had a Stokes radius of 40 Å. In addition, a mutant with the two other CK2 sites mutated to alanines (S185A, A244A) eluted from the gel filtration column at the same position as wild type GGA1. This indicates that these two sites do not have a major effect on the conformation of the molecule.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Relationship of Stokes radius of GGA1 mutants with their ability to bind MPR
GGA1 hinge internal AC-LL sequence 355SLLDDELM362.

To ascertain whether the larger size observed on gel filtration was because of oligomerization or the result of an intramolecular change in conformation, sucrose gradient analysis was done using a 4-20% gradient (1). If the difference in elution position was because of oligomerization, the species with the 60 Å Stokes radii would be expected to have a higher sedimentation coefficient. However, both forms of GGA1 behaved similarly on sucrose gradients having a sedimentation coefficient of ~3.2 S (Svedberg units) (Fig. 4D) as observed previously (1). These data indicate that the increased Stokes radius is because of an intramolecular conformation change.

Dephosphorylation of GGA1 Favors Interaction with AP-1-- Because GGAs and AP-1 do not associate with each other in cytosol (2), but do interact on the membrane (12), we tested whether the dephosphorylation event on the membrane favored this interaction. Purified GGA1 was incubated with various amounts of PP2A and then assayed for binding to GST-AP-1gamma ear (residues 703-822). As shown in Fig. 5A, PP2A treatment of GGA1 resulted in a marked increase in binding to the gamma -appendage of AP-1 (Fig. 5A).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5.   Dephosphorylated GGA1 binds gamma -appendage of AP-1 with increased avidity. A, aliquots (10 µg) of purified His-myc-GGA1 were incubated with or without PP2A at the indicated amounts for 3 h at 30 °C in 50 µl of buffer G. The PP2A/mock treated samples were then incubated with 50 µg of GST-AP-1gamma ear (residues 703-822) pre-bound to glutathione-Sepharose beads for 4 h at 4 °C. The beads were washed and the bound protein was eluted by boiling in SDS sample buffer. 40% of the eluted protein was subjected to SDS-PAGE on a 10% gel and stained with Coomassie Brilliant Blue followed by destaining with 30% methanol, 10% acetic acid. The His-myc-GGA1 bands were quantitated using a Kodak densitometer, calculated as the percentage increase relative to the mock treated sample and plotted against the amount of PP2A using "KaleidaGraph." B, 500 µg of Sf9 cell lysates expressing wild type myc-GGA1 or two mutants (myc-GGA1 D358A and myc-GGA1`Swi'*) were incubated with 50 µg of GST-AP-1gamma ear fusion peptide pre-bound to glutathione-Sepharose beads for 4 h at 4 °C. 10% of the pellets and 1% of the supernatants were subjected to SDS-PAGE followed by Western blotting with anti-c-myc mAb to detect the bound GGA1 protein. All experiments were repeated three or more times. Asterisk (*), in the GGA1 `Swi' mutant residues 358DDELM362 of the GGA1 hinge are substituted by the corresponding hinge residues 370HQDLA374 of GGA2 (11). P, pellet; S, supernatant.

GGA1 is phosphorylated in at least two other sites in addition to serine 355.2 We therefore determined whether dephosphorylation of serine 355 alone could cause the same effect as dephosphorylation of the full-length GGA1 molecule. This was done by analyzing mutants that cannot be phosphorylated at serine 355 because of a disrupted CK2 consensus sequence at that site (11). Both mutants bound AP-1 with much higher avidity than the wild type GGA1, indicating that phosphorylation at serine 355 modulates the interaction with AP-1 (Fig. 5B). Similar results were obtained using endogenous GGA1 from bovine adrenal cytosol (data not shown).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The data presented in this study provide insight into the dynamic nature of the phosphorylation of GGAs 1 and 3 and how this serves to regulate the function of these proteins. We have previously reported that CK2-mediated phosphorylation of serine 355 in the hinge domain of GGA1 results in autoinhibition because of binding of an internal AC-LL motif in the hinge to the ligand binding site in the VHS domain (11). We also showed that GGA1 (as well as GGAs 2 and 3) interacts directly with AP-1 and that AP-1 contains an associated CK2 that can phosphorylate serine 355 to induce autoinhibition (12). The current findings extend these observations in several significant ways. First, we document that the cytosolic form of GGA1 is phosphorylated in both COS cells and Sf9 cells and that the molecule becomes dephosphorylated upon membrane recruitment. This dephosphorylation is prevented by low levels of okadaic acid that have been reported to selectively inhibit PP2A (15, 16). Furthermore, nanogram amounts of purified PP2A dephosphorylate GGA1. Together these findings implicate PP2A as the phosphatase responsible for the dephosphorylation of GGA1 that occurs upon membrane recruitment. A likely source of the PP2A are the two MPRs that have been reported (and confirmed by us) to have this phosphatase bound to their cytoplasmic tails (17). Second, we show that dephosphorylation of GGA1 causes three measurable effects: a change in conformation to a larger or "open" form, an increase in binding avidity toward AC-LL ligands, and an increase in binding to the gamma  appendage of AP-1. Whereas GGA1 has at least three sites of phosphorylation,2 we found that the phosphorylation status of serine 355 was the key determinant in all three effects.

The large conformational change in GGA1 that is induced by dephosphorylation of serine 355 is very striking. The increase in Stokes radius of ~20 Å may be contributed by the flexible hinge domain as it moves out of the VHS ligand binding groove that it had previously occupied in the phosphorylated/autoinhibited state. This would explain that mechanism of release of the autoinhibition. The unoccupied VHS ligand binding groove will now be available for interacting with the AC-LL on the MPR cytoplasmic tail (18, 19). It has been reported that a phosphoserine in the CI-MPR COOH-terminal sequence DDpSDEDLLHI enhances binding to the VHS domain by fitting into the binding groove for the AC-LL motif (20). Serine 355 is located two residues further upstream from the AC-LL motif in the GGA1 hinge. Therefore at this point it is not clear whether the phosphorylated serine 355 binds directly to the VHS domain or induces a conformational change that favors binding of the downstream AC-LL motif to the VHS binding groove. Either way, phosphorylation of serine 355 enhances binding of the hinge motif to the VHS domain, resulting in a "closed" or autoinhibited form of the molecule. When this occurs, the region of the hinge that interacts with the gamma  appendage of AP-1 appears to become less accessible because binding to the gamma  appendage is greatly impaired.

We have previously proposed a working model for how the GGAs may cooperate with AP-1 in the packaging of MPRs into clathrin-coated transport carriers at the TGN (11, 12). The current findings allow us to refine and extend the model as depicted in Fig. 6. Autoinhibited phosphorylated GGAs 1 and 3 are recruited from the cytosol onto the Golgi via ARF·GTP (21) where they encounter the MPRs on smooth membranes (panels A and B). PP2A bound to the cytoplasmic tails of the MPRs could dephosphorylate the GGAs, triggering a large intramolecular rearrangement whereby the flexible hinge domain moves out of the VHS ligand binding groove (panel C). This relieves the autoinhibition and allows the unoccupied VHS to interact with the AC-LL motifs of the MPRs (panel D). The GGA-MPR complex could then interact with AP-1 in forming clathrin-coated buds via binding of the GGA hinge to the AP-1 gamma -appendage (panel E). This association could possibly be stabilized by common binding partners, including gamma -synergin (7, 22) and clathrin (9, 21). The AP-1-associated CK2 could then act on GGAs 1 and 3 and the MPRs (12, 23). Phosphorylation of the GGA hinge will restore the ability of the internal AC-LL motif to bind to the VHS domain, thereby serving as a competitive inhibitor to release the bound MPR (panel F). The displaced MPR can then bind to AP-1. This binding will be enhanced by phosphorylation of CK2 sites present in the cytoplasmic tails of both MPRs (24-27) an event that is closely associated with their exit from the TGN (28). At some point during this process, the µ1 subunit of AP-1 is also phosphorylated (27). This causes a conformational change that enhances binding to the MPR cytoplasmic tails. The combination of increased ligand binding avidity by AP-1 with impaired ligand binding by GGAs 1 and 3 would favor the directed transfer of MPR cargo molecules from the GGAs to AP-1. As the clathrin-coated bud extends and incorporates more AP-1 molecules, the MPRs become trapped in the CCVs. The phosphorylated GGAs 1 and 3, on the other hand, would be expected to return to the cytosol (panel G). This could explain why the GGAs have been undetectable in isolated CCVs whereas AP-1 is highly concentrated in these structures (2).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.   A model for phosphoregulation of GGAs 1 and 3 at the TGN. A, cytosolic GGAs 1 and 3 exist in a phosphorylation-induced autoinhibited state (Fig. 1) (11). The autoinhibition is mediated by intramolecular binding of an AC-LL motif in the hinge to the ligand binding groove in the VHS domain (11). The MPRs with PP2A bound to their cytoplasmic tails (17) are present on the smooth membranes of the TGN. B, GGAs 1 and 3 are recruited from the cytosol onto the smooth membranes of the TGN via ARF·GTP (21) where they encounter the cytoplasmic tails of the MPR cargo molecules and PP2A. C, PP2A dephosphorylates GGAs 1 and 3 on the membrane and relieves autoinhibition because of a major conformational change (this study) that moves the hinge away from the ligand binding groove on the VHS. D, the open forms of GGAs 1 and 3 are capable of binding to AC-LL motifs on the cytosolic tails of the MPRs (this study). E, the GGAs encounter AP-1 in the coated membranes where the majority of AP-1 is localized at steady state (12). Within the forming clathrin-coated buds and vesicles at the TGN, GGAs interact with the gamma -appendage of AP-1 via their hinge domain (12). F, AP-1 associated CK2 phosphorylates GGAs 1 and 3 (12) and restores the ability of the AC-LL motif in the hinge to bind to the VHS binding site. This displaces the MPR cargo molecules that can now bind to the AP-1. The AP-1-associated kinase also phosphorylates the CK2 sites on the cytoplasmic tail of the MPR (23). At some point the µ1 subunit of AP-1 undergoes phosphorylation upon recruitment onto the membrane (27). This causes ligand binding sites on the µ1 subunit to become exposed because of a conformational change (27). Phosphorylation of the MPR tail and of the µ1 subunit of AP-1 generates high avidity binding of the receptor tails to AP-1 thereby ensuring directed transfer of MPR cargo molecules from GGAs 1 and 3 to membrane-associated AP-1. Phosphorylation of GGAs 1 and 3 decreases binding avidity for the AP-1 gamma  appendage (this study), resulting in dissociation of the GGA·AP-1 complex. G, as the clathrin-coated vesicle assembly proceeds, the MPR cargo molecules are trapped by AP-1 and incorporated into CCVs. The autoinhibited, phosphorylated GGAs 1 and 3 are released into the cytosol. Key to the symbols is shown at the bottom of the figure.

The rate of formation of the CCVs may be governed by the MPR concentration (cargo load) (29). Once the CCVs bud from the TGN, a cytosolic form of PP2A plays a role in the "uncoating" process by dephosphorylating the µ1 subunit of AP-1 (27). This allows the AP-1 to dissociate from the vesicle following clathrin release (27).

The phosphoregulation of these various protein-protein interactions would be expected to enhance the efficiency of sorting and packaging of cargo molecules into AP-1 transport carriers. It is curious that GGA2, in contrast to GGAs 1 and 3, is not phosphorylated and does not appear to be subject to autoinhibition (11). Perhaps its function is regulated by another form of post-translational modification. Alternatively, GGA2 may somehow act in conjunction with GGAs 1 and 3 on the membrane and not require its own regulation. We plan to explore this issue in further studies.

    ACKNOWLEDGEMENTS

We thank Dr. Rosalind Kornfeld and members of the Kornfeld laboratory for critical reading of the manuscript and helpful comments and H. Bai for the construct myc-GGA1 S185A/S244A.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA08759 (to S. K.).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.

Dagger To whom correspondence should be addressed: Washington University School of Medicine, Division of Hematology, 660 S. Euclid Ave., Campus Box 8125, St. Louis, MO 63110. Tel.: 314-362-8803; Fax: 314-362-8826; E-mail: skornfel@im.wustl.edu.

Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M212543200

2 P. Ghosh and S. Kornfeld, unpublished results.

    ABBREVIATIONS

The abbreviations used are: GGA, Golgi-localizing, gamma -adaptin ear homology domain, adenosine 5'-diphosphate-ribosylation factor-binding protein; AP-1, adaptor protein complex-1; TGN, trans-Golgi network; CCV, clathrin-coated vesicle; MPR, mannose 6-phosphate receptor; CI-MPR, cation-independent mannose 6-phosphate receptor; PP2A, protein phosphatase 2A; CK2, casein kinase 2; AC-LL, acidic cluster dileucines; ARF, adenosine 5'-diphosphate ribosylation factor; GST, glutathione S-transferase; mAb, monoclonal antibody; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Dell'Angelica, E. C., Puertollano, R., Mullins, C., Aguilar, R. C., Vargas, J. D., Hartnell, L. M., and Bonifacino, J. S. (2000) J. Cell Biol. 149, 81-94[Abstract/Free Full Text]
2. Hirst, J., Lui, W. W., Bright, N. A., Totty, N., Seaman, M. N., and Robinson, M. S. (2000) J. Cell Biol. 149, 67-80[Abstract/Free Full Text]
3. Black, M. W., and Pelham, H. R. (2000) J. Cell Biol. 151, 587-600[Abstract/Free Full Text]
4. Zhdankina, O., Strand, N. L., Redmond, J. M., and Boman, A. L. (2001) Yeast 18, 1-18[CrossRef][Medline] [Order article via Infotrieve]
5. Poussu, A., Lohi, O., and Lehto, V. P. (2000) J. Biol. Chem. 275, 7176-7183[Abstract/Free Full Text]
6. Boman, A. L., Zhang, C. J., Zhu, X., and Kahn, R. A. (2000) Mol. Biol. Cell 11, 1241-1255[Abstract/Free Full Text]
7. Takatsu, H., Yoshino, K., and Nakayama, K. (2000) Biochem. Biophys. Res. Commun. 271, 719-725[CrossRef][Medline] [Order article via Infotrieve]
8. Puertollano, R., Aguilar, R. C., Gorshkova, I., Crouch, R. J., and Bonifacino, J. S. (2001) Science 292, 1712-1716[Abstract/Free Full Text]
9. Zhu, Y., Doray, B., Poussu, A., Lehto, V. P., and Kornfeld, S. (2001) Science 292, 1716-1718[Abstract/Free Full Text]
10. Takatsu, H., Katoh, Y., Shiba, Y., and Nakayama, K. (2001) J. Biol. Chem. 276, 28541-28545[Abstract/Free Full Text]
11. Doray, B., Bruns, K., Ghosh, P., and Kornfeld, S. (2002) Proc. Natl. Acad. Sci. 99, 8072-8077[Abstract/Free Full Text]
12. Doray, B., Ghosh, P., Griffith, J., Geuze, H., and Kornfeld, S. (2002) Science 297, 1700-1703[Abstract/Free Full Text]
13. Puertollano, R., van der Wel, N. N., Green, L. E., Eisenberg, E., Peters, P. J., and Bonifacino, J. S. (2003) Mol. Biol. Cell, in press
14. Doray, B., Bruns, K., Ghosh, P., and Kornfeld, S. (2002) J. Biol. Chem. 277, 18477-18482[Abstract/Free Full Text]
15. Bertrand, F., Turowski, P., and Hemmings, B. A. (1997) J. Biol. Chem. 272, 13856-13863[Abstract/Free Full Text]
16. Mumby, M. C., and Walter, G. (1993) Physiol. Rev. 73, 673-699[Abstract/Free Full Text]
17. Varlamov, O., Kalinina, E., Che, F. Y., and Fricker, L. D. (2001) J. Cell Sci. 114, 311-322[Abstract/Free Full Text]
18. Misra, S., Puertollano, R., Kato, Y., Bonifacino, J. S., and Hurley, J. H. (2002) Nature 415, 933-937[CrossRef][Medline] [Order article via Infotrieve]
19. Shiba, T., Takatsu, H., Nogi, T., Matsugaki, N., Kawasaki, M., Igarashi, N., Suzuki, M., Kato, R., Earnest, T., Nakayama, K., and Wakatsuki, S. (2002) Nature 415, 937-941[CrossRef][Medline] [Order article via Infotrieve]
20. Kato, Y., Misra, S., Puertollano, R., Hurley, J. H., and Bonifacino, J. S. (2002) Nat. Struct. Biol. 9, 532-536[Medline] [Order article via Infotrieve]
21. Puertollano, R., Randazzo, P. A., Presley, J. F., Hartnell, L. M., and Bonifacino, J. S. (2001) Cell 105, 93-102[Medline] [Order article via Infotrieve]
22. Page, L. J., Sowerby, P. J., Lui, W. W., and Robinson, M. S. (1999) J. Cell Biol. 146, 993-1004[Abstract/Free Full Text]
23. Meresse, S., Ludwig, T., Frank, R., and Hoflack, B. (1990) J. Biol. Chem. 265, 18833-18842[Abstract/Free Full Text]
24. Le Borgne, R., Schmidt, A., Mauxion, F., Griffiths, G., and Hoflack, B. (1993) J. Biol. Chem. 268, 22552-22556[Abstract/Free Full Text]
25. Mauxion, F., Leborgne, R., Munierlehmann, H., and Hoflack, B. (1996) J. Biol. Chem. 271, 2171-2178[Abstract/Free Full Text].
26. Dittie, A. S., Thomas, L., Thomas, G., and Tooze, S. A. (1997) EMBO J. 16, 4859-4870[Abstract/Free Full Text]
27. Ghosh, P., and Kornfeld, S. (2003) J. Cell Biol. 160, 699-708[Abstract/Free Full Text]
28. Meresse, S., and Hoflack, B. (1993) J. Cell Biol. 120, 67-75[Abstract]
29. Le Borgne, R., and Hoflack, B. (1997) J. Cell Biol. 137, 335-345[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.