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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GGAs (Golgi-localizing,
The family of multidomain proteins named
Golgi-localized, 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 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
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 [ 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
Plasmids encoding GST-CI-MPR (18AA), GST-GGA1 hinge peptide (residues
342-367), and GST-AP-1 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 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
[ 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).
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.
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.
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).
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).
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
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-1
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).
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 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 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 -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
-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
-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)
-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.
-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).
-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
-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.
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.
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.
80 °C for further use. The antibody
affinity column was regenerated by washing with glycine, pH
2.5.
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[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.
View larger version (34K):
[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
[ -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.
View larger version (25K):
[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.
View larger version (40K):
[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.
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.
Relationship of Stokes radius of GGA1 mutants with their ability to
bind MPR
ear (residues 703-822). As shown in
Fig. 5A, PP2A treatment of
GGA1 resulted in a marked increase in binding to the
-appendage of
AP-1 (Fig. 5A).
View larger version (27K):
[in a new window]
Fig. 5.
Dephosphorylated GGA1 binds
-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-1
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-1
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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
appendage of AP-1
appears to become less accessible because binding to the
appendage
is greatly impaired.
-appendage (panel E). This
association could possibly be stabilized by common binding partners,
including
-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 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 -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
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.
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, -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
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 |
3. |
Black, M. W.,
and Pelham, H. R.
(2000)
J. Cell Biol.
151,
587-600 |
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 |
6. |
Boman, A. L.,
Zhang, C. J.,
Zhu, X.,
and Kahn, R. A.
(2000)
Mol. Biol. Cell
11,
1241-1255 |
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 |
9. |
Zhu, Y.,
Doray, B.,
Poussu, A.,
Lehto, V. P.,
and Kornfeld, S.
(2001)
Science
292,
1716-1718 |
10. |
Takatsu, H.,
Katoh, Y.,
Shiba, Y.,
and Nakayama, K.
(2001)
J. Biol. Chem.
276,
28541-28545 |
11. |
Doray, B.,
Bruns, K.,
Ghosh, P.,
and Kornfeld, S.
(2002)
Proc. Natl. Acad. Sci.
99,
8072-8077 |
12. |
Doray, B.,
Ghosh, P.,
Griffith, J.,
Geuze, H.,
and Kornfeld, S.
(2002)
Science
297,
1700-1703 |
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 |
15. |
Bertrand, F.,
Turowski, P.,
and Hemmings, B. A.
(1997)
J. Biol. Chem.
272,
13856-13863 |
16. |
Mumby, M. C.,
and Walter, G.
(1993)
Physiol. Rev.
73,
673-699 |
17. |
Varlamov, O.,
Kalinina, E.,
Che, F. Y.,
and Fricker, L. D.
(2001)
J. Cell Sci.
114,
311-322 |
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 |
23. |
Meresse, S.,
Ludwig, T.,
Frank, R.,
and Hoflack, B.
(1990)
J. Biol. Chem.
265,
18833-18842 |
24. |
Le Borgne, R.,
Schmidt, A.,
Mauxion, F.,
Griffiths, G.,
and Hoflack, B.
(1993)
J. Biol. Chem.
268,
22552-22556 |
25. |
Mauxion, F.,
Leborgne, R.,
Munierlehmann, H.,
and Hoflack, B.
(1996)
J. Biol. Chem.
271,
2171-2178 |
26. |
Dittie, A. S.,
Thomas, L.,
Thomas, G.,
and Tooze, S. A.
(1997)
EMBO J.
16,
4859-4870 |
27. |
Ghosh, P.,
and Kornfeld, S.
(2003)
J. Cell Biol.
160,
699-708 |
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 |