From the Institute for Cancer Research, The Norwegian
Radium Hospital, Montebello, 0310 Oslo, Norway, § Institut
Curie, Biologie du Cycle Cellulaire et de la Motilité, 75248 Paris Cedex 05, France, and the ¶ Institute of Medical
Biochemistry, University of Oslo, 0317 Oslo, Norway
Received for publication, September 30, 2002, and in revised form, November 4, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies of RII The mechanism by which different extracellular ligands that
mediate their signals through the same second messenger might give rise
to a specific intracellular response has been the subject of intensive
research for several years (1). In the case of cAMP signal
transduction, it has been demonstrated that the subcellular localization of protein kinase A
(PKA)1 is important for the
specificity (1).
PKA is composed of two catalytic (C) subunits and one regulatory dimer
(R2) that in the absence of cAMP form an inactive
heterotetramer (R2C2). Upon binding of cAMP to
the R subunits, the enzyme dissociates and releases two free, active C
subunits (2). The R2 dimer is also implicated in the
targeting of different PKA isoforms to various intracellular locations
and to specific substrates through interactions with protein kinase
A-anchoring proteins (3). Four different isoforms of the R subunit have
been identified as products of separate genes in mammalian cells, and
they have been termed RI Studies of several human cell lines have revealed that the PKA type
II Ricin belongs to a family of plant and bacterial toxins that enter
cells via the endocytic pathway. The toxin is transported retrogradely
through the Golgi to the endoplasmic reticulum (ER) before it enters
the cytosol, where it inhibits protein synthesis (11, 12). Ricin
consists of an A-chain and a B-chain that are linked by a disulfide
bridge. The B-chain binds to terminal galactose in both glycolipids and
glycoproteins at the plasma membrane, whereas the A-chain enzymatically
inhibits the protein synthesis after entry into the cytosol (12).
Because ricin binds to both glycolipids and glycoproteins at the plasma
membrane, it will be endocytosed by any vesicle that pinches off. Once
ricin is endocytosed, it can be transported through the endosomal
compartments, recycled back to the plasma membrane, delivered to the
lysosomes, or transported retrogradely to the TGN and to the ER (11,
12).
In this study, we took advantage of a RII Materials--
[3H]Leucine and
Na235SO4 were purchased from
Amersham Biosciences. Na125I was purchased from DuPont.
Hygromycin B was bought from Roche Molecular Biochemicals (Mannheim,
Germany) and Mowiol was obtained from Calbiochem. Cy3-labeled goat
anti-rabbit, FITC-labeled goat anti-mouse, and FITC-labeled goat
anti-rabbit were purchased from Jackson ImmunoResearch (West Grove,
PA). Fetal calf serum (FCS), RPMI 1640 medium, RPMI 1640 medium without
sulfate, and streptomycin were bought from Invitrogen and
protein A-Sepharose was purchased from Pharmacia.
8-(4-Chlorophenylthio)-cAMP (8CPT-cAMP), aprotinin, cycloheximide,
HEPES, lactose, MESNA, phenylmethylsulfonyl fluoride, poly(D-lysine) (Mr = 150,000),
ricin, and ricin B-chain were obtained from Sigma Chemical. Rabbit
anti-human RII Cells and Cell Culture--
A human B-lymphoid cell line (Reh)
stably transfected with pMep4 vector (clone pMep) or RII Measurement of Protein Synthesis--
The cells were washed
twice with HEPES medium (bicarbonate-free Eagle's minimum essential
medium buffered with 20 mM HEPES to pH 7.4), and then
incubated with the same medium for 30 min at 37 °C. The samples were
then incubated in the presence or absence of 350 µM
8CPT-cAMP for 30 min before 1, 10, and 100 ng/ml of ricin were added to
the cells, which were further incubated for 30 min at 37 °C. The
cells were incubated thereafter with HEPES medium containing 1 µCi/ml
[3H]leucine for 20 min at 37 °C, extracted with 5%
(w/v) trichloroacetic acid for 10 min followed by a brief wash
with the same solution. Subsequently, cells were dissolved in 0.1 M KOH, and the acid-precipitable radioactivity was
measured. The results are presented as percentage of radioactivity
incorporated in cells incubated without toxin. The concentration of
ricin required to inhibit the protein synthesis by 50% was chosen as a
measure of the sensitivity of cells to ricin. Variation between
duplicate measurements was less than 15%.
Sulfation of Ricin Sulf-1 and Sulf-2--
Recombinant ricin
A-sulf-1 and ricin A-sulf-2, modified to contain a tyrosine sulfation
site and both a tyrosine sulfation site and three overlapping
N-glycosylation sites, respectively, were expressed,
purified, and reconstituted with ricin B-chain (ricin sulf-1 and ricin
sulf-2, respectively) according to the procedure described previously
(14). The cells were washed twice in sulfate-free RPMI 1640 medium that
contained 2 mM L-glutamine, and then incubated
with 0.1 mCi/ml Na235SO4 in the
same medium for 3 h. The cells were then incubated in the presence
or absence of 350 µM 8CPT-cAMP and/or 20 µg/ml cycloheximide for 30 min at 37 °C, before ricin sulf-1 or ricin sulf-2 (~300 ng/ml) was added. The incubation was continued for 2 h at 37 °C. The cells were then washed twice for 5 min at
37 °C with HEPES medium that contained 0.1 M lactose
followed by cold PBS (140 mM NaCl and 10 mM
Na2HPO4, pH 7.2). The cells were thereafter
lysed (lysis buffer, 0.1 M NaCl, 10 mM
Na2HPO4, 1 mM EDTA, 1% (v/v)
Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1 mM aprotinin, pH 7.4), and centrifuged at 5000 rpm for 10 min at 4 °C. The supernatant was immunoprecipitated overnight at
4 °C with rabbit anti-ricin antibodies immobilized on protein A-Sepharose. The beads were then washed twice with PBS containing 0.35% (v/v) Triton X-100, and the immunoprecipitated material was
analyzed by SDS-PAGE (12%) under reducing conditions.
SDS-PAGE--
SDS-PAGE was carried out in the presence of
Immunofluorescence Microscopy--
For analysis of ricin
distribution, ricin was labeled with Cy5 (Amersham Biosciences)
according to the manufacturer's instructions. The coverslips were
coated with poly(D-lysine) (Mr = 150 000) as described previously (17). The cells were washed twice with HEPES medium before addition of Cy5-labeled ricin (~1000 ng/ml). After incubating the cells for 30 min at 37 °C, they were washed with cold PBS and further incubated with 3% (w/v) paraformaldehyde in
PBS for 15 min at room temperature. The cells were then washed 3 times
with PBS before incubation with 0.1% (v/v) Triton X-100 dissolved in
PBS for 5 min at room temperature. Subsequently, the cells were washed
in PBS, and incubated with PBS containing 5% (v/v) FCS for 30 min. The
permeabilized cells were incubated with rabbit anti-human RII
For analysis of PKA distribution versus centrosomal marker,
cells were fixed in 4% paraformaldehyde in PBS for 20 min at 37 °C,
rinsed twice in PBS, and incubated for 10 min with 50 mM
ammonium chloride in PBS. Subsequently, cells were permeabilized with
0.1% Triton X-100 in PBS with 0.2% BSA. Primary antibodies were
diluted in PBS containing 3% BSA to concentrations of 140 ng/ml for
mAb CTR 453 (a centrosomal marker), 100 ng/ml for rabbit anti-human RII Intracellular Accumulation of Ricin--
Ricin was
125I-labeled according to the procedure described by Fraker
and Speck (18) to a specific activity of 5 × 104
cpm/ng. The intracellular accumulation of 125I-labeled
ricin was measured after 2 h at 37 °C as the amount of toxin
that could not be removed by lactose treatment, as described previously
(19). The cells were preincubated with 350 µM 8CPT-cAMP for 30 min at 37 °C.
Ricin Endocytosis--
The endocytosis of ricin was measured
using the ORIGEN analyzer (IGEN Inc., Rockville, MD). Ricin was labeled
with N-hydroxysuccinimide ester-activated tris(bipyridine)
chelated ruthenium(II) TAG (IGEN Inc.) according to the
manufacturer's instructions and simultaneously biotinylated with
reducible immunopure NHS-SS-Biotin (Pierce). The cells were washed with
HEPES medium and then incubated in the presence or absence of 350 µM 8CPT-cAMP for 30 min at 37 °C, followed by addition
of TAG-labeled ricin (25 ng/ml) to allow endocytic uptake of the toxin
for 30 min at 37 °C. Half of the samples were then treated with 0.1 M MESNA for 1 h on ice (20), and the other half of the
samples was washed in cold PBS. The cells were then lysed (lysis
buffer, 100 mM NaCl, 5 mM MgCl2, 50 mM HEPES, and 1% (v/v) Triton X-100) for 10 min on ice.
Ricin that is both TAG-labeled and biotinylated can be detected in the lysate by using streptavidin-conjugated beads (Dynal, Oslo, Norway) and
the ORIGEN analyzer (IGEN Inc.). The endocytosis of TAG-labeled ricin
was measured as the amount of toxin that could not be removed by MESNA
treatment as described previously (21).
Miscellaneous--
Detection of PKA subunits by Northern blot
analysis or [32P]8-azido-cAMP photoaffinity labeling and
immunoprecipitation was performed essentially as described previously
(13).
Expression of PKA-RII PKA-RII Intoxication of Lymphocytes with Ricin Is Affected by RII Sulfation of Ricin Sulf-1 in Cells with or without RII Sulfation and Glycosylation of Ricin Sulf-2 in Cells with and
without RII Intracellular Accumulation of Ricin in Cells with and without
RII Colocalization of Ricin and RII
It has been reported that both the type II In the present study, we have investigated the influence of the
Golgi-associated type II The first indication that RII It has previously been shown that addition of 8-bromo-cAMP to
Madin-Darby canine kidney cells gives a selective stimulation of the
transport of apically internalized ricin to the Golgi apparatus (25)
(Fig. 9). However, in those cells, we
cannot ascribe this regulation to a Golgi-located PKA. We here
demonstrate that the sulfation of ricin is increased in cells
expressing RII-deficient B lymphoid cells and
stable transfectants expressing the type II
regulatory subunit
(RII
) of cAMP-dependent protein kinase (PKA), which is
targeted to the Golgi-centrosomal area, reveal that the presence of a
Golgi-associated pool of PKA type II
mediates a change in
intracellular transport of the plant toxin ricin. The transport of
ricin from endosomes to the Golgi apparatus, measured as sulfation of a
modified ricin (ricin sulf-1), increased in RII
-expressing cells
when PKA was activated. However, not only endosome-to-Golgi transport,
but also retrograde ricin transport to the endoplasmic reticulum (ER),
measured as sulfation and N-glycosylation of another
modified ricin (ricin sulf-2), seemed to be increased in cells
expressing RII
in the presence of a cAMP analog,
8-(4-chlorophenylthio)-cAMP. Thus, PKA type II
seems to be
involved in both endosome-to-Golgi and Golgi-to-ER transport. Because
ricin, after being retrogradely transported to the ER, is translocated
to the cytosol, where it inhibits protein synthesis, we also
investigated the influence of RII
expression on ricin toxicity. In
agreement with the other data obtained, 8-(4-chlorophenylthio)-cAMP and
RII
were found to sensitize cells to ricin, indicating an increased
transport of ricin to the cytosol. In conclusion, our results
demonstrate that transport of ricin from endosomes to the Golgi
apparatus and further to the ER is regulated by PKA type II
isozyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, RI
, RII
, and RII
(1, 2, 4). They all contain two C-terminal cAMP binding sites, a hinge region that
interacts with and inhibits the catalytic subunit, and a dimerization
domain responsible for the interaction between the two regulatory
subunits that make up the regulatory dimer of PKA (2). Whereas RI
subunits are known to be mainly soluble, RII subunits are primarily
associated with cytoskeletal elements and membranes (1, 3).
isozyme (containing RII
and C
) is concentrated in centrosomes and in Golgi-associated compartments (5, 6). In contrast,
PKA type II
(containing RII
) is associated more selectively with
the centrosomal region and not with Golgi structures (5). Based on the
important role of the Golgi apparatus in intracellular transport and
protein sorting and the localization of PKA, which previously has been
implicated in vesicle-mediated protein transport processes (7-10), the
possibility existed that a distinct Golgi-associated pool of PKA type
II
isozyme was involved in regulation of transport through this
organelle. To investigate whether PKA type II
is involved in the
regulation of retrograde transport, we studied the transport of the
plant toxin ricin.
-deficient B lymphoid cell
line, Reh (13), and reintroduced RII
by making stable transfectants
with a Golgi-associated pool of PKA type II
to examine the effect on
intracellular transport. We used two different ricin constructs to
monitor the retrograde toxin transport. Within the Golgi apparatus,
recombinant ricin with a tyrosine sulfation site (ricin sulf-1) becomes
radiolabeled in the presence of radioactive sulfate (14). This has made
ricin sulf-1 a valuable tool to study intracellular transport to the
TGN. A recombinant ricin construct with a sulfation site and three
overlapping N-glycosylation sites (ricin sulf-2) is both
modified in the TGN and N-glycosylated in the ER (14). The
N-glycosylation of the toxin results in a molecular
shift that can be observed on SDS-PAGE. This construct has therefore
been used to study intracellular transport to the ER. As shown in the
present study, the expression of PKA type II
(RII
) on a negative
background in a lymphoid cell line leads to modulation of the
retrograde transport of ricin, indicating a regulatory role for
Golgi-associated PKA type II
on these transport steps.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and RII
polyclonal antibodies and mouse
anti-RII
monoclonal antibodies have been described elsewhere (5).
Medial-Golgi (mAb CTR 433) and centrosomal (mAb CTR 453) markers were
kindly provided by Dr. Michel Bornens (Institute Curie, Paris, France).
The cis Golgi marker GM130 was obtained from BD Biosciences. The trans
Golgi marker TGN46 was obtained from Serotec (Oxford, UK).
under
direction of the human metallothionein IIA promoter (clone RII
) (15)
was maintained under standard conditions (5% CO2 in RPMI
1640 medium containing 5% (v/v) FCS, 2 mM
L-glutamine, and 100 µg/ml streptomycin). Every third
month, the cells expressing RII
were incubated under standard conditions in the presence of 200 µg/ml hygromycin B. On the day that
the experiments were performed, the cells were seeded into Eppendorf
tubes at a density of 8 × 105 cells/tube.
-mercaptoethanol as described previously (16). The gels were fixed
in 4% acetic acid (v/v) and 27% (v/v) methanol for 30 min and then
incubated with 1 M sodium salicylate, pH 5.8, in 2% (v/v)
glycerol for 30 min. The dried gels were then exposed to Kodak XAR-5
films (Eastman Kodak Co.) at
80 °C for autoradiography.
(1:1000) or with mouse mAb CTR 433 (1:10) to label the medial Golgi
compartment, with mouse mAb GM130 (1:1000) to label the cis Golgi, or
with sheep anti-human TGN46 (1:100) to label the trans Golgi in PBS
containing 5% (v/v) FCS for 30 min at room temperature. The cells were
then washed three times for 5 min with PBS containing 5% (v/v) FCS
followed by incubation with FITC-labeled goat anti-mouse antibody
(1:100) to detect CTR 433 and GM130, with Cy3-labeled goat anti-rabbit
antibody (1:500) to detect RII
, or with FITC-labeled donkey
anti-sheep/goat antibody (1:100) to detect TGN46 in PBS containing 5%
(v/v) FCS. After staining, the cells were washed three times for 5 min
with PBS at room temperature, and the coverslips were mounted in
Mowiol. Immunofluorescence microscopy was performed using a Leica
(Wetzlar, Germany) confocal microscope. Images were captured with a
resolution of 1024 × 768 pixels and prepared with the use of
Adobe Photoshop 4.0 (Adobe Systems, Mountain View, CA).
, 500 ng/ml for rabbit anti-human RII
, and 1 µg/ml for mouse anti-RII
mAb and incubated for 1 h at room temperature. Cells were then washed three times in PBST (PBS with 1% Tween 20) to remove
unbound antibodies followed by incubation with fluorochrome-conjugated secondary antibodies (FITC and Texas Red) for 1 h at room
temperature. Finally, the cells were mounted in CITIFLUOR (Citifluor,
London, UK). Confocal microscopy was performed on a Sarastro 2000 confocal microscope (Amersham Biosciences), equipped with an
argon laser (488 to 514 nm wavelength). Ten sections of 0.25 µm
(averaging five full frames of the same section) were scanned, and
stacks of optical sections for each data set were compiled with Voxel View software on an IRIS 4D-70 GT graphics work station (SGI, Mountain
View, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in a RII
-deficient, B Lymphoid Cell
Line, Reh--
To study effects related specifically to expression of
RII
, stably transfected cell lines expressing RII
under control
of the zinc-inducible type IIa metallothionein promoter were made together with control clones transfected with empty plasmid by selection on hygromycin B. Fig. 1 shows
characterization of one clone expressing RII
compared with a control
clone. The RII
transfected clone displayed high levels of RII
mRNA (A) as well as a cAMP-binding protein,
immunoreactive with RII
antibodies and present in both the soluble
S200 and detergent-soluble Tx-100 fractions (B). In
contrast, a control-transfected clone did not have any detectable
RII
mRNA or protein. Both cell clones had equal levels of RII
mRNA. We conclude that the RII
-transfected clone expresses
RII
at quite high levels even in the absence of zinc.
View larger version (35K):
[in a new window]
Fig. 1.
Introduction of RII
by stable transfection in the
RII
-deficient, B lymphoid cell line, Reh.
A, Northern blot analysis of Reh-cell clones selected on
hygromycin B after transfection with RII
-pMEP4 or empty pMEP4
expression vector and incubated in the absence (
) and presence (+) of
75 µM ZnSO4 for 24 h. Total RNA was
extracted in guanidinium isothiocyanate and prepared by CsCl-gradient
centrifugation, subjected to electrophoresis, and blotted onto nylon
membrane. The resulting filter was hybridized with radiolabeled
cDNA probes for RII
and RII
. B, stably transfected
clones were lysed in isotonic sucrose buffer by ultra Turrax
homogenization, nuclei were pelleted at 500 × g, the
postnuclear supernatant was subjected to a 200,000 × g
spin for 1 h to yield a soluble fraction (S200), and a pellet was
solubilized in isotonic sucrose buffer with 0.5% Triton X-100 and
centrifuged at 15,000 × g to remove insoluble material
(Tx-100). The soluble S200 and detergent-soluble Tx-100 fractions were
next subjected to photoaffinity labeling with
[32P]8-azido-cAMP in the presence (+) or absence (
) of
excess cold cAMP to compete binding. Ten percent of the samples were
then boiled directly in SDS-sample buffer (lanes
1, 2, 5, and 6), whereas
the rest were used for immunoprecipitation with antibodies to RI
and
RII
before analysis by SDS-PAGE and autoradiography
(lanes 3, 4, 7, and
8). Arrows indicate RI
(top and
bottom) and RII
(top).
Expressed in Reh Cells Is Targeted to the
Golgi-Centrosomal Region--
It has previously been shown that RII
is localized to the Golgi-centrosomal area in SaOS2 osteosarcoma cells
and in COS-7 cells (5, 6). We have examined, by immunofluorescence and confocal microscopy, whether this was also the case when RII
was
expressed in Reh cells. Double staining of the centrosomal marker CTR
453 (22) (Figs. 2, A,
C, and E) and RII
(Figs. 2, B,
D, and F) demonstrated that RII
was absent in
wild-type cells (Fig. 2B), but appeared in the centrosomal
region when expressed (Fig. 2, D and F
versus C and E). Furthermore, the
distribution of RII
was wider than that of the centrosomal protein
CTR 453 (Fig. 2, F versus E). Because
RII
is targeted to centrosomes and present in Reh cells, as in most
cancer cell lines [Fig. 1; Ref. 5), we next examined the distribution
of RII
versus that of RII
by dual staining and image
overlays (Fig. 2G). Again, RII
(red) was found
in a wider area than RII
(green, overlap seems
yellow), which in separate experiments showed a distribution overlapping well with that of the centrosomal marker, CTR 453 (not
shown). In addition, we performed immunofluorescence studies of the
localization of RII
and the Golgi markers GM130 and TGN46. As shown
in Fig. 3, RII
was partially
colocalized with both Golgi markers. Together, these experiments
demonstrated that clones expressing RII
acquired a new PKA isozyme
that localizes in the centrosomal-Golgi region. To explore the function
of this particular pool of Golgi-associated PKA, we next examined the
intracellular transport of ricin in Reh cells in the presence and
absence of a Golgi-associated pool of PKA type II
.
View larger version (38K):
[in a new window]
Fig. 2.
RII is targeted to
the Golgi-centrosomal region when expressed in an
RII
-deficient, B lymphoid cell line, Reh, and
concentrates in a wider area than RII
. Human
wild-type Reh cells (A and B) and a clone stably
expressing RII
(C to F) were double stained
with a centrosomal marker (mAb CTR 453; A, C, and
E) and hRII
antibodies (B, D, and
F), and confocal images from sections in the centrosomal
area are shown separately in pseudo-color, ranging from no signal
(blue) to high signal (yellow to red).
G, confocal image after double staining with mAb RII
(red) and hRII
Abs (green). Bars:
A to D, 5 µm; E to G, 2 µm. Arrows indicate centrosomes.
View larger version (9K):
[in a new window]
Fig. 3.
RII expressed in an
RII
-deficient, B lymphoid cell line, Reh,
colocalizes with Golgi markers. RII
cells were fixed,
permeabilized, double stained with hRII
antibodies (red,
A and D) and with the cis Golgi marker GM130
(green, B) or the trans Golgi marker TGN46
(green, E), and analyzed in a confocal
microscope. Merged images are shown in C and
F.
Expression--
To investigate whether one or more steps along the
intracellular route followed by ricin are affected by RII
, we tested
the sensitivity to ricin of clone RII
cells with and without the stimulation of PKA and compared it with that of cells deficient in PKA
type II
(clone pMep). To activate PKA, we used a cell-permeable cAMP
analog, 8CPT-cAMP, with high affinity for the type II regulatory subunits (23, 24). As shown in Fig. 4,
cells expressing RII
were ~2-fold more sensitive to ricin than the
RII
-deficient cells, and although addition of 8CPT-cAMP had a
sensitizing effect on the pMep cells, it sensitized the cells
expressing RII
to a much larger extent. Similar experiments with
untransfected cells (Reh), as well as with other clones transfected
with RII
with similar targeting, showed the same pattern of
sensitivity (results not shown). Thus, RII
regulates one or several
steps on the route of ricin to the cytosol in lymphoid cells, although
RII
is not strictly required for ricin intoxication.
View larger version (35K):
[in a new window]
Fig. 4.
Ability of ricin to inhibit protein synthesis
in lymphocytes deficient in (clone pMep) or stably
transfected with RII (clone
RII
) in the presence or absence of
8CPT-cAMP. Cells were incubated in HEPES medium with or without
8CPT-cAMP for 30 min at 37 °C followed by the addition of increasing
concentrations of toxin. Thirty minutes later, protein synthesis was
measured as described under "Experimental Procedures." The figure
shows the average of duplicates from a representative experiment.
--
To
study the transport of ricin from the plasma membrane to the Golgi
apparatus, recombinant ricin sulf-1 that contains a tyrosine sulfation
site was used. The cells were incubated in the presence or absence of
8CPT-cAMP and ricin sulf-1 (Fig. 5). Because the protein synthesis was somewhat stimulated in cells preincubated with 8CPT-cAMP, and an increased transport of newly synthesized proteins through the TGN could in theory result in an
increased competition for sulfation and thus interfere with the assay;
some cells were also incubated with cycloheximide to inhibit protein
synthesis. Fig. 5C shows that the sulfation of ricin in control cells
increased by ~70% in the presence of 8CPT-cAMP, whereas the
sulfation increased by ~120% in the cells expressing RII
.
However, in cells treated with both 8CPT-cAMP and cycloheximide, the
sulfation of ricin increased by ~80% in the control cells and by
~250% in the RII
-expressing cells. This result indicates that
when PKA is activated, the transport of ricin to the Golgi apparatus is
increased to a larger extent in cells expressing RII
than in control
cells.
View larger version (29K):
[in a new window]
Fig. 5.
The effect of 8CPT-cAMP and cycloheximide on
the sulfation of recombinant ricin A chain with a sulfation site
(ricin A-sulf-1) in RII
deficient (clone pMep) and
RII
expressing (clone
RII
) cells. A and
B, autoradiograms from representative experiments with ricin
sulfation in pMep (A) and RII
cells (B)
respectively. Cells were washed in sulfate-free RPMI 1640 medium and
incubated with 0.1 mCi/ml Na235SO4
for 3 h, and then further incubated in the presence or absence of
8CPT-cAMP and/or cycloheximide for 30 min at 37 °C before ricin
sulf-1 was added. The incubation was continued for 2 h at 37 °C
before the cells were washed with lactose and cold PBS before cell
lysis, as described under "Experimental Procedures." Nuclei were
removed by centrifugation, and ricin was immunoprecipitated overnight
at 4 °C with rabbit anti-ricin antibodies immobilized on protein
A-Sepharose. The immunoprecipitated material was analyzed by SDS-PAGE
(12%) under reducing conditions followed by autoradiography.
C, levels of ricin sulfation in the presence of 8CPT-cAMP
are calculated relative to levels in untreated cells, whereas sulfation
levels in cells treated with both cycloheximide and 8CPT-cAMP are
calculated relative to cells incubated with cycloheximide alone by
densitometric scanning of bands. Error bars show half range
between duplicates of lanes from the experiment shown in A
and B. The intensities of the resulting bands were
determined by densitometric quantitation using ImageQuant 5.5 (Amersham
Biosciences).
--
To further investigate the transport of ricin to
the ER, recombinant ricin sulf-2 that contained a tyrosine sulfation
site and three overlapping N-glycosylation sites was used.
When ricin sulf-2 was added to control cells (clone pMep) or to
RII
-expressing cells (clone RII
) in the presence of radioactive
sulfate and immunoprecipitated from cell lysates, two bands were
visible (Figs. 6, A and
B). The upper molecular weight band represents ricin that
has been both sulfated and glycosylated, and the lower molecular weight
band represents ricin that has only been sulfated. As shown in Fig. 6C,
the amount of ricin in the ER, measured as sulfated and
glycosylated ricin relative to the total amount of sulfated ricin,
was significantly increased in cells expressing RII
(clone RII
)
compared with the control cells (clone pMep) when PKA was activated by
8CPT-cAMP. These observations indicate that not only the transport of
ricin to the Golgi apparatus but also the further transport of the
toxin to the ER is increased by RII
in the presence of
8CPT-cAMP.
View larger version (23K):
[in a new window]
Fig. 6.
The effect of 8CPT-cAMP on the sulfation and
glycosylation of recombinant ricin A-chain with sulfation and
glycosylation sites (ricin A-sulf-2) in cells
deficient in (clone pMep) or expressing
RII (clone
RII
). A and
B, autoradiograms from representative experiments with ricin
sulfation and N-glycosylation in pMep (A) and
RII
cells (B), respectively. The higher molecular weight
bands represent ricin A-sulf-2 that has been both sulfated and
glycosylated, whereas the lower molecular weight bands represent ricin
A-sulf-2 that has been sulfated only. C, the intensities of
the upper molecular weight bands were calculated relative to the sum of
both the upper and lower molecular weight bands for the different
conditions listed. The cells were treated as described in the legend to
Fig. 5.
--
The increased sulfation of ricin sulf-1 and ricin sulf-2
observed in cells expressing RII
compared with control cells
deficient in RII
could be caused by an increased binding and
endocytosis of ricin or by an increased endosome-to-Golgi transport. We
therefore investigated the endocytosis of ricin in the presence or
absence of 8CPT-cAMP. Fig. 7 demonstrates
that the accumulation of ricin after 2 h of incubation was not
significantly changed by addition of 8CPT-cAMP or by the expression of
RII
. Similar data were obtained when the cells were incubated with
ricin for 30 min (results not shown). In addition, the binding of ricin
to the plasma membrane was not significantly altered by 8CPT-cAMP or by
expression of RII
(data not shown). Thus, the increased transport of
ricin to the Golgi apparatus cannot be accounted for by a change in the
endocytosis of ricin.
View larger version (20K):
[in a new window]
Fig. 7.
The effect of 8CPT-cAMP and expression of
RII on accumulation of ricin in cells
deficient in (clone pMep) or expressing
RII
(clone
RII
). The intracellular
accumulation of 125I-labeled ricin was measured in the
presence and absence of 8CPT-cAMP after 2 h of incubation at
37 °C as the amount of toxin that could not be removed by lactose
treatment. The error bars show half range between
duplicates from a representative experiment.
in the Golgi Area--
As
evident from Figs. 2 and 3 and previous reports (5), RII
mainly
exhibits a perinuclear, Golgi-associated localization that is
detergent-extractable, indicating membrane-associated localization. To
investigate whether RII
is associated with ricin-containing structures, immunofluorescence studies were performed using antibodies raised against human RII
(Fig.
8A, visualized by Cy3-labeled secondary antibodies, red), Cy5-labeled ricin (Fig.
8B, blue), and the medial Golgi marker CTR 433 (Fig. 8C, visualized by FITC-labeled secondary antibodies,
green). Both RII
and ricin seemed colocalized (Fig.
8F) and were also found to partly colocalize with the medial Golgi marker (Fig. 8, D and E, respectively).
View larger version (19K):
[in a new window]
Fig. 8.
Localization of RII
(A) and ricin (B) in the Golgi
area (C). D to F, merged
images of A and C, B and C,
and A and B, respectively. Lymphoid Reh cells
stably transfected with RII
were incubated with Cy5-labeled ricin
(~1000 ng/ml) for 30 min at 37 °C. The cells were then fixed,
permeabilized, stained with antibodies against RII
and a medial
Golgi marker (CTR 433), and analyzed in a confocal microscope.
and II
regulatory
subunits are associated with centrosomes (5). Because the previous
experiment showed colocalization between ricin and RII
, we
investigated the distribution and localization of ricin in the
centrosomal area. We showed that whereas minor amounts of RII
are
present in centrosomes, no ricin was detected in this region (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
regulatory subunit of PKA on the intracellular transport of ricin. RII
was expressed on a negative background, and 8CPT-cAMP was used to activate PKA. Because phenotypic differences between clones may arise (e.g. relating to
incorporation in the genomic DNA), we investigated several clones
expressing RII
with similar results. Different clones transfected
with empty vector displayed a phenotype similar to that of wild-type cells.
might be involved in regulation of
intracellular transport was found by investigating the entry of the
plant toxin ricin into the cytosol, measured as ricin toxicity. Even
when PKA was not activated by addition of external 8CPT-cAMP, the cells
expressing RII
were about 2-fold more sensitive to ricin than the
control cells. The most likely explanation seems to be that there is a
certain level of endogenous cAMP that partly activates PKA. This
explanation was strengthened by the finding that the transfected cells
were about 4-fold more sensitive to ricin than the control cells when
PKA was activated by addition of external 8CPT-cAMP. Also, the control
cells were shown to be slightly more sensitive to ricin when PKA was
activated. This might be caused by the activation of other isozymes of
PKA that also regulate intracellular transport but, apparently, less
efficiently than PKA type II
. The PKA type II
might be a better
regulator than the other PKA isozymes because it is closer to the
vesicular route of ricin transport into the Golgi apparatus. It might
be a question of the local concentration of PKA whether it serves as a
good regulator.
compared with control cells when PKA was activated by
externally added 8CPT-cAMP. This result indicates that RII
is a
strong regulator of the transport of ricin from the plasma membrane to
the Golgi apparatus. Earlier studies have demonstrated that calmodulin
(26) and calcium (27) can modulate the transport of ricin from
endosomes to the Golgi apparatus in other cell lines (Fig. 9). Clearly, different factors are involved in the regulation of retrograde transport.
View larger version (14K):
[in a new window]
Fig. 9.
Model of ricin endosome to Golgi
transport. There is a well-established Rab9-dependent
transport from late endosomes (LE) to the Golgi apparatus of
furin and the M6PR. TGN38, Shiga toxin B, and ricin seem to be
transported to the Golgi apparatus from early/recycling endosomes
(EE/RE). As indicated, several molecules
implicated in the transport of ricin from the endosomal compartment to
the Golgi apparatus have been identified.
Interestingly, confocal microscopy demonstrated a localization of both
ricin and RII in the Golgi area. The difference in ricin sulfation
was much larger when both cell types were preincubated in the presence
of cycloheximide. Such experiments were performed because the protein
synthesis was somewhat stimulated in cells incubated with 8CPT-cAMP
(data not shown), and an increased transport of newly synthesized
proteins through the TGN in theory could result in an increased
competition for sulfation. Even though it cannot be excluded that
cycloheximide might result in an increased transport of ricin to the
Golgi apparatus, the increased sulfation of ricin that was observed in
cells expressing RII
compared with the control cells in the presence
of 8CPT-cAMP and cycloheximide strongly supports the notion of a
regulatory role of RII
in intracellular transport from the plasma
membrane to the Golgi apparatus.
The increased transport of ricin to the Golgi apparatus in cells
expressing RII could be caused by increased binding and endocytosis
of the toxin. However, no significant stimulation of the binding or the
endocytosis of the toxin after 30 min or 2 h of incubation were
observed in the RII
-expressing cells, strongly indicating a
selective regulatory role of RII
in the transport of ricin from the
endosomal compartments to the Golgi apparatus and further to the
ER.
At the moment, we can only speculate about the molecular mechanism of
the regulation of the endosome to Golgi transport of ricin by PKA type
II. There are several examples of the importance of phosphorylation
for transport. For example, it has been shown that the activity of PKA
has an effect on the in vitro association of ARF1
to Golgi membranes (28). It is possible that upon stimulation, the
Golgi-associated PKA type II
phosphorylates membrane proteins in
neighboring compartments that recruit cytosolic proteins involved in
the trafficking of ricin between endocytic organelles and the Golgi
apparatus. Another possibility is that PKA type II
is important for
the fusion of incoming vesicles with the Golgi apparatus.
Transport of ricin not only to the TGN, where the sulfotransferase is
located (29), but also from the Golgi apparatus to the ER, measured as
ricin that has been both sulfated and glycosylated, was shown to
increase in cells expressing RII relatively to the control cells in
the presence of 8CPT-cAMP. An increased amount of glycosylated ricin
could have been a result of the stimulated transport to the Golgi
apparatus of this toxin. However, the fraction of ricin that has been
both sulfated and glycosylated compared with the total amount of
sulfated ricin is also increased in cells expressing RII
in the
presence of 8CPT-cAMP, thus indicating an additional regulatory role of
RII
in the transport of ricin from the Golgi to the ER.
In conclusion, our results indicate that the Golgi-associated type
II regulatory subunit of PKA regulates endosome-to-Golgi and
Golgi-to-ER transport of ricin in lymphocytes.
![]() |
FOOTNOTES |
---|
* This work was supported by the Norwegian Research Council, The Norwegian Cancer Society, Novo Nordic Research Foundation, Anders Jahre's Foundation, and Jeanette & Søren Bothners legacy.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. Tel.:
47-22-93-42-94; Fax: 47-22-50-86-92; E-mail:
ksandvig@radium.uio.no.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M209982200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKA, protein kinase A; TGN, trans Golgi network; ER, endoplasmic reticulum; ricin sulf-1, recombinant ricin with a tyrosine sulfation site; ricin sulf-2, recombinant ricin construct with a sulfation site and three overlapping N-glycosylation sites; FITC, fluorescein isothiocyanate; FCS, fetal calf serum; 8CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; MESNA, 2-mercaptoethanesulfonic acid; mAb, monoclonal antibody; PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Skalhegg, B. S., and Tasken, K. (2000) Front. Biosci. 5, D678-D693[Medline] [Order article via Infotrieve] |
2. | Francis, S. H., and Corbin, J. D. (1994) Annu. Rev. Physiol. 56, 237-272[CrossRef][Medline] [Order article via Infotrieve] |
3. | Colledge, M., and Scott, J. D. (1999) Trends Cell Biol. 9, 216-221[CrossRef][Medline] [Order article via Infotrieve] |
4. | Oyen, O., Myklebust, F., Scott, J. D., Hansson, V., and Jahnsen, T. (1989) FEBS Lett. 246, 57-64[CrossRef][Medline] [Order article via Infotrieve] |
5. | Keryer, G., Skalhegg, B. S., Landmark, B. F., Hansson, V., Jahnsen, T., and Tasken, K. (1999) Exp. Cell Res. 249, 131-146[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Martin, M. E.,
Hidalgo, J.,
Vega, F. M.,
and Velasco, A.
(1999)
J. Cell Sci.
112,
3869-3878 |
7. |
Pimplikar, S. W.,
and Simons, K.
(1994)
J. Biol. Chem.
269,
19054-19059 |
8. | Hansen, S. H., and Casanova, J. E. (1994) J. Cell Biol. 126, 677-687[Abstract] |
9. | Mostov, K. E., and Cardone, M. H. (1995) Bioessays 17, 129-138[Medline] [Order article via Infotrieve] |
10. |
Eker, P.,
Holm, P. K.,
van Deurs, B.,
and Sandvig, K.
(1994)
J. Biol. Chem.
269,
18607-18615 |
11. |
Sandvig, K.,
and van Deurs, B.
(1996)
Physiol. Rev.
76,
949-966 |
12. |
Sandvig, K.,
and van Deurs, B.
(2000)
EMBO J.
19,
5943-5950 |
13. |
Tasken, K.,
Skalhegg, B. S.,
Solberg, R.,
Andersson, K. B.,
Taylor, S. S.,
Lea, T.,
Blomhoff, H. K.,
Jahnsen, T.,
and Hansson, V.
(1993)
J. Biol. Chem.
268,
21276-21283 |
14. |
Rapak, A.,
Falnes, P. O.,
and Olsnes, S.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3783-3788 |
15. |
Carlson, C. R.,
Witczak, O.,
Vossebein, L.,
Labbé, J.-C.,
Skålhegg, B. S.,
Keryer, G.,
Herberg, F. W.,
Collas, P.,
and Tasken, K.
(2001)
J. Cell Sci.
114,
3243-3254 |
16. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
17. | Huang, W. M., Gibson, S. J., Facer, P., Gu, J., and Polak, J. M. (1983) Histochemistry 77, 275-279[Medline] [Order article via Infotrieve] |
18. | Fraker, P. J., and Speck, J. C., Jr. (1978) Biochem. Biophys. Res. Commun. 80, 849-857[Medline] [Order article via Infotrieve] |
19. | Sandvig, K., and Olsnes, S. (1979) Exp. Cell Res. 121, 15-25[Medline] [Order article via Infotrieve] |
20. | Smythe, E., Redelmeier, T. E., and Schmid, S. L. (1992) Methods Enzymol. 219, 223-234[Medline] [Order article via Infotrieve] |
21. |
Skretting, G.,
Torgersen, M. L.,
van Deurs, B.,
and Sandvig, K.
(1999)
J. Cell Sci.
112,
3899-3909 |
22. | Bailly, E., Doree, M., Nurse, P., and Bornens, M. (1989) EMBO J. 8, 3985-3995[Abstract] |
23. | Ogreid, D., Ekanger, R., Suva, R. H., Miller, J. P., and Doskeland, S. O. (1989) Eur. J. Biochem. 181, 19-31[Abstract] |
24. | Ogreid, D., and Doskeland, S. O. (1981) FEBS Lett. 129, 287-292[CrossRef][Medline] [Order article via Infotrieve] |
25. | Llorente, A., van Deurs, B., and Sandvig, K. (1998) FEBS Lett. 431, 200-204[CrossRef][Medline] [Order article via Infotrieve] |
26. | Llorente, A., Garred, Ø., Holm, P. K., Eker, P., Jacobsen, J., van Deurs, B., and Sandvig, K. (1996) Exp. Cell Res. 227, 298-308[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Lauvrak, S. U.,
Llorente, A.,
Iversen, T. G.,
and Sandvig, K.
(2002)
J. Cell Sci.
115,
3449-3456 |
28. |
Martin, M. E.,
Hidalgo, J.,
Rosa, J. L.,
Crottet, P.,
and Velasco, A.
(2000)
J. Biol. Chem.
275,
19050-19059 |
29. |
Leitinger, B.,
Brown, J. L.,
and Spiess, M.
(1994)
J. Biol. Chem.
269,
8115-8121 |