2549
Journal of Cell Science 112, 2549-2557 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0494
Phorbol ester promotes endocytosis by activating a factor involved in
endosome fusion
Alejandro Aballay1, Philip D. Stahl1,* and Luis S. Mayorga2
1Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA
2Instituto de Histologia y Embriologia, Facultad de Ciencias Medicas, Universidad Nacional de Cuyo - CONICET, Mendoza,
Argentina
*Author for correspondence (e-mail: [email protected])
Accepted 28 May; published on WWW 7 July 1999
SUMMARY
Previous studies indicate that a zinc- and phorbol esterbinding factor is necessary for in vitro endosome fusion and
for the effect of Rab5 on endosome fusion. Rab5 is a small
GTPase that regulates membrane fusion between early
endosomes derived from either receptor-mediated
endocytosis or fluid-phase endocytosis. In its GTP-bound
form, Rab5 promotes endocytosis and enhances fusion
among early endosomes. To determine if PMA stimulates
endocytosis by activating a factor required for endosome
fusion, we overexpressed wild-type Rab5, a dominant
negative mutant (Rab5:S34N), and a GTPase deficient
mutant (Rab5:Q79L) in BHK-21 cells. The phorbol ester
PMA stimulates endocytosis and increases the number and
the size of endocytic vesicles, even in the presence of
Rab5:S34N. Zinc depletion with N,N,N′,N′-tetrakis-(2pyridylmethyl)ethylenediamine (TPEN) and addition of
calphostin C (CPC), an inhibitor of PKC that interacts with
zinc and phorbol ester binding motifs, inhibited both basal
and Rab5-stimulated fluid phase endocytosis. These two
reagents also inhibited the size and number of endocytic
vesicles promoted by Rab5. These results suggest that PMA
stimulates endocytosis by regulating the dynamics of the
early endosome compartment.
INTRODUCTION
of cell surface receptors in a process that can involve both
protein phosphorylation by PKC and receptor removal from the
cell surface by enhanced endocytosis. Thus, PMA has been
demonstrated to stimulate the transcytosis and apical recycling
of the polymeric immunoglobulin receptor (pIgR) in MDCK
cells, potentially involving either the α and/or ε isozymes
(Cardone et al., 1994). PMA has also been suggested to induce
rapid endocytosis and down modulation of the chemokine
receptor CXCR4 that is required, together with CD4, for the
entry of T cell line-adapted HIV-1 (Signoret et al., 1997).
Phorbol ester-binding factors are also involved in other steps
of intracellular trafficking. The export of the vesicular
stomatitis virus glycoprotein from the endoplasmic reticulum
requires a phorbol ester binding protein (Fabbri et al., 1994).
In addition, Simon et al. (1996a,b) have shown that the
generation of vesicles from purified Golgi fractions requires a
phorbol ester-binding factor. Interestingly, both groups have
reported that a kinase activity is not necessary for the factor(s)
effect. Consistent with these results, we have shown that a
kinase activity is probably not directly involved in the effect of
the phorbol ester-binding factor acting downstream of Rab5 in
endosome fusion (Aballay et al., 1998).
Rab5 is a limiting regulator of early endosome fusion, a
critical step along the endocytic pathway (Bucci et al., 1992).
In BHK-21 cells, overexpression of wild-type Rab5
(Rab5:WT) leads to increased early endosome fusion and
Endocytosis is an essential process for cell function. There are
at least two types of endocytosis: receptor-mediated
endocytosis and fluid-phase endocytosis. Endosomal vesicles
derived from both types of endocytosis fuse with a common
endosomal sorting compartment and appear to share common
components. Interestingly, factors that affect endosome fusion
regulate the kinetics and extent of endocytosis. For example,
the small GTPase Rab5 that regulates membrane fusion
between early endosomes derived from either receptormediated endocytosis or fluid-phase endocytosis, strongly
affect the internalization of material from the extracellular
compartment (Bucci et al., 1992; Li et al., 1994).
In previous studies we have shown that a zinc- and phorbol
ester-binding factor is necessary for in vitro endosome fusion
(Aballay et al., 1995, 1997). In addition, we have shown that
a phorbol ester factor is required downstream of Rab5 during
endosome fusion (Aballay et al., 1998).
Several groups have reported that phorbol ester can strongly
affect endocytosis. Using human neutrophils, it has been shown
that phorbol 12-myristate 13-acetate (PMA) or diacylglycerol
can stimulate endocytosis (Keller, 1990). Recently, it has been
shown that a phorbol ester-binding protein is important for
RacC-mediated phagocytosis (Seastone et al., 1998). Phorbol
esters are also believed to regulate the movement of a number
Key words: PMA, Zinc, Rab5, Rab protein, PKC
2550 A. Aballay, P. D. Stahl and L. S. Mayorga
elevated endocytosis whereas overexpression of the dominant
negative Rab5 mutant (Rab5:S34N) results in decreased early
endosome fusion and reduced endocytosis (Bucci et al., 1992;
Li and Stahl, 1993; Stenmark et al., 1994). Interestingly, the
GTPase deficient mutant (Rab5:Q79L) increases endosome
fusion (Stenmark et al., 1994; Barbieri et al., 1996),
indicating that GTP hydrolysis is not required for the process.
In this context, it has been proposed that Rab5 in its GTPbound form, recruits rabaptin-5 to the membrane. This
binding itself retards GTP hydrolysis by blocking the
activation of the GTPase activity of rab5 by GAPs. Thus, the
docking/fusion machinery would remain bound to Rab5
allowing the fusion to occur (Rybin et al., 1996). Moreover,
a connection between Rab5, Ras and phosphatidylinositol 3kinase (PI 3-kinase) has been suggested (Li et al., 1995, 1997;
Simonsen et al., 1998).
Since Rab5 is a rate limiting regulator of endosome fusion
and since a phorbol ester binding-protein acts downstream of
Rab5 in the process, we decided to study whether the effects
of phorbol ester on endocytosis could be caused by the
activation of the factor acting in endosome fusion. We took
advantage of the Sindbis virus system to overexpress in BHK21 cell monolayers Rab5:WT, and both the dominant negative
Rab5:S34N and the GTPase deficient mutant Rab5:Q79L. We
also generated stable cell lines overexpressing GFP-Rab5:WT
and GFP-Rab5:S34N. We found that inhibitors of the phorbol
ester binding-domains block the Rab5:WT and Rab5:Q79L
effects on endocytosis. The phorbol ester PMA, on other hand,
can overcome the inhibitory effect on endocytosis caused by
the overexpression of Rab5:S34N. In addition, PMA increased
both the size and number of GFP-Rab5:WT positive endocytic
structures. These results indicate that PMA promotes
endocytosis and alters the morphology of the endocytic
compartment by stimulating a factor required for endosome
fusion after Rab5 activation.
MATERIALS AND METHODS
Chemicals
Dextran Texas Red, Lyso Tracker, and N,N,N′,N′-tetrakis-(2pyridylmethyl) ethylenediamine (TPEN), were obtained from
Molecular Probes, Portland, OR, USA. The vector pEGFP-C1 was
purchased from Clontech, Palo Alto, CA, USA. All other unspecified
reagents were purchased from Sigma Chemical Co., St Louis, MO,
USA.
Plasmid constructions
To generate stable cell lines expressing GFP-Rab5:WT and GFPRab5:S34N, Rab5:WT and Rab5:S34N cDNAs were amplified by
PCR from cDNA clones (Li and Stahl, 1993) and subcloned as
HindIII-BamHI fragments into a pEGFP-C1 vector. All coding
regions were sequenced to confirm the absence of PCR induced
errors.
Recombinant Sindbis virus production
The cDNAs of Rab5:WT, Rab5:S34N, and Rab5:Q79L were
subcloned into the unique XbaI restriction site of the Sindbis vector
Toto1000:3′2J (Li and Stahl, 1993). The plasmids were then linearized
by XhoI digestion and used as a template for in vitro transcription with
SP6 RNA polymerase. The resulting RNA transcripts were used for
transfection of BHK-21 cell monolayers using LipofectAmine reagent
(Life Technologies, Inc). The cells were maintained in serum-free α-
minimal essential medium (α-MEM) containing 3% fetal bovine
serum (FBS) at 37°C. At 24 hours post-transfection, the medium
containing the released viruses was harvested and titered on fresh
BHK-21 monolayers. The virus titers were generally between 107 and
109 plaque-forming units (p.f.u.) per ml. Virus stocks were aliquoted
and frozen at −70°C and thawed just before use.
Cell culture, transfection and treatments
CHO cells were grown in T75 flasks for 24 hours before transfection.
After washing once with serum-free α-MEM, the cells were
transfected with equal amounts of pEGFP-Rab5:WT and pEGFPRab5:S34N plasmids. LipofectAmine reagent (Life Technologies,
Inc) was used for transfections according to the manufacturer’s
instructions. Clones were isolated by growth in medium containing
G418.
BHK-21 cells were grown to confluence as monolayers and
maintained in 35-mm dishes. The growth medium was α-MEM
containing 10% fetal bovine serum. The infection with recombinant
Sindbis viruses and the expression of Rab5:WT and its mutant
derivatives was carried out as described (Li and Stahl, 1993). At 4 hours
postinfection, the medium was replaced with α-MEM supplemented
with N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN),
calphostin C (CPC), phorbol 13-monoacetate (PA), or phorbol 12myristate 13-acetate (PMA) as required. Following incubation for 20
minutes at 37°C, endocytosis was quantitated as described below.
Fluid phase endocytosis assay
Endocytosis was carried out as described earlier (Li et al., 1994, 1995,
1997). Briefly, BHK-21 cell monolayers in 35-mm dishes were
washed three times with serum-free α-MEM, and horseradish
peroxidase (HRP) uptake was initiated by addition of 1 ml of α-MEM
containing 1 mg/ml HRP and 1% bovine serum albumin. Following
uptake and removal of excess HRP, the cells were then lysed in icecold PBS containing 5 mM EGTA and 0.1% Triton X-100 and the
lysates were assayed for horseradish peroxidase activity. The products
were quantified by measuring OD490nm in a Bio-Rad microplate
reader. Protein content was determined by the Bio-Rad protein assay
according to the manufacturer’s instructions.
Electron microscopy
BHK-21 cells were grown to 50% confluence in α-MEM containing
10% fetal bovine serum. The transfection with recombinant Sindbis
viruses and the expression of Rab5:WT and its mutant derivatives was
carried out as described (Li and Stahl, 1993). At 4 hours postinfection,
the medium was replaced by α-MEM supplemented with TPEN,
calphostin C, chelerythrine, or PMA as required. Incubation
proceeded for 30 minutes at 37°C. The cells were then allowed to
internalize (1 hour or 5 minutes) colloidal gold particles (20 nm of
diameter) coated with bovine serum albumin. After incubation, the
cells were washed twice with HB-EGTA buffer (250 mM sucrose, 0.5
mM EGTA, 20 mM Hepes-KOH, pH 7.0), fixed with 2%
glutaraldehyde in 0.1 M cacodylate buffer, pH 7.0, embedded in
plastic, and processed for electron microscopy. Quantitation was
carried out by counting the vesicles containing gold particles in 20
cells for each set of experimental conditions.
Confocal microscopy
CHO cells stably expressing GFP-Rab5 were grown on 15-mm square
coverslips in 35-mm dishes for 24 hours, washed three times with
serum-free α-MEM and treated with or without 100 nM PMA for 20
minutes at 37°C followed by 0.5 mg/ml Dextran Texas Red for 5
minutes or 0.5 µM Lyso Tracker for 10 minutes. Cells were then
washed twice with PBS and fixed with 2% paraformaldehyde in PBS
for 15 minutes at room temperature. The coverslips were mounted on
glass slides in 50% glycerol in PBS. Confocal imaging was performed
using a Zeiss axiovert microscope and a Bio-Rad confocal scanning
imaging system.
Phorbol ester promotes endocytosis 2551
RESULTS
Phorbol ester stimulates fluid phase endocytosis
and overrides the inhibitory effect of Rab5:S34N
Incubation of cells with PMA (100 nM) for 20 minutes
stimulated horseradish peroxidase (HRP) endocytosis by nearly
2-fold. Moreover, the extend of stimulation reached was similar
to that observed in cells overexpressing Rab5:WT (Fig. 1A). To
better characterize the stimulatory effect of PMA on
endocytosis, the cells were preincubated in the presence of
increasing concentrations of PMA or PA (a less active PMA
analog). Fig. 1B shows that the simulatory effect of PMA
plateaus at 200 nM and that the less active analog, PA was nearly
inactive in stimulating endocytosis at the same concentrations,
indicating that the stimulatory effect of PMA is neither due to
the lipidic nature of the compound nor to the solvent.
Previous observations indicate that PMA can overcome the
inhibitory effect of Rab5:S34N in endosome fusion, suggesting
that the phorbol ester is acting downstream of Rab5 (Aballay
et al., 1998). Confluent BHK-21 cells monolayers were
infected with recombinant Sindbis virus expressing
Rab5:S34N. At 4 hours postinfection, the cells were treated
with 100 nM PMA or PA. HRP uptake was then assayed. HRP
uptake recorded following overexpression of Rab5:S34N was
around 50% of that observed in the vector infected control cells
(Fig. 1C). Rab5:S34N mediated inhibition of endocytosis was
reversed by PMA but not by phorbol 13-monoacetate. These
results suggest that activation of the phorbol ester-binding
protein acting downstream Rab5 causes a dramatic effect on
endocytosis. The protein kinase C family (PKC) are among the
best characterized proteins that bind phorbol esters; however,
a kinase activity is probably not involved in the effect of PMA
on endosome fusion (Aballay et al., 1997). To examine whether
the effect of PMA was mediated by activation of a PKC, we
performed additional experiments using specific inhibitors of
PKC activity: Staurosporine, chelerythrine, and Ro 31-8220.
As shown in Fig. 1D, pretreatment with the inhibitors had no
effect on the PMA-stimulatory effect on endocytosis. Similar
results were obtained by Seastone et al. (1998) in phagocytosis
and suggest that a PKC-like kinase activity is neither required
by phagocytosis nor by endocytosis.
Morphological alteration of early endosomes
revealed by electron and confocal microscopy
We next examined the effect of PMA on the morphology of
endocytic organelles. To visualize the endocytic structures,
cells were allowed to internalized 20 nm gold for 1 hour. PMA
causes a significative increase in the number and size of
endosomes in BHK cells. Using the Sindbis virus, we observed
that the overexpression of Rab5:S34N reduces the size and the
number of endocytic vesicles (Table 1). Fig. 2 shows that PMA
is capable of inducing the formation of large endocytic
structures in cells overexpressing Rab5:S34N. The large
endocytic vesicles were also observed when cells were allowed
to internalize 20 nm gold for 5 minutes (Fig. 2B, insert),
indicating that the structures stimulated by PMA correspond to
early endosomes. The quantification of three independent
experiments (Table 1) suggests that PMA was not only capable
HRP uptake (%)
HRP uptake (%)
HRP uptake (%)
HRP uptake (%)
A
Fig. 1. Effect of PMA on fluid phase endocytosis.
B
(A) Confluent BHK-21 cells monolayers were mock
100
infected (Control) or infected with recombinant
150
Sindbis viruses encoding Rab5:WT (Rab5). At 4
75
hours postinfection, control and infected cells were
PMA
incubated for 30 minutes in medium containing 100
100
PA
nM PMA (PMA). Horseradish peroxidase (HRP)
50
uptake (60 minutes) was determined as described in
Materials and Methods. Values are expressed as a
50
25
percentage of control HRP uptake. The small bars
represent the calculated standard deviations of five
independent experiments. (B) HRP uptake was
0
0
0
25
50
100
200
determined in BHK-21 cells in the presence of
Control
Rab5
PMA
[Phorbol esters] (nM)
increasing concentration of PMA and the less active
analogue PA. To better visualized the stimulatory
effect of PMA observed in panel A, the uptake
under control conditions was subtracted. Data are
C
D
expressed as a percentage of the maximal value
observed. The data are representative of two
150
150
independent experiments. (C) Confluent BHK-21
cells monolayers were infected with Sindbis viruses
alone (Vector) or infected with recombinant Sindbis
100
100
viruses encoding Rab5:S34N (Rab5:S34N). At 4
hours postinfection, the cells were incubated for 30
50
50
minutes in the presence or absence of 100 nM PMA.
Values are expressed as a percentage of the HRP
uptake in the presence of the vector. The small bars
0
0
represent the calculated standard deviations of five
PMA
+
+
+
+
Vector Vector Rab5:S34N Rab5:S34N
Staurosporine
+
independent experiments. (D) HRP uptake was
+
+
Chelerythrine
+
PMA
PMA
Ro 31-8220
determined in BHK-21 cells in the presence of 100
+
nM PMA alone or 100 nM PMA plus 10 µM
staurosporine, 10 µM chelerythrine, or 10 µM Ro 31-8220. Values are expressed as a percentage of control HRP uptake. The small bars
represent the standard deviations of five independent experiments.
2552 A. Aballay, P. D. Stahl and L. S. Mayorga
Fig. 2. PMA expands early endocytic
compartment in BHK cells
overexpressing Rab5:S34N. BHK-21
cells were infected with recombinant
Sindbis viruses encoding Rab5:S34N.
At 4 hours postinfection cells were
incubated for 30 minutes in the
presence (B) or absence (A) of 100
nM PMA. Subsequently, cells were
allowed to internalize 20 nm gold for 1
hour or 5 minutes (B insert). Cells
were then fixed and processed for
electron microscopy. Bar, 0.5 µm.
of increasing the size of endocytic structures but also their
number. Table 1 also shows that the inhibitor of the PKC
catalytic activity, chelerythrine, has no effect on the PMA
stimulated structures. The morphological data are consistent
with the increase of HRP uptake observed biochemically.
Inhibitors of the PMA-binding domains block Rab5
stimulated endocytosis
The results obtained indicate that PMA alters endocytosis by
targetting a factor involved in endosome fusion. According to
our previous results, this factor acts after Rab5 activation. A
Phorbol ester promotes endocytosis 2553
Table 1. Quantification of endocytic vesicles in cells treated
with PMA
<100 nm
>100 nm
Vector
PMA
Rab5:S34N
Rab5:S34N + PMA
PMA + Chelerythrine
2.56±0.9
4.58±0.6
0.35±0.2
4.04±0.3
4.10±0.5
1.20±0.9
4.80±0.8
0.10±0.1
4.66±0.7
4.00±0.4
BHK-21 cells were infected with either Sindbis virus alone or Sindbis virus
encoding Rab5:S34N. The cells were incubated for 30 minutes in the
presence or absence of 100 nM PMA or in the presence of 100 nM PMA plus
10 µM chelerythrine. The cells then were allowed to internalize 20 nm gold
for 1 hour, fixed, and processed for electron microscopy. The number of
vesicles containing gold in 20 cells per experimental condition was counted.
The number of vesicles per 10 µm2 was classified according to their size. The
data represent the means ± s.d. of three independent experiments.
Electron microscopic analysis of the inhibition of
endocytosis: effects of TPEN and CPC
To confirm the effects of TPEN and CPC at the morphological
level, the endosomal compartment was examined in cells
TPEN
150
100
50
0
Vector
200
Rab5
Rab5:Q79L
B
no additions
HRP uptake (%)
prediction would be that inhibition of this factor would block
endocytosis even in cells expressing active Rab5. To test this
hypothesis, we examined the effect of reagents that inhibits
PMA-binding proteins on cells overexpressing Rab5:WT and
Rab5:Q79L. The phorbol ester-binding motifs have been well
characterized and it has been shown that they contain a
cysteine-rich region that coordinates zinc (Quest et al., 1994)
and that point mutations completely abolish both the zinc and
the phorbol ester binding activities (Kazanietz et al., 1995). We
have previously shown that the zinc chelator N,N,N′,N′tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) blocks the
PMA effect on endosome fusion, possibly by altering the active
conformation of PMA-binding proteins (Aballay et al., 1997).
We examined the effect of the zinc chelator TPEN and
calphostin C (CPC), a membrane permeant reagent that
inhibits PKC by irreversibly oxidizing the phorbol ester
binding site in a light-dependent manner (Bruns et al., 1991).
Confluent BHK-21 cell monolayers were infected with
recombinant Sindbis virus encoding wild-type Rab5 or the
GTPase deficient mutant Rab5:Q79L. At 4 hours postinfection,
the cells were treated with 50 µM TPEN or 2 µM CPC and the
HRP uptake was assayed. As expected, overexpression of
Rab5:WT and Rab5:Q79L led to an increase in the
internalization rate of the endocytic marker (Fig. 3A and B).
TPEN treatment inhibited HRP uptake stimulated by the
overexpression of both Rab5:WT and Rab5:Q79L. Basal
endocytosis, observed in the presence of vector alone, was also
inhibited (Fig. 3A). Rab5-stimulated endocytosis was also
sensitive to CPC treatment. As CPC is activated by light, we
challenged the cells in darkness to test the specificity of the
inhibitor. Fig. 3B shows that when cells were incubated with
CPC in the dark, no inhibition was observed. When exposed
to light, CPC treatment inhibited basal endocytosis observed
in the presence of the vector, and both wild-type Rab5:WT- and
Rab5:Q79L-stimulated HRP uptake (Fig. 3B). Since
Rab5:Q79L is constitutively activated and will bypass the
activation steps, the inhibition of endocytosis in cells
overexpressing the Rab5 mutant suggests that a PMA-binding
protein is acting after the activation of Rab5.
A
no additions
HRP uptake (%)
Size of vesicles
200
+CPC
150
+CPC (dark)
100
50
0
Vector
Rab5
Rab5:Q79L
Fig. 3. Inhibition of Rab5-stimulated HRP uptake by TPEN and
CPC. Confluent BHK-21 cells monolayers were infected with
Sindbis viruses alone or with recombinant Sindbis viruses encoding
either Rab5:WT or Rab5:Q79L. At 4 hours postinfection cells were
incubated for 30 minutes in the presence or absence of the following
reagents, (A) 50 µM TPEN and (B) 2 µm CPC (light and dark).
Subsequently, cells were allowed to internalize HRP for 1 hour.
Values are expressed as a percentage of control HRP uptake. The
small bars represent the calculated standard deviations of three
independent experiments.
overexpressing Rab5:WT and Rab5:Q79L. Overexpression of
Rab5:WT and Rab5:Q79L produces large endocytic vesicles
(Fig. 4A and D). When cells expressing either Rab5:WT or
Rab5:Q79L were treated with 50 µM TPEN (Fig. 4B and E)
or 2 µM CPC (Fig. 4C and F) for 20 minutes before allowing
the cells to internalize 20 nm gold, the large endosomes loaded
with gold disappeared. In the quantified experiments shown in
Table 2, cells expressing Rab5:WT or Rab5:Q79L contain
more vesicles, which are also larger compared with control
cells. This positive regulation by Rab5:WT and Rab5:Q79L
was abolished by the treatment with TPEN or CPC.
Rab5 presence in PMA-induced large endosomes
To further characterize the large vesicles induced by PMA,
we generated CHO cells lines stably expressing GFPRab5:WT (sCHO-GFP-Rab5:WT) and GFP-Rab5:S34N
(sCHO-GFP-Rab5:S34N). When sCHO-GFP-Rab5:WT cells
were treated with PMA, the number and the size of Rab5
positive endocytic structures were increased (Fig. 5D and J).
The sCHO-GFP-Rab5:WT cells were also allowed to
2554 A. Aballay, P. D. Stahl and L. S. Mayorga
Fig. 4. CPC and TPEN inhibited the endocytic vesicles expanded by Rab5. BHK-21 cells were infected with recombinant Sindbis viruses
encoding Rab5:WT (A,B,C) or Rab5:Q79L (D,E,F). At 4 hours postinfection cells were incubated 30 minutes without any additions (A and D)
or in the presence of 50 µM of TPEN (B and E) or 2 µM of CPC (C and F). Subsequently, cells were allowed to internalize 20 nm gold for 1
hour. Cells were then fixed and processed for electron microscopy. Bar, 0.5 µm.
Table 2. Quantification of endocytic vesicles in cells
expressing Rab5:WT and Rab5:Q79L: effects of TPEN
or CPC
Size of vesicles
<100 nm
>100 nm
Vector
Rab5:WT
Rab5:WT + TPEN
Rab5:WT + CPC
Rab5:Q79L
Rab5:Q79L + TPEN
Rab5:Q79L + CPC
2.56±0.9
7.50±0.1
0.70±0.3
0.39±0.1
5.30±0.2
0.80±0.3
0.70±0.1
1.20±0.9
6.30±0.6
1.20±0.5
0.50±0.3
3.50±0.1
0.90±0.4
0.10±0.1
BHK-21 cells were infected with either Sindbis viruses alone or Sindbis
viruses encoding either Rab5:WT or Rab5:Q79L. The cells were incubated
for 30 minutes in the presence or absence of 50 µM TPEN or 2 µM CPC. The
cells were then allowed to internalize 20 nm gold for 1 hour, fixed, and
processed for electron microscopy. The number of vesicles containing gold in
20 cells per experimental condition was counted. The number of vesicles per
10 µm2 was classified according to their size. The data represent the means ±
s.d. of three independent experiments.
internalize Dextran Texas Red for 5 minutes (Fig. 5B and E).
Merged images shown in Fig. 5F indicate that the endosomal
marker used is localized inside the GFP-Rab5:WT
endosomes induced by PMA. To study the effect of PMA on
late endocytic structures, the cells were incubated in the
presence of Lyso Tracker, a lysosomal membrane permeant
marker. Merged images shown in Fig. 5L indicate that the
enlarged structures promoted by PMA do not correspond to
late endocytic structures, suggesting that the PMA effect is
restricted to the endosomal compartment.
Western blot analysis shows that Rab5:S34N appears as both
cytosolic and membrane associated and PMA has no effect on
its distribution (data not shown). In sCHO-GFP-Rab5:S34N
cells, PMA has no effect on the Rab5 negative mutant
distribution either (Fig. 6A and D). However, PMA was
capable of inducing large structures that were loaded with
Dextran-Texas Red (Fig. 6E and F arrows). The endosomes
induced by PMA do not contain Rab5:S34N. These results
Phorbol ester promotes endocytosis 2555
DISCUSSION
Fig. 5. PMA induced early endocytic vesicles are GFP-Rab5:WT
positive. CHO cells stably expressing GFP-Rab5:WT were incubated
in the presence (D,E,F,J,K,L) or absence (A,B,C,G,H,I) of 100 nM
PMA for 1 hour. The cells were allowed to internalize 0.5 mg/ml
Dextran Texas Red for 5 minutes (B and E) and they were also
marked for 10 minutes with 0.5 µm Lyso Tracker (H and K).
(C,F,I,L) Merged images. The cells were fixed and processed for
confocal microscopy. Bar, 10 µm.
suggest that the PMA induced structures correspond to early
endosomes and that PMA overcomes the inhibitory effect of
Rab5:S34N, perhaps by increasing the size and the number of
vesicles containing endogenous Rab5.
Fig. 6. PMA induced early endocytic vesicles are GFPRab5:S34N negative. CHO cells stably expressing
GFP-Rab5:S34N were incubated in the presence
(D,E,F) or absence (A,B,C) of 100 nM PMA for 1 hour.
The cells were allowed to internalize 0.5 mg/ml
Dextran Texas Red for 5 minutes (B and E).
(C and F) Merged images. The cells were fixed and
processed for confocal microscopy. Arrows indicate the
large structures loaded with Dextran-Texas Red that
were induced by PMA. Bar, 10 µm.
In this study we have shown that PMA stimulates fluid phase
endocytosis and causes the formation of large early endosomes
in BHK-21 cells. Similar alterations of the endocytic pathway
have been observed by overexpression of a GTPase defficient
mutant of Rab5 that specifically activates the endosome fusion
process (Bucci et al., 1992). Using a cell free system, in a
previous report, we have indicated that a phorbol ester binding
protein is required for endosome fusion after Rab5 activation
(Aballay et al., 1998). Hence, in intact cells, PMA may activate
the factor that promotes endosome fusion and mimic the effect
of Rab5 in endocytosis. Similar to the results obtained using
the in vitro endosome fusion assay, a PKC-like activity is not
required for the PMA effect on endocytosis as shown by the
lack of effect of staurosporine, chelerythrine, and Ro 31-8220.
To assess whether the PMA effect was related to Rab5
function, the Sindbis virus system was used to alter the
endocytosis in BHK-21 cells by overexpressing Rab5:WT,
Rab5:S34N, and Rab5:Q79L. The phorbol ester PMA
stimulates endocytosis in a concentration dependant manner
and to the same extent as Rab5. The biochemical and
morphological data show that PMA activation was able to
overcome the inhibitory effect of the dominant negative mutant
Rab5:S34N. In addition, inhibitors of the PMA-binding
domains block basal endocytosis and both Rab5:WT- and
Rab5:Q79L-stimulated endocytosis. Since Rab5 is a rate
limiting regulator of early endosome fusion, this coordinate
regulation of endocytosis by Rab5 and PMA is consistent with
the hypothesis that PMA promotes endocytosis by activating a
factor that is required downstream Rab5 on endosome fusion.
Moreover, under the conditions used, inhibitors and activators
of phorbol ester binding factors affect mostly early endosomes.
Thus, the inhibitors of the phorbol ester binding domains block
the expansion of the endosomal compartment caused by Rab5
and the large vesicles formed in the presence of PMA were
loaded with early endocytic fluid phase markers such as
colloidal gold particles and dextran. More acidic vesicles
marked by Lyso Tracker were not affected.
The nature of the phorbol ester binding factor is presently
unknown. Several proteins that contain phorbol ester-binding
2556 A. Aballay, P. D. Stahl and L. S. Mayorga
domains interact with small GTPases. N-Chimaerin is a GAP
for Rac, a small GTPase that regulates actin organization
(Ahmed et al., 1993). In yeast, Rho1 in its active form binds
and regulates the kinase activity of PKC1 (Kamada et al.,
1996). Recently, it has been shown that RacC stimulates
phagocytosis in Dictyostelium and that its effect could be
blocked by preincubating the cells with Calphostin C (Seastone
et al., 1998), suggesting that a phorbol ester-binding protein
acting downstream RacC is necessary for phagocytosis in
Dictyostelium. Consistent with our results, this group has also
shown that PMA activates a phorbol ester-binding protein,
perhaps lacking PKC activity (Seastone et al., 1998).
Moreover, it has been shown that Rho and Rab family members
coordinately regulate the phorbol ester-induced reorganization
of stress fibers and local adhesions (Imamura et al., 1998).
Recently, two exchange factors, containing PMA-binding
domains, for Ras and Rap1 have been cloned (Ebinu et al.,
1998; Kawasaki et al., 1998). However, none of the proteins
that interact with Rab5 are known to possess phorbol ester
binding region although some of them contain zinc-binding
domains. The Rab5 homologue Vps21p interacts with the zincbinding protein Vps8p (Horazdovsky et al., 1996). EEA1 is a
hydrophilic protein present in cytosol and early endosome
membrane fractions. At its C terminus, the protein contains a
cysteine-rich motif that is crucial for its colocalization with the
GTPase deficient mutant of Rab5 (Stenmark et al., 1996). In
addition, it has been shown that EEA1 directly interacts with
both the PI(3)K product phosphatidylinositol-3-phosphate and
Rab5, linking PI(3)K function to Rab5 regulation of endosome
fusion (Simonsen et al., 1998). The same group has also shown
that EEA1 is a crucial downstream Rab5 effector (Cristoforidis
et al., 1999). It is unlikely that EEA1 would interact with
phorbol esters. The known phorbol-ester binding domains are
highly homologous. A sequence alignment between the rat
PKCα zinc- and phorbol ester- binding domains and both the
FYVE finger (Mackay and Crossley, 1998) and the C2H2 zincbinding domains from EEA1 does not show significant
similarity (Blast 2.0.6). We have presented evidences that the
PMA-binding factor is sensitive to zinc depletion (Aballay et
al., 1997). However, we cannot rule out the possibility that
TPEN could be targeting EEA1. It would be interesting to
assess whether EEA1-dependent fusion, as described by
Cristoforidis et al., is sensitive to zinc chelators. Stenmark et
al. (1995) have shown that Rab5 in its GTP-bound form binds
Rabaptin-5. This protein inhibits Rab5-GAP activity of
membranes and may regulate the GTP-GDP cycle of Rab5
(Rybin et al., 1996). Recently, Horiuchi et al. (1997) have
described a novel Rab5 GDP/GTP exchange factor and Xiao
et al. (1997) have shown that the tuberous sclerosis 2 gene
product, tuberin, functions as a Rab5 GAP. None of these
proteins contains a zinc binding domain and presumably do not
bind phorbol esters. Thus, the phorbol ester-binding factor may
be acting downstream of these proteins or may interact with
Rab5 in conjunction with them.
The best characterized proteins that bind phorbol esters
belong to the PKC family. PKC and PKC-like molecules have
been conspicuously implicated in both the exocytic and
endocytic pathways. As mentioned before, PMA is believed to
regulate the movement of a number of cell surface receptors
(Cardone et al., 1994; Signoret et al., 1997). Moreover, two
laboratories have directly implicated phorbol ester binding
factors in the exocytic pathway, using in vitro assays, and they
have also reported that PKC-like kinase activity is not
necessary for the factor(s) effect (Fabri et al., 1994; Simon et
al., 1996a,b). In addition, Alonso et al. (1998) have shown that
calphostin C induces disassembly of the Golgi complex by a
mechanism that does not require PKC-like activity. Consistent
with the results observed in the exocytic pathway, a kinase
activity is probably not directly involved in the effect of the
phorbol ester-binding factor acting downstream of Rab5, as
inferred from the lack of effect of several inhibitors of the
catalytic activity of PKC. Similar results have been obtained in
experiments on phagocytosis in Dictyostelium (Seastone et al.,
1998). Undoubtedly phorbol ester will affect a large set of
factors in intact cells; however, the effects in endocytosis are
probably related to the activation of a factor acting in
endosome fusion.
The fact that several intracellular transport steps involve
PMA-regulated factors raises the possibility that a family of
these proteins will be necessary for transport. The treatment of
cells with PMA could alter several steps of intracellular
trafficking. However, we have shown that a protein acting
downstream Rab5 at the endosome fusion level (Aballay et al.,
1998) is being activated by the phorbol ester and that the PMA
effect on endocytosis is restricted to the endosomal
compartment. Further investigation is necessary to identify the
PMA-binding protein regulating the endocytosis.
We thank Alejandra Challa for excellent technical assistance with
the electron microscopy. This work was partly supported by an
International Research Scholar of the Howard Hughes Medical
Institute, and by grants of the National Science FoundationCONICET International Program, CONICET, CIUNC, Fundacion
Antorchas, and PDS grants NIH. A.A. is supported by Pew and
CONICET fellowships.
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