Department of Molecular Physiology and Biological Physics, University of Virginia Medical School, Charlottesville, VA 22908, USA
* Author for correspondence (e-mail: ado2t{at}virginia.edu)
Accepted 23 April 2003
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Summary |
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Key words: NDP kinase, NM23, Microtubules, Rac
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Introduction |
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At the molecular level, the finding that NM23-H1 (human NDP kinase A)
interacts with Tiam1, a Rac guanine nucleotide exchange factor, and
downregulates Tiam1-Rac1 signaling, implied that it could affect remodeling of
the actin cytoskeleton (Otsuki et al.,
2001). More recently, Palacios et al. demonstrated that
constitutively activated ARF6 binds NM23-H1 and recruits NM23-H1 to cell
junctions (Palacios et al.,
2002
). The presence of NM23-H1 at these sites facilitates
dynamin-dependent endocytosis and downregulates Rac1 activity. Additionally,
NM23-H2 (human NDP kinase B) was found to be linked to ß-integrins
through integrin cytoplasmic domain associated protein 1-
(ICAP1-
) (Fournier et al.,
2002
), which inhibits activation of Rac1 and Cdc42 GTPases during
integrin-mediated cell adhesion (Degani et
al., 2002
). Taken together, these reports hint at the multiple
ways by which NDP kinases A and B could affect cell adhesion, signaling and
motility. One important question is whether activation of surface receptors
that trigger signal transduction pathways can modulate the subcellular
distribution of the essentially cytosolic NDP kinases A and B in a manner that
is spatially and temporally consistent with a role in signaling. In the
present study, we examined the ability of NDP kinases A and B to respond to
activation of receptor tyrosine kinases and G-protein-coupled receptors
(GPCRs) by monitoring their spatial distribution in NIH-3T3 fibroblasts. These
experiments allowed us to identify two distinct pools of NDP kinase in these
cells: one population is rapidly translocated to the cell periphery when
receptors are activated, whereas a second pool binds constitutively to
microtubule-associated vesicles. Vesicular NDP kinase is released by
nucleotides, with GTP being more efficient than ATP; this suggests that NDP
kinase might associate with intracellular vesicles when GTP levels are low, in
order to provide the substrate used by the many GTPases that control
intracellular trafficking.
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Materials and Methods |
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The monoclonal antibody specific for NDP kinase A (NM301) was from Santa
Cruz Biotechnology; this antibody does not recognize NDP kinase B.
Immunofluorescence staining of NDP kinase was also performed with a polyclonal
antibody, Ab-1 (Labvision, Freemont, CA), raised to a homologous inner region
of human NDP kinases A and B (amino acids 86-102). This antibody reacts with
two proteins with the relative mobility of NDP kinase A and B in immunoblots
of lysates from NIH-3T3 cells; no cross-reactivity with other proteins was
detected (not shown). Although Ab-1 yielded similar results to NM301, it often
stained cell nuclei, in agreement with the finding that NDP kinase B can
localize to the nucleus (Kraeft et al.,
1996; Pinon et al.,
1999
; Barraud et al.,
2002
). Like other rodent cells
(Kimura et al., 2000
;
Barraud et al., 2002
), NIH-3T3
fibroblasts express much higher levels of NDP kinase B than NDP kinase A (not
shown). Therefore, the signal obtained with Ab-1 is presumably dominated by
the B isoform. NDP kinase was detected in immunoblots with a rabbit polyclonal
antibody that recognizes mammalian NDP kinases A and B (a generous gift from
I. Lascu, Université de Bordeaux). Other antibodies used were:
polyclonal anti-Rab4 (StressGen), polyclonal anti-Tiam1 (Santa Cruz),
monoclonal anti-Rac (clone 23A8, Upstate Biotechnology) monoclonal anti-HA
epitope tag (12CA5, Exalpha), monoclonals anti-LAMP-1, anti-kinesin heavy
chain and Na+,K+-ATPase (1D4B, SUK4 and
5,
respectively, from the Developmental Studies Hybridoma Bank, Iowa) and
monoclonal anti-
-tubulin (clone DM1A, Sigma-Aldrich). Texas
Red-transferrin and all secondary antibodies (Texas Red or Cy2 conjugates,
multiple labeling grade) were from Jackson Immunoresearch Laboratories.
Specificity of immunofluorescence labeling was established by pre-incubation
with excess antigen, which effectively blocked cell staining. The secondary
antibodies did not stain cells in the absence of primary antibodies.
Cell culture and transfection
NIH-3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium
pyruvate, 100 U l1 penicillin and 100 µg
ml1 streptomycin at 37°C in a humidified 5%
CO2 atmosphere. Cells were subcultured at 50-70% confluence; assays
were performed on cultures derived from the same stock, between passages 2 and
8. Transfections were performed using LipofectAMINETM 2000 as recommended
by the manufacturer.
Immunofluorescence
Cells were grown on No. 1 thickness acid-washed glass coverslips. For
experiments, cells 30-40% confluent were placed in serum-free medium for 16-24
hours and then treated with 10 ng ml1 EGF, 10 nM bombesin or
10% serum for the times specified in the figure legends. Treatment with 30 mM
BDM was performed in serum-free medium supplemented with 0.5% bovine serum
albumin (BSA) and 10 mM glucose; cells were then stimulated with EGF for 2
minutes.
Cells were fixed for 10 minutes at room temperature in 4%
paraformaldehyde/0.1% glutaraldehyde. Free aldehyde groups were quenched with
NaBH4, and cells were then stained as described
(Pinon et al., 1999). Briefly,
after a wash with PBS, cells were transferred to a blocking solution composed
of 0.1% saponin and 3% BSA in PBS. This same buffer was used to dilute primary
and secondary antibodies. Coverslips were incubated overnight with primary
antibodies at 4°C, washed extensively with PBS and incubated for 45
minutes at room temperature with secondary antibodies. After washes with PBS,
coverslips were mounted in Mowiol (Calbiochem) containing 2%
n-propylgallate. To stain F-actin, rhodamine-conjugated phalloidin
(0.05 ng ml1) was added to the secondary antibody solution.
The coverslips were examined on a Nikon Diaphot microscope equipped with a
40x oil immersion objective. Digital images were obtained with a Nikon
CoolPix 990 camera. The phase-contrast images of the immunolabeled cells were
routinely collected to confirm the identification of specific cell features.
Figures were assembled using Adobe Photoshop software. Results shown are
representative of 4-25 independent experiments.
For fluorescence ratio imaging, NIH-3T3 cells transfected with GFP were fixed and immunostained with NM301 followed by Texas-Red-conjugated anti-mouse IgG. Paired images of GFP and Texas Red fluorescence were acquired under conditions designed to avoid pixel saturation, inspected to verify alignment and saved as TIFF (8-bit) files. Ratio images (NDP kinase A/GFP) were obtained with Scion Image (Scion). Line profiles obtained with Maxim DL 2.12 (Diffraction Limited) were used for quantitative analysis of fluorescence values in digitized images.
Measurements of the diameters of NDP-kinase-labeled vesicles were performed in images of cells immunolabeled with Ab-1 or NM301. Histograms were constructed using a bin size of 0.5 µm.
Subcellular fractionation
Membrane preparation
Isolation of the particulate fraction from quiescent and serum stimulated
cells was performed by the method of Del Pozo et al.
(Del Pozo et al., 2002) with
slight modifications. Briefly, serum-deprived cells in 10 cm dishes were
treated with medium containing 0% or 10% serum for 10 minutes, rinsed with PBS
and incubated in ice-cold lysis buffer (10 mM Tris, pH 7.4 with HCl, 1.5 mM
MgCl2, 5 mM KCl, 1 mM DTT, 0.2 mM sodium vanadate, 1 mM PMSF, 1
µg ml1 each aprotinin and leupeptin) for 5 minutes. Cells
were scraped, homogenized by 15 passes in a Dounce homogenizer and the lysates
were centrifuged at 700 g for 3 minutes. The supernatants were
spun for 15 minutes at 167,000 g in a Beckman Airfuge; the
cytosolic fraction was removed, the membrane pellet was washed once with lysis
buffer. Samples containing equal amounts of protein were solubilized in
SDS-PAGE sample buffer with 20 mM DTT, alkylated with 60 mM iodoacetamide and
resolved in 15% or 4-20% minigels. Proteins were transferred to nitrocellulose
and immunoblotted with antibodies to Rac and NM23 as described previously
(Otero, 1997
). Immunoblotting
with the plasma membrane marker Na+,K+-ATPase was used
to verify equal loading of control and serum-treated samples. Results are
expressed as the proportion of the protein detected in control membrane
fractions. Proteins were detected by chemiluminescence and quantified using
Scion Image.
Isolation of microtubule-associated endocytic vesicles and
proteins
Endocytic vesicles associated with endogenous microtubules were isolated
essentially as described by Wolkoff and colleagues
(Goltz et al., 1992;
Oda et al., 1995
). Briefly,
80% confluent cells in 15 cm plates were washed with PBS, scraped into 0.6 ml
MEPS buffer (35 mM PIPES pH 7.1 with NaOH, 5 mM MgSO4, 5 mM EGTA,
200 mM sucrose, 1 mM DTT) containing protease inhibitors (2 mM PMSF, 1 mM
benzamidine, 2 µg ml1 leupeptin) and homogenized by 10-14
passes through a 27G needle. Homogenates were centrifuged at 1000
g for 10 minutes to sediment nuclei and large debris. The
post-nuclear supernatant was centrifuged at 40,000 g for 20
minutes. The supernatant (small vesicles and cytosol) was incubated at
37°C for 30 minutes with 20 µM Taxol (and nucleotides when indicated)
to polymerize tubulin. The resulting microtubules and associated vesicles were
pelleted at 16,000 g for 30 minutes at 4°C. The pellets
were brought to the original volume with MEPS, taxol was added and the samples
were centrifuged at 16,000 g. After a second wash, pellets
were solubilized in SDS-PAGE sample buffer and analysed as described above.
Results shown are representative of 3-12 separate experiments.
To determine whether NDP kinase is initially associated with vesicles or soluble tubulin, the supernatant of the 40,000 g step was centrifuged at 230,000 g for 1 hour in a TLA 100.3 rotor (Beckman Coulter) to sediment membrane vesicles. The supernatant was incubated with taxol as described above to polymerize soluble tubulin, and centrifuged at 16,000 g. Both pellets were washed twice with MEPS and analyzed by immunoblotting.
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Results |
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In cells treated with EGF, NDP kinases are present not only in the cytosol and in vesicular structures but also at well-defined areas of the cell cortex. Fig. 1c shows several features of NDP kinase localization in cells exposed to EGF for 2 minutes; similar results were obtained with 5 nM PDGF and with 10% fetal bovine serum (not shown). In stimulated cells, NDP kinase staining is apparent in the submembranous space of cell protrusions such as ruffling lamellipodia; the large, phase-bright vesicles labeled by NDP kinase in quiescent cells are still prominent after stimulation with EGF (Fig. 1c,d). Translocation of NDP kinase to ruffles is a rapid and transient event, being evident within 1 minute of exposure to growth factors (Fig. 2) and becoming less pronounced after 30 minutes of continued stimulation (data not shown).
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Areas heavily stained for NDP kinase were not always phase dark; by the
same token, NDP kinase did not accumulate in all phase-dense regions
(Fig. 1c,d, arrowheads),
suggesting that volume effects are not the main reason for its enrichment in
specific areas. Nevertheless, given that NDP kinases A and B are abundant
proteins, we tested the possibility that the increased fluorescence signal
observed in ruffles and around vesicles reflected the increased thickness in
these locations and not actual accumulation of the antigen. To measure the
enrichment of NDP kinase around vesicles and in ruffles, we examined its
distribution in cells transfected with GFP, a soluble protein that behaves as
a volume marker for the cytosol (Kaksonen
et al., 2000) and analysed the data by fluorescence ratio imaging.
Fig. 2 shows a GFP-expressing
cell stained with the antibody specific for NDP kinase A after a short (1
minute) exposure to EGF. Although the signal for both GFP and NDP kinase A is
strong in the cell body, the two patterns are mostly distinct. NDP kinase A
(Fig. 2a) is concentrated at
ruffling lamellipodia and forms a ring around one of several perinuclear
vesicles seen in the phase-contrast image
(Fig. 2c). By contrast, GFP is
not present in ruffles and is excluded from, but does not accumulate around,
vesicles (Fig. 2b). The ratio
image (Fig. 2d) illustrates the
high ratios of NDP kinase A to GFP in the ruffling edge (15:1 20:1)
and at the rim of the vesicle seen in Fig.
2a (2:1 3:1), and reveals a feature that is not obvious
from the fluorescent image (Fig.
2a), namely that other vesicles are faintly positive for NDP
kinase A. This analysis is consistent with a genuine enrichment of NDP kinase
around vesicles as well as in ruffling membranes.
Comparison of Figs 1 and 2 shows that the monoclonal antibody specific to NDP kinase A and the polyclonal antibody that recognizes both NDP kinase A and B yield similar results, suggesting that these two isoforms coexist within the same areas of a cell. Therefore, in the following sections we refer to NDP kinase A and B jointly as NDP kinase.
NDP kinase is translocated to ruffles in response to bombesin, a GPCR
agonist
To determine whether the intracellular distribution of NDP kinase could be
changed by stimulation of membrane receptors other than receptor tyrosine
kinases (RTKs) such as the EGF and PDGF receptors, we also examined the
localization of NDP kinase in cells stimulated by bombesin, which acts through
a GPCR (Kjöller and Hall,
1999). Exposure of quiescent cells for 5-10 minutes to bombesin
induced membrane ruffles that stained brightly for NDP kinase
(Fig. 3a). Thus, stimulation of
GPCRs, as well as RTKs, triggers the translocation of NDP kinase to the cell
cortex.
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Membrane ruffling results from profound changes in the organization of the
actin cytoskeleton, with increased accumulation of F-actin at the cortex
(Ridley et al., 1992). To
determine whether the translocation of NDP kinase to ruffles is related to the
redistribution of F-actin or actin binding proteins, we labeled stimulated
cells with phalloidin and anti-NDP kinase.
Fig. 3b shows the distribution
of F-actin in a cell incubated with bombesin; similar results were obtained in
cells treated with EGF. There is some overlap between NDP kinase staining and
the intense F-actin signal at the edge of cells and at the tip of ruffles but
the NDP kinase signal in ruffles typically forms broad patches that extend
towards the center of the cell (Fig.
3a, arrowheads), well beyond the areas rich in F-actin. There is
little or no co-localization of the two proteins elsewhere, with NDP kinase
being absent from the stress fibers detected by phalloidin and the focal
adhesions outlined by an antibody to vinculin (not shown). Notably, the
vesicles labeled by NDP kinase antibodies
(Fig. 3a, arrows) do not stain
with phalloidin. In vitro studies with the purified proteins show no
interaction between actin and NDP kinase (not shown). Thus, it appears that
localization of NM23 to the cell periphery does not depend on a direct
interaction with F-actin. To verify this hypothesis, we used BDM, which
reduces ruffling (Rottner et al.,
1999
) but does not interfere with actin polymerization
(Cramer and Mitchison, 1995
).
When cells were treated with BDM prior to stimulation with EGF, NDP kinase
remained in the cell body (Fig.
4a), whereas significant amounts of F-actin were observable at the
cortex (Fig. 4b). BDM has no
effect on the enzymatic activity of purified erythrocyte NDP kinase (B. K.A.P.
and A.d.S.O., unpublished). Taken together, these data imply that NDP kinase
does not interact directly with F-actin and is translocated to the cell
periphery through a mechanism distinct from binding to microfilaments. Given
that BDM is a low-affinity myosin ATPase inhibitor, it is conceivable that a
member of the large myosin family is involved on NDP kinase translocation. We
are currently exploring this possibility.
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Rac controls the translocation of NDP kinase to ruffles in response
to activation of membrane receptors
Activation of Rac, a member of the Rho family of small GTPases that
regulates the formation of lamellipodia and membrane ruffles, is a shared step
in the response of fibroblasts to stimulation of different types of receptors
(Kjöller and Hall, 1999).
Our observations in cells responding to growth factors and bombesin suggest
that localization of NDP kinase to the cell periphery is linked to a common
event, possibly Rac activation. To test this hypothesis, we examined the
distribution of NDP kinase in cells expressing a constitutively activated form
of Rac1, RacG12V. Expression of RacG12V
(Fig. 5a) induced extension of
lamellipodia in unstimulated, serum-starved cells as expected
(Ridley et al., 1992
) and also
promoted the appearance of NDP kinase at the periphery
(Fig. 5b). By contrast, NIH-3T3
cells expressing the dominant negative Rac mutant RacT17N did not extend
lamellipodia in response to EGF (Fig.
5d) and showed the perinuclear staining for NDP kinase typical of
quiescent cells (Fig. 5e). The
numbers and appearance of NDP kinase-labeled intracellular vesicles were
similar in cells expressing Rac mutants and in neighboring untransfected cells
(Fig. 5b,e), indicating that
Rac controls only the recruitment of NDP kinase to the cortex.
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Tiam1 is not involved in NDP kinase translocation
Although NDP kinase and activated Rac co-localize in lamellipodia
(Fig. 5a,b, arrow), NDP kinase
does not bind directly to Rac (Otsuki et
al., 2001) (K.A.P. and A.d.S.O., unpublished). Therefore, its
connection to activated Rac is likely to be mediated by another cell
component. NDP kinase A associates with a Rac1-specific nucleotide exchange
factor, Tiam1 (Otsuki et al.,
2001
; Palacios et al.,
2002
). However, the idea of a Tiam1-mediated redistribution of NDP
kinase to the cortex as a response to growth factors and bombesin is not
supported by our results. Fig.
6 shows that there is limited overlap of the signals for NDP
kinase A and Tiam1 at ruffles following exposure to EGF for 2 minutes. In
particular, NDP kinase was visible throughout ruffling areas in cells, whereas
Tiam1 staining was confined to small areas of the cell periphery
(Fig. 6). This result is
consistent with previous work showing that Tiam1's ability to translocate to
membranes and to act as a GEF for Rac is markedly reduced in cells cultured in
low or no serum media (Michiels et al.,
1997
; Bourguignon, 2000). Thus, under our experimental conditions,
Tiam1 is not likely to be responsible for the translocation of NDP kinase to
the cell cortex.
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Cell stimulation promotes association of NDP kinase with
membranes
The cortical staining of stimulated cells with NDP kinase antibodies might
reflect an actual translocation from the cytosol to the plasma membrane, as
observed with activated Rac. To test this hypothesis, we compared the
distribution of Rac and NDP kinase by two independent approaches. First, we
performed a line scan analysis of images of cells treated with EGF and
fluorescently stained for endogenous Rac and NDP kinase.
Fig. 7A shows that the
fluorescence intensities for both NDP kinase and Rac are correlated only in
peripheral ruffles. The two proteins do not co-localize elsewhere in the
lamellipodium and, as seen also in Fig.
3b, the signal for NDP kinase, but not Rac, increases across a
large patch immediately behind the ruffling region. Neither protein localizes
to less active areas of the plasma membrane, indicating that the appearance of
cortical NDP kinase is associated with ruffling activity. To confirm this
result, we also compared the NDP kinase content of membrane fractions isolated
from quiescent and serum-stimulated cells. Immunoblotting with antibodies to
Rac and NDP kinase shows that the amounts of both proteins in the particulate
fraction are similarly increased (3.5 times) following serum stimulation,
whereas the plasma membrane marker Na+K+-ATPase remains
unchanged, as expected (Fig.
7B).
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Overall, these results demonstrate that, in NIH-3T3 cells, stimulation of surface receptors that lead to Rac activation triggers the translocation of a pool of NDP kinase to the cell periphery without affecting vesicular NDP kinase. The shift of NDP kinase towards the cell cortex is rapid, being evident within 1 minute of treatment with EGF, and results in extensive co-localization of NDP kinase and activated Rac at lamellipodia but does not involve a direct interaction with Rac, Tiam1 or F-actin.
Characterization of the vesicles labeled by NDP kinase
The results above show that the Rac-related events that lead to the dynamic
targeting of NDP kinase to ruffles differ considerably from the biochemical
processes responsible for its constitutive association with vesicles. To
define more clearly the mechanism by which NDP kinase assembles around
vesicles, we first characterized their morphology and protein composition.
Fig. 8A shows the size
distribution of NDP-kinase-labeled vesicles, which covers a range of 0.7-6.9
µm with a median diameter of 2.3 µm (n=209). There is no
statistically significant difference (P=0.19) between the size
distribution of NDP-kinase-labeled vesicles in resting and stimulated cells.
The large size and variable diameter of these vesicles, as well as lack of
overlap of NDP kinase staining with internalized transferrin (not shown),
indicate that they do not represent small, uniform sized clathrin-coated pits.
Although NDP-kinase-labeled vesicles have many characteristics of the
macropinosomes formed by the closing of ruffles (broad size distribution,
appearance in phase-contrast optics)
(Swanson and Watts, 1995),
they lack basic features of these structures. For instance, if NDP kinase were
associated with macropinosomes, one would expect to see an increase in the
number of labeled vesicles following Rac activation, which stimulates
macropinocytosis in murine fibroblasts
(Ridley et al., 1992
), yet we
observed no significant effects of Rac activation on vesicle-associated NDP
kinase. More importantly, the structures coated by NDP kinase are not
accessible to a fluorescent endocytic tracer (Texas Red dextran) even after an
18 hour chase (not shown), and therefore do not appear to be involved in fluid
phase uptake.
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The vesicles labeled by NDP kinase are also distinct from structures
containing Rab4 (data not shown), a marker of early endosomes
(Van der Sluijs et al., 1991).
However, double labeling of quiescent NIH-3T3 fibroblasts for NDP kinase and
the lysosome-associated membrane protein 1 (LAMP-1) sometimes shows a
significant overlap of the two signals in phase-bright vesicles. NDP kinase
accumulates around the cytoplasmic surface of the vacuoles, whereas LAMP-1
labels the lumen (Fig. 8Ba,b).
Because LAMP-1 is a membrane protein, its apparent localization in the lumen
of late endosomes is likely to be a consequence of smaller vesicles being
present in the lumen of the larger vacuoles. Indeed, occasionally, the
LAMP-1-positive vacuoles labeled by NDP kinase are very large and contain
clusters of smaller, round vesicles (Fig.
8Bc,d). The morphological features and strong labeling with
anti-LAMP-1 suggests that these structures are multivesicular bodies (MVBs),
which are part of the late endosomal compartment and participate in the
sorting of endocytosed proteins and lipids
(Felder et al., 1990
). However,
late endosomes and MVBs should accumulate internalized fluorescent dextran,
whereas the vesicles labeled by NDP kinase do not. Thus, the
NDP-kinase-positive vesicles, including those with multivesicular appearance,
belong to an atypical intracellular compartment that is not active in the
classical endocytic pathway.
NDP-kinase-labeled vesicles co-localize with microtubules
MVB-like organelles that are not involved in fluid phase uptake have been
identified in neurons through their association with a member of the kinesin
superfamily of microtubule motors, KIFC2
(Saito et al., 1997). Given
that NDP kinase co-localizes partially with the microtubular network in
epithelial cells (Pinon et al.,
1999
) and the centrosome of C6 glioma cells
(Roymans et al., 2001
), we
investigated the relationship between microtubules and the vesicles coated
with NDP kinase by double labeling with antibodies to
-tubulin and NDP
kinase (Fig. 9). Although
treatment with EGF induces pronounced accumulation of NDP kinase in ruffles,
microtubules are largely absent from ruffling areas (not shown). However, the
signals for the two proteins overlap distinctly around the vesicles labeled by
NDP kinase, indicating that the vesicles interact simultaneously with
microtubules and NDP kinase. The co-localization of tubulin and NDP kinase at
the periphery of vesicles is not a result of serum deprivation because it is
also observed in cells cultured in complete medium
(Fig. 9). Depolymerization of
microtubules with nocodazole eliminates association of tubulin and NDP kinase
with large intracellular vesicles (Fig.
9). Thus, clustering of NDP kinase at the periphery of vesicles
depends on an intact microtubular network.
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NDP kinase is associated with membranes that bind to microtubules in
vitro
Our observations are suggestive of an interaction between NDP kinase and
microtubule-bound vesicles. To determine whether the overlap in staining
reflects a physical association between NDP kinase and microtubule-associated
vesicles, we took advantage of a cell-free assay that reproduces the in vivo
association between endosomes and microtubules
(Goltz et al., 1992;
Oda et al., 1995
), thus
putting the morphological results to a biochemical test. A fraction containing
light membranes and cytosol was obtained from NIH-3T3 cells. Endogenous
tubulin was polymerized with taxol, and pelleted at g forces
sufficient to sediment microtubules and associated structures, but not
isolated membranes. The pellet (MT/Ves) was washed extensively and subjected
to immunoblot analysis. The MT/Ves fraction contains NDP kinase, tubulin, Rab4
and LAMP-1 (Fig. 10),
indicating that both early and late endosomal membranes associate with
microtubules under our conditions. Stimulation of cells with EGF does not
affect the amount of NDP kinase, tubulin or LAMP-1 in the MT/Ves fraction but
eliminates Rab4 from the pellet (Fig.
10). Translocation of Rab4 from endosomes to the cytosol in
response to stimulation was previously reported in adipocytes treated with
insulin (Cormont et al., 1993
)
and our data show that EGF has the same effect in fibroblasts. The lack of
effect of EGF on the levels of NDP kinase bound to the MT/Ves fraction is
consistent with our observations that the association of NDP kinase with
vesicles is not altered by extracellular stimulation
(Fig. 9) or by expression of
activated or dominant-negative Rac (Fig.
5).
|
NDP kinase co-immunoprecipitates from cell lysates with tubulin
(Lombardi et al., 1995;
Roymans et al., 2001
) and
co-localizes partially with microtubules in intact cells
(Pinon et al., 1999
), although
purified NDP kinase does not bind directly to microtubules
(Melki et al., 1992
). To
determine whether the NDP kinase pool bound to the MT/Ves pellet was
associated with tubulin prior to the formation of microtubules, the
postnuclear supernatant was centrifuged at 230,000 g to
sediment membranes. The supernatant (cytosol) was removed, incubated with
taxol to polymerize soluble tubulin and centrifuged at 16,000
g. When both pellets were washed and examined for their NDP
kinase content by immunoblotting, we found that NDP kinase is present in the
membrane pellet but not in the microtubule pellet (not shown). Thus, the NDP
kinase in the MT/Ves pellet derives from membranes, not the tubulin component.
This result demonstrates that the association of NDP kinase with the MT/Ves
fraction is specific and not due to trapping of this abundant cytosolic
protein in the microtubule pellet.
Nucleotides release NDP kinase from microtubule-bound endosomes
Formation of taxol-stabilized microtubules is usually performed in the
presence of GTP, even though (at appropriate concentrations) taxol alone is
sufficient to induce complete polymerization
(Oda et al., 1995). Indeed,
inclusion of 1 mM GTP in the incubation with Taxol does not affect the amount
of tubulin in the MT/Ves pellet; neither does it affect the association of
early and late endosomal membranes with the microtubule pellet, as indicated
by the unchanged levels of Rab4 and LAMP-1
(Fig. 11A). By contrast, GTP
induces a striking release of NDP kinase from the pellet; ATP also reduces the
association of NDP kinase with MT/Ves, but is noticeably and consistently
(n=6) less effective than GTP
(Fig. 11A). Thus, NDP kinase
is released from MT/Ves through a nucleotide-sensitive site that shows some
specificity for the base.
|
The nucleotide-induced release of NDP kinase from the MT/Ves fraction
qualitatively resembles the behavior of the molecular motors dynein and
kinesin, whose binding to microtubules and associated endosomal vesicles is
also responsive to nucleotides (Oda et
al., 1995). However, we find that the amounts of dynein and
kinesin associated with the MT/Ves fraction are not altered by 1 mM GTP or ATP
(not shown). Presumably the interaction of NDP kinase with MT/Ves is more
sensitive to nucleotides than that of molecular motors, so the nucleotide
concentration used in this work is too low to cause elution of significant
amounts of dynein and kinesin. For instance, 10 mM ATP releases only 50% of
bound dynein and kinesin from an analogous fraction obtained from rat liver
(Oda et al., 1995
), with GTP
being slightly less effective. Thus, the dissociation of NDP kinase from the
MT/Ves pellet by nucleotides is not linked to the release of motor proteins
and their cargo.
NDP kinases bind nucleotides with affinities in the range of 10-200 µM,
and guanine nucleotides are somewhat preferred over other substrates
(Schaertl et al., 1998;
Cervoni et al., 2001
;
Schneider et al., 2002
).
Therefore, the simplest explanation for the preferential release of NDP kinase
from the MT/Ves fraction by GTP is that it involves binding of the nucleotide
directly to its catalytic site. However, it is also possible that the release
of NDP kinase from the MT/Ves fraction is secondary to the interaction of GTP
with a highly selective binding site, perhaps a GTP-binding protein, and that
the effect of ATP is indirect, requiring its conversion into GTP. To
distinguish between these possibilities, we examined the effects of the
guanine nucleotide analogs guanosine-5'-O-(3-thio)triphosphate
(GTP
S), guanylyl-imidodiphosphate (GMP-PNP) and
guanosine-5'-O-(2-thio)diphosphate (GDPßS) on the
association of NDP kinase with the MT/Ves fraction. If release of NDP kinase
by GTP is related to a GTP-binding protein, the activating GTP analogs
GTP
S and GMP-PNP are expected not only to act alike but also to have
opposite effects to those of GDPßS, which locks GTP-binding proteins in
the inactive form. However, if the release is mediated by the catalytic site
of NDP kinase, the thiophosphate analogs GTP
S and GDPßS, which are
substrates for NDP kinases (Schaertl et
al., 1998
), should behave similarly to GTP. Imidodiphosphate
analogs bind to NDP kinases with low affinity and are not substrates
(Cervoni et al., 2001
), so
GMP-PNP should have modest effects, if any, on the retention of NDP kinase by
the MT/Ves fraction. As seen in Fig.
11B, 1 mM GTP
S and GDPßS, but not GMP-PNP, are as
effective as GTP in eluting NDP kinase, suggesting that the nucleotide binding
site involved is that of NDP kinase.
![]() |
Discussion |
---|
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---|
Although we used two different antibodies as tools, a monoclonal specific
to NDP kinase A and a polyclonal that recognizes both NDP kinase A and B both
highlight the same structures. This is not surprising, given that the bulk of
mammalian NDP kinases A and B are present in cells as stable mixed hexamers
(Gilles et al., 1991). This
finding is also consistent with a report that isoforms A and B are similarly
distributed in the cytoplasmic space under normal culture conditions
(Pinon et al., 1999
);
nevertheless, the possibility that the ratio of one isoform to another might
change substantially as a function of the compartment examined
(Barraud et al., 2002
) cannot
be overlooked. Another caveat is that the antibodies used here were tested
against NDP kinase A and B, which are by far the most abundant isoforms in
mammals (Lacombe et al.,
2000
). Given that some NDP kinase proteins are yet to be isolated,
it is possible (although unlikely) that our results reflect the localization
of cross-reacting NDP kinase isoforms that are expressed at low levels and
undetectable by immunoblotting but might be concentrated in specific cell
locations; a definite answer will have to await the purification of all NDP
kinase isoforms and development of high-affinity isoform-specific tools.
Translocation of molecules into ruffles is often achieved by binding to
proteins enriched in these structures, such as F-actin or activated Rac1.
However, NDP kinase does not bind to F-actin or Rac in vitro. NDP kinase also
does not carry consensus sequences found in Rac effectors, Rac GEFs or in Rac
GTPase activating proteins. Therefore, its connection to Rac activation is
probably indirect. Otsuki et al. reported that NDP kinase A associates with
the Rac1 nucleotide exchange factor Tiam1 and that overexpression of NDP
kinase A inhibits the activating effect of Tiam1 on Rac
(Otsuki et al., 2001).
However, our experiments show that, in serum-starved NIH-3T3 cells stimulated
briefly with EGF, ruffles that are strongly stained for NDP kinase contain
little Tiam1, suggesting that association with Tiam1 does not mediate
translocation of NDP kinase A upon Rac activation. Recently, Palacios et al.
demonstrated that, in MDCK epithelial cells, the small GTPase ARF6 mediates
the recruitment of heterologously expressed human NDP kinase A to areas of
cell-cell contact (Palacios et al.,
2002
). Once translocated to these sites, NDP kinase A would
facilitate dynamin-dependent endocytosis and downregulate Rac activation by
binding Tiam1. We are currently investigating whether an analogous interaction
with ARF6 can mediate the rapid translocation of endogenous NDP kinase to the
cortex of stimulated NIH-3T3 fibroblasts.
NDP kinase B was also found to be directed to ruffles, through interaction
with ICAP1- (Fournier et al.,
2002
). However, we use adherent cells and ICAP1-
reportedly
affects cell morphology and behavior only during cell adhesion
(Degani et al., 2002
), so a
similar mechanism is not likely to be responsible for the rapid translocation
of NDP kinase in response to EGF and bombesin reported here.
The marked effect of BDM on NDP kinase localization
(Fig. 4) suggests yet another
alternative, namely that a member of the myosin family might be involved.
However, because BDM is also known to affect calcium homeostasis and
microtubule function (Kiehart, 1999), the precise mechanism of its action
requires careful investigation. Despite the uncertainty as to the underlying
mechanism, the demonstration that a proportion of cellular NDP kinases is
quickly redistributed following cell stimulation and Rac activation strongly
supports previous data indicating that these proteins are involved in
signaling (reviewed in Otero,
2000).
The finding that, in NIH-3T3 cells, NDP kinase localizes constitutively to
the surface of intracellular vesicles was surprising. Based on their
inaccessibility to a fluid phase uptake marker, we conclude that these
structures are not macropinosomes. Some of the structures ringed by NDP kinase
in quiescent cells contain LAMP-1, a marker of late endosomes/lysosomes;
occasionally, the NDP kinase/LAMP-1-positive vesicles resemble multivesicular
bodies, which are part of the late endosomal compartment. However, double
labeling with markers for endocytic compartments indicates that the vesicles
labeled by NDP kinase antibodies are distinct from early and recycling
endosomes, and lack fundamental features of late endosomes and MVBs. Thus,
most of the NDP-kinase-positive vesicles are not conventional endocytic
organelles and might be part of the secretory pathway. Although the analysis
of the protein composition of the vesicles coated by NDP kinase is currently
being performed, the possibility of a link between NDP kinase and secretory
vesicles is backed by recent work showing that, in neurons, NDP kinases
localize to membranes from the Golgi and endoplasmic reticulum, and to
vesicles budding from the trans-Golgi
(Barraud et al., 2002).
The morphological evidence presented here points to an interaction between
NDP kinase and discrete vesicles that associate with microtubules. The
co-localization of NDP kinase and tubulin seen by immunofluorescence is not
coincidental: a separate experimental approach using a cell-free assay
demonstrates that the overlap reflects a physical association between NDP
kinase, stabilized microtubules and membrane vesicles. The tight binding of
NDP kinase to this fraction and the full release induced by GTP and other
substrates suggest that the interaction is specific. We hypothesize that the
dynamic association of NDP kinase with microtubule-bound vesicles in
fibroblasts is relevant to the operation of the multiple GTPases that control
intracellular membrane transport. Namely, in vivo, NDP kinase might associate
with microtubule-bound vesicles when GTP levels are low, using nucleoside
triphosphates such as ATP to phosphorylate GDP, whereas a rise in GTP levels
leads to its release. Our data agree with studies by other groups implicating
NDP kinase in intracellular vesicle trafficking processes. Thus, NDP kinase B
is a component of isolated phagosomes
(Garin et al., 2001) and the
Drosophila homolog of NDP kinase, Awd, regulates dynamin-dependent
synaptic vesicle recycling through a mechanism that requires its intrinsic NDP
kinase activity, presumably GTP regeneration
(Krishnan et al., 2001
).
Facilitation of dynamin-based endocytosis by NM23-H1 was also reported for
mammalian cells (Palacios et al.,
2002
). Recently, Baillat et al.
(Baillat et al., 2002
)
demonstrated a direct interaction between NDP kinase and dynamin I, as well
NDP kinase and phocein, which is homologous to the
subunits of
clathrin adaptor subunits, supporting the idea that NDP kinase plays a role in
vesicular traffic.
![]() |
Acknowledgments |
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