From the Department of Pharmacy, University of Sydney, New South Wales 2006, Australia
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ABSTRACT |
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Ral-A is a Ras-related GTP-binding protein that
has been suggested to be the downstream target of Ras proteins and is
involved in the tyrosine kinase-mediated, Ras-dependent
activation of phospholipase D. We reported recently that Ral-A purified
from human erythrocyte membrane binds to calmodulin in a
Ca2+-dependent manner at a calmodulin
binding domain identified near its C-terminal region (Wang, K. L.,
Khan, M. T., and Roufogalis, B. D. (1997) J. Biol.
Chem. 272, 16002-16009). In this study we show the enhancement
of GTP binding to Ral-A by Ca2+/calmodulin. The stimulation
up to 3-fold by calmodulin was Ca2+-dependent,
with half-maximum activation occurring at 180 nM calmodulin and 80 nM free Ca2+ concentration. The present
work supports a regulatory role of Ca2+/calmodulin for the
activation of Ral-A and suggests a possible direct link between signal
transduction pathways of Ca2+/calmodulin and Ral-A proteins.
Calmodulin is a ubiquitous, highly conserved Ca2+
sensor protein that translates the Ca2+ signal into a wide
variety of cellular processes. In response to an increase in the
intracellular concentration of Ca2+, calmodulin undergoes a
conformational change that results in binding to its target proteins
and acts as a trigger to modify their functions. Calmodulin-binding
proteins therefore play an important role in intracellular
Ca2+ signaling and in various physiological functions,
including glycogen metabolism, secretion, muscle contraction,
actin/cytoskeletal organization, and cell division (1, 2). Ras proteins
are integral to signal transduction of extracellular signals to the nucleus, thus regulating a diverse spectrum of intracellular processes (3, 4). Accumulating evidence indicates the existence of a direct link
between signaling pathways of Ca2+/calmodulin and Ras
proteins. RasGRF, a neuronal Ras-guanine nucleotide releasing factor,
has been shown to be activated in response to Ca2+ via
direct binding of calmodulin (5). In rat pheochromocytoma PC12 cells,
Ca2+ influx through voltage-sensitive Ca2+
channels induces neurite growth via activation of Ras, which in turn
activates the serine/threonine protein kinase, Raf (6, 7).
Peppelenbosch et al. (8) have reported that epidermal growth
factor-induced Ca2+ influx is mediated by Rac proteins.
Rapid activation of Rap1 in human platelets has been shown to be
mediated by an increased intracellular Ca2+ concentration
(9). In addition, an increasing number of Ras-related proteins and Ras
protein effectors have been identified as calmodulin-binding proteins,
supporting the involvement of Ca2+/calmodulin in regulation
of Ras-related GTPase function. IQGAP1, a novel RasGAP-related and
Ras-binding protein, has been found to bind to calmodulin and the
interaction between IQGAP1 and Cdc42 is modulated by
Ca2+/calmodulin (10).
Ral proteins represent a distinct family of Ras-related GTP-binding
protein. RalGDS was found to interact with ras p21
specifically and to function as an effector target in Ras signaling
pathways, inducing cellular transformation in parallel with activation
of the Raf/mitogen-activated protein kinase cascade (11-13). Ral-A has
been shown to be involved in the tyrosine kinase-mediated activation of
phospholipase D, suggesting that the signaling pathway from Ras to Ral
leads to the regulation of phospholipid metabolism (14). Ral may also
have a functional role in regulating the cytoskeleton through its
interaction with the effector protein RLIP (Ral interacting protein)
and Cdc42 (15). In addition, the diverse subcellular localization of
Ral-A, not only in plasma membrane, but also in endocytotic vesicles,
synaptic vesicles (16, 17), and specialized secretory organelles (18),
suggests that Ral proteins may be involved in exo- or endocytosis and
membrane traffic. However, the physiological activation of Ral by
extracellular stimuli is largely unknown.
We have reported that Ral-A, purified from human erythrocyte plasma
membrane, can bind to calmodulin in a
Ca2+-dependent manner, and its calmodulin
binding domain has been identified and characterized near its
C-terminal region (19). The Ca2+/calmodulin regulation of
Ral-A was strongly supported by the evidence that calmodulin could
block Ral-A phosphorylation by PKA,1 PKG, and PKC and that
Ral-A was phosphorylated by CaM kinase II in
vitro.2 In this study we
report the effect of Ca2+/calmodulin on GTP binding
activity of Ral-A. Up to 3-fold
Ca2+/calmodulin-dependent stimulation of GTP
binding to Ral-A was observed. The results raise the possibility that
calmodulin may be a potential effector for Ral-A GTPase activation and
serve as a molecular switch in response to changes of intracellular Ca2+ concentration over the physiological range.
Materials--
Packed red blood cells were obtained from the Red
Cross blood bank, Sydney (New South Wales, Australia).
[ Protein Purification--
Ral-A protein was purified from human
red blood cell plasma membrane as described previously (19). Briefly,
calmodulin-depleted human plasma membranes were prepared according
to the method of Wang et al. (20). The membrane solute in
200 mM KCl, 1 mM MgCl2, 200 µM CaCl2, 20 mM Tris-HCl, pH 7.4, 0.55% (w/v) Triton X-100, and 20% (v/v) glycerol was applied to a
calmodulin agarose column and the column was washed extensively with
washing buffer (200 mM KCl, 1 mM
MgCl2, 200 µM CaCl2, 20 mM Tris-HCl, pH 7.4, 0.1% (w/v) Triton X-100, and 20%
(v/v) glycerol) until no protein was detected in the wash. The protein
was eluted from the column by a gradient of increasing concentrations
of EDTA from 2 to 5 mM in the elution buffer (200 mM KCl, 20 mM Tris-HCl, pH 7.4, 0.1% (w/v)
Triton X-100, and 20% (v/v) glycerol). The fractions were analyzed by
6-14% SDS-polyacrylamide gel electrophoresis and stored at
Free Ca2+ Concentration Measurement--
The
fractions containing purified Ral-A were pooled. To determine the
precise concentration of EDTA in the protein sample, excess
CaCl2 was added to the sample and Mag-Fura-2, a
Ca2+-sensitive fluorescence probe, was used to measure the
free Ca2+ available. This method is based on the fact that
Mag-Fura-2 displays shifts in its excitation spectra upon calcium
binding and the ratio of the fluorescence intensities at the
characteristic maxima for completely bound and unbound forms is
indicative of the concentration of Ca2+ present (21). The
fluorescence ratio measurements of Mag-Fura-2 (I331/I383) in the sample
buffer were conducted using a Luminescence Spectrometer (Perkin-Elmer
model LS 50B). Excitation was measured at 331 nm when Mag-Fura-2 was
completely bound to calcium, and 383 nm when Mag-Fura-2 was unbound to
calcium, with emission at 510 nm. Ca2+ concentration was
calculated from the ratio
(I331/I383) and
calibration data using the program in the "Intracellular Biochemistry
Application" (Perkin-Elmer model LS 50B). EDTA concentration in the
protein sample was then obtained by subtracting the measured free
Ca2+ concentration from the total added CaCl2
concentration. When the Ca2+ dose-response curve for
calmodulin stimulation of Ral-A GTP binding activity was performed,
various external CaCl2 concentrations were added to yield
the free Ca2+ concentrations required, as calculated by a
computer program from Fabiato et al. (22). This program
calculates the free ionic concentrations resulting from specified total
concentrations of cations (Ca2+ and Mg2+) and
ligands (EDTA) that have been used in the assay.
Guanine Nucleotide Binding--
GTP binding activity of Ral-A
was determined using a spin column filtration assay. Approximately 1.2 ml of Sephadex G-25 filtration matrix pre-equilibrated in 200 mM KCl and 20 mM Tris-HCl, pH 7.4, was added to
individual spin columns. The assembly was then centrifuged at 800 × g in a Jouan CR 4-11 centrifuge (St-Nazaire Cedex,
France) with a E4 swinging bucket rotor for 5 min. A second
centrifugation is necessary to remove remaining buffer. 200 µl of
Ral-A preparation (about 20 pmol) was incubated for the required time
at 37 °C with 1 µCi of [ Since Ral-A, purified from human erythrocyte membrane, has been
described as a Ca2+/calmodulin-binding protein (19), we
investigated a possible role of Ca2+/calmodulin to modulate
the GTP binding of Ral-A. To test this hypothesis
[
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]GTP (3000 Ci/mmol) was purchased from NEN Life
Science Products. Calmodulin was obtained from Calbiochem.
Calmodulin-agarose was from Sigma. Mag-Fura-2 tetrapotassium salt was
obtained from Molecular Probes (Eugene, OR). Sephadex G-25 was
purchased from Pharmacia Biotech (Uppsala, Sweden). Spin columns were
obtained from Promega (Madison, WI). Other materials and chemicals were
the highest grade available from commercial sources.
80 °C.
-32P]GTP (3000 Ci/mmol)
in the assay buffer containing 200 mM KCl, 20 mM Tris-HCl, pH 7.4, 5 mM MgCl2,
and required CaCl2 in the absence and in the presence of
calmodulin. Controls were obtained by omission of Ral-A in the assay
mixture. Aliquots of 60 µl were withdrawn in triplicate and directly
applied to the center of the spin column matrix. The assembly was
subjected to centrifugation at 800 × g for 5 min at
4 °C. Unincorporated free [
-32P]GTP remains in the
matrix and is effectively removed from [
-32P]GTP-bound
Ral-A. 5 ml of scintillation fluid was added to the flow-through
collected from the spin columns, and the radioactivity of
[
-32P]GTP-bound protein was measured by scintillation
counting in a liquid scintillation analyzer (1900A, TRI-CARB, Packard
Pty. Ltd.). The [
-32P]GTP binding activity of Ral-A
was determined as the radioactivity of the sample minus the
radioactivity of the relevant control and expressed as nanomoles of
[
-32P]GTP/milligrams of protein.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]GTP binding activity of Ral-A was determined
using a spin column filtration assay in the absence and presence of
calmodulin. Although a nitrocellulose filtration assay is commonly used
to measure [
-32P]GTP binding activity of Ras-related
GTPases, spin column chromatography eliminates the possibility that
calmodulin at high concentration might alter Ral-A binding to
nitrocellulose membranes and generate false results. As shown in Fig.
1A, the presence of
Ca2+/calmodulin enhances GTP binding to Ral-A
significantly. The increase in GTP binding to Ral-A in the presence of
240 nM calmodulin required Ca2+, as the
omission of Ca2+ completely abolished the stimulatory
effect of calmodulin. Ca2+ alone does not affect GTP
binding to Ral-A, indicating that GTP binding of Ral-A is not directly
activated by Ca2+, but rather requires calmodulin in its
Ca2+-bound form. Fig. 1B shows that the binding
of GTP to Ral-A in the presence and absence of
Ca2+/calmodulin is time-dependent and that
Ca2+/calmodulin stimulates the initial rate of GTP
binding.
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Fig. 1.
Ca2+/calmodulin enhancement of
[32P]GTP binding to Ral-A. GTP binding to Ral-A was
determined using a spin column filtration assay as described under
"Experimental Procedures." 200 µl of Ral-A preparation (about 20 pmol) was incubated with 1 µCi of [ -32P]GTP (3000 Ci/mmol) in the assay buffer in the presence or absence of required
CaCl2 or calmodulin concentrations. After an appropriate
time at 37 °C, aliquots of 60 µl were withdrawn in triplicate and
directly applied to the spin column. The flow-through was collected
from the spin columns, and [
-32P]GTP binding to Ral-A
was measured by scintillation counting. A shows
[
-32P]GTP binding to Ral-A after 90 min in the
presence 3.688 mM EDTA, 240 nM calmodulin, or
1.1 µM Ca2+ (final free concentration), as
indicated. B shows the time dependence of
[
-32P]GTP binding to Ral-A in the presence and absence
of Ca2+/calmodulin.
, absence of
Ca2+/calmodulin;
, presence of 240 nM calmodulin and final free Ca2+ of 1.1 µM. Results represent the mean of three independent
experiments and are expressed as [
-32P]GTP
binding ± S.D.
To assess how varying the concentration of GTP affects
[-32P]GTP binding to Ral-A in response to
Ca2+/CaM, an experiment was performed in which unlabeled
GTP at high concentration (0.5 µM) was displaced by
addition of increasing amounts of [
-32P]GTP. Fig.
2 shows that Ca2+/CaM
enhances the [
-32P]GTP binding over the concentration
range examined. Based on Fig. 1B, the
[
-32P]GTP binding stoichiometry of Ral-A is calculated
to be 0.11 mol of GTP-bound per mol of Ral-A and 0.2 mol of GTP-bound
per mol of Ral-A in the absence and presence of Ca2+/CaM,
respectively. However, the stoichiometry could be underestimated in the
current experimental conditions, since Fig. 2 shows the [
-32P]GTP binding of Ral-A has not reached saturation
over the [
-32P]GTP concentration range examined.
Nevertheless, accumulating evidence shows that only a small amount of
Ral, in the order of 10%, is in the active GTP-bound form in intact
cells (23, 24). Based on these observations we suggest that most of the
Ral-A purified from human erythrocyte may be in its inactive GDP-bound form and Ca2+-dependent calmodulin binding to
Ral-A may accelerate the displacement of the bound GDP, thereby
promoting GTP binding. The displacement of GDP may be incomplete under
the conditions used.
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To further assess the influence of Ca2+ on calmodulin
stimulation of GTP binding, a Ca2+ dose-response curve was
carried out. Since Ral-A was purified by elution from a calmodulin
agarose column by a gradient EDTA elution buffer, the EDTA
concentration in Ral-A was required for the accurate determination of
free Ca2+ and was determined as described under
"Experimental Procedures." A computer program (22) was then used to
calculate the free Ca2+ concentration in the final assay
buffer when EDTA, Ca2+, and Mg2+ were present.
Fig. 3A shows the -fold
calmodulin stimulation of Ral-A GTP binding activity as a function of
increasing concentration of free Ca2+. In the presence of
240 nM calmodulin, half-maximum stimulation was achieved
when the free Ca2+ concentration was about 80 nM. With increasing concentration of calmodulin in the
presence of saturating free Ca2+ (1.1 µM), a
stimulation of GTP binding activity up to 3-fold was observed, as shown
in Fig. 3B. Approximately 180 nM calmodulin was
required to induce half-maximum stimulation. The data in Fig. 3,
A and B, was fit to the Hill equation
B = Bmax[C]/(KA
+ [C]
), where the Hill coefficient
for
free Ca2+ is 0.244, and
for calmodulin is 0.352. The
negative cooperativity allows sensitivity of response over a wide
concentration range (Fig. 3, A and B,
insets). These results are consistent with the known
requirement of Ca2+ and calmodulin for the interaction
between calmodulin and most of its target proteins (25, 26). For
example, in vitro study shows the concentration of
calmodulin producing half-maximal activation of immobilized CaM kinase
II in response to Ca2+ spikes was 271 nM for
subunit and 70 nM for
subunit, respectively (27).
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It is known that guanine nucleotide exchange factors, such as RalGAP, RalGDS, and RalGDI, are responsible for direct interaction with and regulation of the "on" and "off" status of Ral protein through the conformational transitions induced by the cycle of GTP/GDP exchange and GTP hydrolysis. Although Hinoi et al. (28) have demonstrated that post-translational modification of Ral enhances the action of RalGDS to stimulate GDP/GTP exchange on Ral, the modes of activation and action of RalGDS or RalGAP have not yet been clarified. The Ca2+/calmodulin effect on GTP binding activity of Ral-A differs from that of Ral-A regulatory factors. The maximum stimulation of GTP binding by Ca2+/calmodulin of about 3-fold is lower than the enhanced proportion of Ral-A GTP-bound form induced by RalGDS in COS-7 cells (11). RalGAP partially purified from human platelet cytosol also stimulates the intrinsic GTPase activity of Ral-A by at least 6-fold (29). These results indicate that activation by Ca2+/calmodulin may be less effective than those of Ral-A regulatory factors and thus it might act in conjunction with regulatory factors in vivo.
The sensitivity of GTP binding of Ca2+ (half-maximal activation at 80 nM) is within the range of the increase in Ca2+ from a resting level of about 20 nM to levels of 1 µM or more in cells responding to incoming signals. Calmodulin, acting as a Ca2+-sensor protein, is present in large excess in cells, although the free concentration of Ca2+-bound calmodulin is limiting and essentially all the Ca2+-bound calmodulin present in the cell must be bound to targets (30). The very low physiological levels of free Ca2+-bound calmodulin indicates that small changes in the affinity of a typical target could significantly affect the level of its activity at a submaximal intracellular Ca2+, as demonstrated recently for smooth muscle myosin light chain kinase activity (31). Recent evidence supports the notion that the sensitivity of Ral-A to Ca2+ has potential physiological significance. Wolthuis et al. (24) reported that an elevation of intracellular Ca2+ through Ca2+ influx stimulated by ionomycin, or release from intracellular Ca2+ stores induced by thapsigargin, induced a rapid activation of Ral in platelets. In addition, Hofer et al. (32) found that endogenous levels of activated GTP-bound Ral (Ral-GTP) was increased by treatment with the Ca2+ ionophore ionomycin in Rat fibroblasts. However, the Ca2+ sensitivity of Ral activation has not yet been reported in these papers. Although the precise mechanism of Ral activation by Ca2+ is unknown, it is speculated that Ca2+-dependent calmodulin binding to Ral may have a direct effect on Ral activation (24).
Recently, a growing number of calmodulin binding proteins have been
identified among Ras-related GTPases, including members of the Rad
family of GTPases (including Rad, Gem, and Kir) and Rin family of
GTPases (including Rin, Rit, and Ric) (33-36). However, [3H]GTP or [3H]GDP binding activity of
GST/Rad was not affected by calmodulin, neither was its GTP hydrolysis
activity (34), and calmodulin inhibits GTP binding to GST/Kir and
GST/Gem (33). Furthermore, the effects of calmodulin on Rad, Kir, and
Gem were seen in the absence of Ca2+ (33, 34). Park
et al. (37) have reported that the interaction of
Ca2+/calmodulin with Rab3A induces its dissociation from
synaptic membranes. The same effect can be achieved by RabGDI (37), but compared with Ca2+/calmodulin with less effect and a less
stringent requirement for GDP than that of RabGDI (37). Similarly to
this finding, our work has shown that the stimulatory effect of
calmodulin on Ral-A GTP binding is
Ca2+-dependent and appears to be less effective
than that of Ral-A regulatory effectors, suggesting that the regulation
by Ca2+/calmodulin and GTP/GDP exchange factors of this Ras
protein may occur by a different mechanism and possibly in conjunction
with other Ral effectors. While Ca2+/CaM enhances the
initial rate and apparent sensitivity of GTP binding to Ral-A, it needs
to be determined whether this occurs via enhancement of GDP/GTP
exchange, by increases in the number and/or affinity of GTP binding
sites, or by displacement by calmodulin of an autoinhibitory domain
from the active site (38). This work shows a direct functional
regulation of Ral-A by Ca2+/calmodulin in the
physiological range and suggests a potential role of Ral-A in
Ca2+-regulated pathways.
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ACKNOWLEDGEMENTS |
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We thank Professor Richard Christopherson and Dr. Michael Morris for helpful discussion.
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FOOTNOTES |
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* This work was supported by the National Health and Medical Research Council of Australia.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.
Present address: The Victor Chang Cardiac Research Institute, 384 Victoria St., Darlinghurst, NSW 2010, Australia.
§ To whom correspondence should be addressed: Dept. of Pharmacy, A15, University of Sydney, NSW 2006, Australia. Tel.: 61-2-9351-2831; Fax: 61-2-9351-4447; E-mail: basilr{at}pharm.usyd.edu.au.
2 K. L. Wang and B. D. Roufogalis, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PK, protein kinase; GAP, GTPase-activating protein; GDS, GDP dissociation stimulator; GDI, GDP dissociation inhibitor; CaM, calmodulin; CaM kinase II, Ca2+/calmodulin-dependent protein kinase; GST, glutathione S-transferase.
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