(Received for publication, October 3, 1996, and in revised form, December 10, 1996)
From the Groupe de Biophysique-Equipe 2, Ecole Polytechnique, F-91128 Palaiseau Cedex, France and § Laboratoire d'Enzymologie du Centre National de la Recherche Scientifique, F-91198 Gif-sur-Yvette and Institut Jacques Monod, Université Paris 7, F-75251 Paris 05, France
This work describes the in vitro properties of full-length CDC25Mm (1262 amino acid residues), a GDP/GTP exchange factor (GEF) of H-ras p21. CDC25Mm, isolated as a recombinant protein in Escherichia coli and purified by various chromatographic methods, could stimulate the H-ras p21·GDP dissociation rate; however, its specific activity was 25 times lower than that of the isolated catalytic domain comprising the last C-terminal 285 residues (C-CDC25Mm285) and 5 times lower than the activity of the C-terminal half-molecule (631 residues). This reveals a negative regulation of the catalytic domain by other domains of the molecule. Accordingly, the GEF activity of CDC25Mm was increased severalfold by the Ca2+-dependent protease calpain that cleaves around a PEST-like region (residues 798-853), producing C-terminal fragments of 43-56 kDa. In agreement with the presence of an IQ motif on CDC25Mm (residues 202-229), calmodulin interacted functionally with the exchange factor. Depending on the calmodulin concentration an inhibition up to 50% of the CDC25Mm-induced nucleotide exchange activity on H-ras p21 was observed, an effect requiring Ca2+ ions. Calmodulin also inhibited C-CDC25Mm285 but with a ~100 times higher IC50 than in the case of CDC25Mm (~10 µM versus 0.1 µM, respectively). Together, these results emphasize the role of the other domains of CDC25Mm in controlling the activity of the catalytic domain and support the involvement of calmodulin and calpain in the in vivo regulation of the CDC25Mm activity.
The mouse CDC25Mm 1 protein is a
guanine nucleotide exchange factor (GEF) regenerating the active form
of H-ras p21, the complex with GTP (1-3). Homologous
products were found in rat (p140-rasGRF) (4) and human (H-GRF) (5, 6).
These rasGEFs have been described to be specific for the central
nervous system (4-9). Some evidence has also been reported for the
existence of full-length and truncated forms of these exchange factors
in other tissues (10, 11). Experiments in vivo suggest that
the upstream connection of this GEF involves G-protein-coupled
receptors (9, 12, 13) and not hormone-receptor-bound tyrosine kinases
via the adaptor protein GRB2, as has been found for SOS, a ubiquitous rasGEF (14-16). CDC25Mm contains in the N-terminal moiety
two domains of pleckstrin homology (PH1 and PH2), one of DBL homology
(DH) and a coiled-coil region that follows the PH1 domain
(cf. Ref. 17). PH domains are frequently present in
signaling proteins and represent regions of interactions with specific
ligands such as the -subunits of heterotrimeric G-proteins (18,
19) and phospholipids (20). Coiled-coils are specific tertiary
structures involved in protein interactions (21) and the DH is a domain
sharing similarity with a GDP/GTP exchange factor of members of the Rho
family (2, 4, 22, 23). Farnsworth et al. (24) reported that
in vivo the activity of the homologous p140-rasGRF from rat
brain is enhanced by raising the calcium concentration, an effect
associated with the binding of calmodulin, and that p140-rasGRF and
calmodulin form a stable complex. A direct action of calmodulin was
supported by the presence in the N-terminal region of
CDC25Mm of an IQ domain, a sequence frequently found in
proteins interacting with calmodulin (25, 26). Very recent experiments
in vivo have indicated that PH1, coiled-coil and IQ domains
act cooperatively to facilitate the activation of p140-rasGRF by
calcium (17). Moreover, the presence in p140-rasGRF of two adjacent
PEST sequences has suggested potential cleavage by the
Ca2+-dependent protease calpain (26).
Concerning in vitro properties, whereas C-terminal catalytic
fragments spanning 256 to 488 amino acid residues have been
biochemically characterized (3, 5, 27, 28), little is known about the
functional properties in vitro of the full-length molecule
comprising 1262 (CDC25Mm) or 1244 (p140-rasGRF) amino acid
residues, of which the purification has yet to be reported.
Therefore, with the aim at deepening our knowledge of the mechanisms controlling the activation of H-ras p21, we have produced and purified the full-length CDC25Mm as recombinant protein in Escherichia coli and characterized its properties under well defined conditions in vitro. The purified GEF shows a specific activity much lower than its isolated catalytic domain or the C-terminal half-molecule; it is inhibited by calmodulin and is specifically cleaved by calpain.
The
entire open reading frame of murine CDC25Mm gene
from a BamHI-EcoRI fragment of pHC28 (22) was
cloned in a pGEX2TH in which the HindIII site had been
replaced by a SalI site. A BamHI-SalI fragment, containing the total CDC25Mm gene, was
cloned in BamHI-SalI of pMAL-c2 (New England
Biolabs) and expressed in E. coli strain SCS1 as N-terminal
fusion with the maltose-binding-protein (MBP), comprising a Xa-specific
cleavage site. Cell cultures (1.5 liters) were grown at 30 °C in LB
medium containing 50 µg/ml ampicillin. The induction was started with 0.1 mM isopropyl--D-thiogalactopyranoside at
a cell density of 0.2 A600 units and continued
at 24 °C to a density of 1.5 A600 units.
After centrifugation (7,000 × g for 10 min), the
resuspended pellet was sonicated 5 times for 15 s in buffer A (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 7 mM ME) containing 10% glycerol, 1 mM EDTA, and
1 mM Pefablock-SC and centrifuged for 20 min at 25,000 × g at 4 °C, a temperature at which all the subsequent
purification steps were carried out. The supernatant was loaded on a
6-ml ResourceQ column (fast protein liquid chromatography system,
Pharmacia Biotech Inc.) equilibrated with buffer A and eluted with the
same buffer at a flow rate of 3 ml/min. Unlike the bulk of E. coli proteins, the largest portion of MBP-CDC25Mm was
not retained on the resin. The nonretained active fractions were mixed
with 10 ml of amylose-resin (New England Biolabs) equilibrated in
buffer A and gently shaken for 30 min. After centrifugation at low
speed (2,500 × g for 2 min), the supernatant was
discarded, and the resin mixed again with 50 ml of buffer A was
centrifuged. This step was repeated four times. Finally,
MBP-CDC25Mm was removed from the amylose resin by two
washes with 10 ml of buffer A containing 10 mM maltose.
After centrifugation, the combined supernatants were passed on a HiTrap
heparin column of 5 ml (Pharmacia) that was step-eluted with 250, 400, 600, and 1000 mM NaCl solutions in 25 mM
Tris-HCl, pH 7.5, and 7 mM ME (flow rate: 5 ml/min) under control of the fast protein liquid chromatography system.
MBP-CDC25Mm emerged at 600 mM NaCl. After
dialysis against buffer A plus 50% glycerol, the purest fractions on
SDS-PAGE, were stored at
20 °C. Their activity was stable for at
least 6 months.
The dissociation rates of the
p21·[3H]GDP complexes were measured kinetically at
30 °C after addition of a 500-fold excess of unlabeled nucleotide
using the nitrocellulose binding assay. The labeled
p21·[3H]GDP was prepared by incubation for 5 min at
30 °C in 100 µl of buffer B (50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 100 mM NH4Cl and 0.5 mg·ml1 bovine serum albumin), containing 2 µM p21, 3 mM EDTA, and 6 µM
[3H]GDP (350 Bq·pmol
1, DuPont NEN). Then,
3 mM MgCl2 was added. The reaction mixture for
the dissociation experiments contained, in buffer B, 0.1-0.2 µM p21·[3H]GDP and either
MBP-CDC25Mm, GST-C-CDC25Mm631, or
C-CDC25Mm285, calmodulin, calpain, CaCl2, or
EGTA as indicated in the legends to figures. The concentration of
glycerol carried over from the CDC25Mm storage buffer was
maximally 10%. An equal amount of storage buffer was added to the
control. For calpain treatment see legend to Fig. 4 or the text in the
relative section of "Results." At the given times, the samples
(5-10 µl) were filtered through nitrocellulose discs (Sartorius
11306, 0.45 µm), washed twice with 3 ml of ice-cold 50 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, and 100 mM NH4Cl, and the retained p21-bound
radioactivity was measured in a liquid scintillation counter
LKB/Pharmacia, model Wallac 1410.
Other Materials and Methods
C-CDC25Mm285 and human c-H-ras p21 were isolated and purified as described (27, 29). C-CDC25Mm631 was produced in E. coli, as GST fusion and purified by affinity chromatography on glutathione-Sepharose, under the same conditions as reported for C-CDC25Mm285 by Jacquet et al. (27). This was followed by chromatography on HiTrap Heparin column (see above). Stepwise elution of C-CDC25Mm631 took place at 400 mM NaCl. Calmodulin and calpain were purchased from Sigma. SDS-PAGE was carried out using a 10%/0.25% acrylamide/bisacrylamide gel and stained with Coomassie Blue. The anti-C-CDC25Mm285 antibodies were produced in rabbit, and those anti-MBP were purchased from New England Biolabs. Protein concentration was determined by the Bio-Rad assay, using bovine serum albumin as a standard. The concentration of p21 was checked by [3H]GDP binding. To determine the percentage of protein components, Coomassie Blue-stained gels were analyzed with the Apple scanner system, and the results were evaluated with the Scan Analysis software.
Cloning of the full-length
CDC25Mm gene in pGEX resulted in low expression and
little soluble product, whereas the use of a pMAL vector allowed the
production of ~1 mg of CDC25Mm/1 liter of cell culture at
a A600 cell density of 1.5 units. More than 60%
of the produced CDC25Mm remained soluble after a 20-min
centrifugation at 25,000 × g. The temperature of
induction was important; incubation at 24 °C gave higher
concentrations of soluble CDC25Mm than at 37 °C. On
SDS-PAGE the MBP-fused product showed the expected apparent molecular
mass of 188 kDa and corresponded, after purification, to the slowest
migrating band (~50% of the total proteins) (Fig. 1A, lane 4). In the purified
preparations, Western blot with anti-C-CDC25Mm285
antibodies revealed only one band corresponding to the molecular mass
of the MBP-fused full-length protein (188 kDa) (Fig. 1B, lane 2) while most other SDS-PAGE bands were responsive to
anti-MBP antibodies (Fig. 1B, lane 1). Since no
proteolysis was detectable in the cell extract, these bands consist of
N-terminal incomplete translational products of
MBP-CDC25Mm. Accordingly, a second passage on amylose resin
gave the same gel pattern without any further purification. Also the
use of other chromatographic techniques such as ionic exchange
(ResourceQ, Pharmacia, at pH from 7.0 to 9.5) and hydrophobic resins
(Pharmacia kit), or of hydroxylapatite and filtration methods
(Superdex, Pharmacia and Microcon-100, Amicon) did not improve
purification. Together, these properties show that our
CDC25Mm preparations were not contaminated by C-terminal
fragments and contained little, if any, E. coli proteins.
Protein concentration by means of Aquacide II or Amicon ultrafiltration
was avoided, since it caused loss of activity, likely due to
aggregation. Cleavage of the fused MBP by factor Xa led to degradation
of CDC25Mm; therefore, we have used the fused protein.
C-CDC25Mm631 was obtained >90% pure as GST-fused protein.
C-CDC25Mm285 was homogeneous on SDS-PAGE, as reported
(27).
Comparison of the Activities of Full-length CDC25Mm, C-CDC25Mm631, and C-CDC25Mm285
Experiments in vivo suggest
that CDC25Mm has a constitutive GEF activity (9). In line
with this, the [3H]GDP/GDP exchange rate of
p21·[3H]GDP in vitro was enhanced by the
purified full-length CDC25Mm (Fig.
2A). The stimulation of the p21·GDP
dissociation rate constant increased linearly with increasing the
CDC25Mm concentration (Fig. 2B). Fig.
2C shows that the C-terminal half-molecule C-CDC25Mm631 displayed a constitutive GEF activity several
times higher than the full-length molecule. Also in this case,
increasing concentrations of C-CDC25Mm631 enhanced linearly
the GDP/GDP exchange activity of H-ras p21 (Fig.
2D). Similar results were obtained by determining the
GDP/GTP exchange rate (not shown).
The activity of CDC25Mm and C-CDC25Mm631 was
then compared with that of the C-terminal fragment
C-CDC25Mm285, the best studied catalytic fragment of
CDC25Mm (3, 27), slightly longer than the shortest
catalytic domain isolated so far (last 256 C-terminal residues, ref
28). C-CDC25Mm285 was by far the most active of the three
rasGEF forms. At a concentration of 0.2 µM, the
activities of CDC25Mm and C-CDC25Mm631 were 4.4 and 23%, respectively, that of C-CDC25Mm285 (Fig.
3). This reveals a negative influence on the activity of the C-terminal domain by other domains of CDC25Mm.
CDC25 Mm is specifically cleaved by calpain around a PEST region
Cheney and Mooseker (26) identified in the homologous p140-rasGRF from rat two adjacent PEST sequences located between residues 792 and 836. PEST sequences have been proposed to confer rapid degradation to numerous proteins via the Ca2+-dependent protease calpain (30, 31). To determine whether this was also the case for CDC25Mm, the action of calpain was examined by determining its proteolytic effect and the possible consequences on the GEF activity. On Western blot (Fig. 4A), we observed with anti-C-CDC25Mm285 antibodies that digestion of CDC25Mm by calpain induced the appearance of two to four C-terminal fragments with a molecular mass between 43 and 56 kDa. Digestion of CDC25Mm at 30 °C proceeded rapidly: >90% in 2 min with 1 µM calpain and 50% in 30 min with 0.05 µM calpain. No cleavage of C-CDC25Mm285 by calpain could be detected.
As shown in Fig. 4B, calpain-treated CDC25Mm showed a ras GEF activity ~3 times as strong as that of the untreated CDC25Mm. This effect, clearly dependent on the production of C-terminal CDC25Mm fragments with constitutive activities higher than the activity of the full-length molecule, further confirms the negative influence of the N-terminal moiety on the activity of the C-terminal catalytic domain and suggests a regulatory role by proteases on the activity of this rasGEF.
Calmodulin Action on CDC25Mm ActivityFarnsworth
et al. (24) observed that H-ras p21 activation by
p140-rasGRF in vivo was enhanced by raising the
concentration of Ca2+, a mechanism mediated by calmodulin,
since this ubiquitous protein, known to be involved in cellular
processes controlled by Ca2+-dependent
signaling (cf. Refs. 32 and 33), was coprecipitated with
p140-rasGRF. The fact that CDC25Mm has the same recognition
sequence for calmodulin (IQ motif, residues 202-229), as found in
p140-rasGRF (residues 198-225T) (26) suggests a productive interaction
also between CDC25Mm and calmodulin. As shown in Fig.
5, in our in vitro system
Ca2+-calmodulin was found to inhibit the rasGEF activity of
CDC25Mm. With increasing concentrations of calmodulin, the
inhibition leveled at ~50% the rasGEF activity, the concentration of
calmodulin inducing half maximum inhibition (IC50) being
~0.1 µM (Fig. 5, inset).
In experiments not shown, we have observed that the treatment of CDC25Mm by calpain leading to N-terminal and C-terminal-truncated fragments abolished the inhibition of the GEF activity induced by 1 µM calmodulin. Thus, one can conclude that the inhibitory effect of calmodulin on the catalytic activity of the C-terminal domain is due to an intramolecular mechanism originating from the N-terminal moiety.
As shown in Fig. 6, the omission of Ca2+ and
the addition of 0.1 mM EGTA led to a progressive slight
decrease of the calmodulin inhibition on the CDC25Mm
activity, the obtained values displaying a marked variability. Only at
1 mM EGTA, the calmodulin effect was completely
abolished.
Ca2+-calmodulin also inhibited the GEF activity of
C-CDC25Mm285, but at a concentration about 100 times higher
than in the case of the full-length molecule (IC50 = ~10
versus 0.1 µM, respectively) (Fig.
7). Calmodulin had no effect on the intrinsic GDP
exchange activity of H-ras p21.
The biochemical characterization of purified full-length CDC25Mm shows that its noncatalytic moiety, comprising two PH, one DH, one coiled-coil and one IQ domain, has a regulatory function on the GEF activity of CDC25Mm. In fact, the specific activity of the full-length protein is ~4% that of the isolated catalytic C-terminal fragment of 285 residues. The observation that the C-terminal half-molecule shows an activity, whose extent lies between the activity of the full-length CDC25Mm and that of C-CDC25Mm285, further confirms a regulatory function of the noncatalytic moiety of the molecule and supports the existence in the cell of mechanisms activating this GEF. This is in agreement with experiments in vivo showing that serum stimulation of NIH3T3 transformants leads to an increase in Ras·GTP only in cells expressing the entire CDC25Mm but not in cells expressing smaller C-terminal forms of CDC25Mm (9, 22). Thus, the N-terminal region is essential for the response to serum. In line with this, is also the observation that the C-terminal truncated CDC25Mm lacking the last 600 amino acids behaves as dominant negative to the response to serum of the full-length molecule (9).
The specific cleavage by calpain around the PEST region increases the GEF activity due to the production of C-terminal fragments. The observation that the induced activity is of the same range as the activities of C-CDC25Mm631 and the p140-rasGRF C-terminal fragment of 456 residues (4) suggests that most noncatalytic domains participate in regulating the activity of the catalytic C-terminal conserved region. This indicates that the signal activating the productive interaction between the CDC25Mm C-terminal region and H-ras p21 evokes a global effect on the CDC25Mm molecule.
It is known that the activity of calpain can be coordinated with that of calmodulin, as shown by the frequent presence on calmodulin-binding proteins of a PEST motif and the functional relationship between calpain action and calmodulin binding site (cf. Ref. 34). In a large number of proteins (cf. Refs. 30 and 31), PEST sequences appear to represent a signal for rapid degradation by non-ubiquitin-mediated proteolysis that can involve calpain. Interestingly, in the mouse embryo brain a 58-kDa form of CDC25Mm was described, whereas in the more slowly metabolizing adult brain only the full-length 140-kDa protein could be detected (7). In the human brain, besides the full-length 140-kDa H-GRF, shorter forms of 43-50 kDa were reported (5) that might also be present in tissues other than brain (11). The results of our work show that C-terminal CDC25Mm fragments of 43-56 kDa can originate from the action of calpain. Therefore, as reported in this work, activation of the GEF activity by calpain appears to be mediated by C-terminal fragments that are more active than the full-length molecule. In vivo, an eventual action of calpain could contribute to modulate the activity of CDC25Mm. In this context, it is worth mentioning that the expression of C-CDC25Mm285 in fibroblasts was found to enhance the GDP/GTP exchange activity on H-ras p21 and the tumor formation in the nude mice (35). Moreover, the homologous Saccharomyces cerevisiae rasGEF Cdc25p, containing a cyclin destruction box, has been described to display a rapid metabolism (half-life time, ~20 min) due to proteolytic degradation (36). Whether proteases also act on the mammalian rasGEF in vivo remains to be determined, since the half-life of CDC25Mm in neuronal cells is still unknown.
From our results, Ca2+-calmodulin interacts efficiently with CDC25Mm, inhibiting the rasGEF activity with a IC50 of 0.1 µM, an effect dependent on Ca2+, corresponding approximately to an equimolar ratio between calmodulin and CDC25Mm. The course of inhibition with increasing amounts of calmodulin suggests a noncompetitive effect on the GEF activity of CDC25Mm. Hence, binding of calmodulin and p21 to CDC25Mm should concern distinct sites. The weak interaction of calmodulin with the catalytic domain of CDC25Mm suggests the possibility that in the three-dimensional conformation the IQ sequence at the N-terminal region (residues 202-229) and the C-terminal catalytic domain are vicinal.
Calmodulin is in general known to activate calmodulin-binding proteins. However, in some cases such as calmodulin-kinase II, calmodulin-kinase IV, myosin light chain kinase and calcineurin, incubation with Ca2+/calmodulin has been shown to induce inactivation (cf. Refs. 37 and 38). It is interesting to mention that calmodulin-kinase IV interacts with calmodulin with high affinity, comparable to that of CDC25Mm (37), differently from calmodulin-kinase II which is inhibited only by high concentration of calmodulin (38).
Noteworthy is the relatively high concentration of EGTA required for abolishing the Ca2+-calmodulin-dependent effect. Several recent studies have indicated that the conformation of the N-terminal and C-terminal calcium binding helix-loop-helix motifs of calmodulin depends on whether calcium-is bound or not (cf. Ref. 39). Since the affinity of EGTA for calcium is several orders of magnitude higher than that of calmodulin, this finding suggests that, when occupied, the Ca2+-binding site of calmodulin is little accessible to the chelating agent.
From experiments in vivo (24), one would rather expect that Ca2+-calmodulin activates CDC25Mm. This difference with our results in vitro suggests the existence of other components affecting the action of calmodulin on CDC25Mm in the cell implying a more complex calmodulin-regulated mechanism. The same authors (24) report that calmodulin-bound p140-rasGRF isolated from stimulated cells and calmodulin-free p140-rasGRF isolated from unstimulated cells display the same GEF activity. The different experimental conditions may be the reason for the discrepancy between these and our results.
In conclusion, our work supports a regulatory role of the N-terminal
moiety of CDC25Mm on its catalytic domain and suggests that
calmodulin and calpain act as modulators of the CDC25Mm
activity in the cell. Very recently, Mattingly and Macara (13) have
reported on the basis of experiments in vivo a
phosphorylation-dependent activation of CDC25Mm
exchange factor by muscarinic receptors and G-protein -subunits. The relationship between this effect and the
calmodulin-dependent regulation remains an open
question.
We are indebted to Dr. D. R. Lowy for sending the CDC25Mm gene cloned in pHC28 and Drs. J. B. Créchet, M.C. Parrini, C. Giglione, and I. M. Krab for fruitful discussion and advice.