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Biochemistry and Biophysics |
Laboratory of Cellular Carcinogenesis and Tumor Promotion [P. S. L., J. W. K., P. M. B.], and Laboratory of Experimental Carcinogenesis [S. H. G.], National Cancer Institute, Bethesda, Maryland 20892-4255, and Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada [D. A. B., J. C. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The GEFs are proteins that catalyze the dissociation of GDP from Ras GTPases, to allow the formation of the active GTP-bound conformation (7) . Examples of GEFs for Ras include the well-characterized GEF SOS (8) and the calcium-dependent RasGRF (9) . RasGRP represents the prototype of a new class of GEFs that is composed of at least three members. RasGRP was the first member characterized as a GEF for Ras (5) . The related coding sequence HDC25L, described in 1997 as a potential Ras activator (10) , was later shown to be a GEF for Rap1 and has been referred to as CalDAGI (11) or GRP2 (12) . In a recent study, Clyde-Smith et al. (13) described an alternative spliced variant of this Rap GEF, named RasGRP2, which possesses GEF activity for N-Ras, K-Ras, and Rap1. RasGRP2 is located in chromosome 11p13, a region frequently amplified in human tumors (10 , 13) . Finally, the KIAA0846 sequence (14) was reported to encode a GEF (CalDAGIII or GRP3), which can activate both Ras and Rap1 (12 , 15) . We refer to this third member of the family as RasGRP3. Despite the differences in substrate activity, all of the GRP members share similar overall domain structure. They have the Ras GEF signature motif CDC25 (Ras GEF of Saccharomyces cerevisiae), a pair of atypical EF-hands (a calcium-binding motif), and the C1 domain.
Recent studies have revealed that RasGRP functions as a critical molecule in thymocyte differentiation and T-cell activation, linking the T-cell receptor and diacylglycerol messengers to Ras signaling (16 , 17) . Moreover, phorbol esters can directly interact with RasGRP with nanomolar affinity, promote the EF activity, and thereby activate Ras signaling in T cells. In this study, we present evidence that RasGRP3 is also a high-affinity receptor for phorbol esters. Not only did RasGRP3 bind phorbol esters with nanomolar affinity but also its GEF activity for Ras was increased by phorbol ester treatment in the intact cell. In addition, RasGRP3 redistributed to particulate compartments in response to phorbol ester treatment. Taken together, these findings suggest that RasGRP3 serves as a high-affinity target for the tumor-promoting phorbol esters, inducing PKC-independent activation of Ras. The widespread tissue distribution of RasGRP3 and its ability to modulate the small GTPase Ras may be relevant in the context of the phorbol ester tumor-promoting activity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
HEK-293 cells were propagated in Eagle’s MEM adjusted to contain 0.1
mM nonessential amino acids (American Type Culture
Collection, Manassas, VA) and supplemented with 10% heat-inactivated
horse serum (Life Technologies, Gaithersburg, MD). Cell transfections
with pQBI25 or RasGRP3-pQBI25 constructs were performed using
LipofectAMINE Plus (Life Technologies). For generation of stable
transformants expressing either RasGRP3-GFP or the GFP protein alone,
cells were selected in the presence of 500 µg/ml of the antibiotic
G-418 (Life Technologies) for 2 weeks. For generation of stable cell
lines of RasGRP3 in rat2 fibroblasts, cells were infected with the
RasGRP3-Puro virus and selected in the presence of puromycin.
Binding of [3
H]PDBu.
Binding of [3
H]
[3
H]PDBu (777 Gbq/mmol; New England Nuclear,
Boston, MA) was measured as described elsewhere (18)
. The
assay mixture contained 50 mM Tris-HCl (pH 7.4), 1 mg/ml
IgG, 0.1 mM CaCl2, RasGRP3 protein,
and the corresponding lipid mixture. Incubations were carried out at
18°C. Nonspecific binding was measured using an excess of PDBu (30
µM), and specific binding was done in triplicate at each
ligand concentration (0.31–20 nM). Recombinant RasGRP3
produced in Escherichia coli was used for the assays.
Briefly, BL-21 bacteria, transformed with the RasGRP3-pMAL-p2
construct, were induced using 0.3 mM
isopropyl-ß-D-thiogalactopyranoside for 2 h
at 37°C. The bacterial culture was then centrifuged for 20 min at
4,000 x g at 4°C, and the pellet was
resuspended in column buffer [20 mM Tris-HCl (pH
7.4), 200 mM NaCl, 1 mM
EDTA, 10 mM ß-mercaptoethanol, and protease
inhibitors] and frozen overnight at -20°C. After thawing, cell
lysis was completed by six 15-s pulses of sonication over 2 min.
The lysate was centrifuged at 4°C for 20 min at 14,000 x g, and the supernatant was used as the crude extract
for the subsequent purification. The MBP-RasGRP3 fusion protein was
purified using an amylose resin according to the manufacturer’s
instructions (New England Biolabs). Depending on the batch, 2–10 µg
of partially purified protein per tube were used for the binding
assays.
Lipid Preparation.
Sonicated dispersions of phosphatidylserine were prepared in 50
mM Tris-HCl (pH 7.4). For preparation of large unilamellar
vesicles, mixtures of lipids (POPC, POPS, and POPA; Avanti Polar
Lipids, Alabaster, AL) in chloroform containing added traces of
[14C]DPPC (4.0 Gbq/mmol; Amersham Pharmacia
Biotech, Piscataway, NJ) were dried under nitrogen. Lipids were then
resuspended in 170 mM sucrose and 20 mM
Tris-HCl (pH 7.4). Aliquots of lipid (500 µl) were vortexed for 2
min, subjected to three freeze-thaw cycles, and then extruded 40 times
through two stacked 0.1-µm pore polycarbonate filters using a
Liposofast microextruder (Avestin, Ottawa, Canada) to form large
unilamellar vesicles. The final lipid concentration was calculated from
the amount of [14C]DPPC included in the lipid
mixture.
ERK Activation Assays.
Cells were grown in 60-mm dishes and serum-starved overnight before
treatment with different concentrations of PMA or vehicle for 15 min at
37°C. When indicated, a pretreatment with the PKC inhibitor
GF-109203X (5 µM) was performed 30 min before the
addition of PMA. Cells were washed twice in 1x Dulbecco’s PBS and
harvested in 60 µl of lysis buffer [50 mM Tris-HCl (pH
7.4), 1 mM EDTA, and protease inhibitors]. After a 8-s
sonication pulse, a sample of 20 µg total protein was prepared in 2x
Laemmli buffer and boiled for 5 min. Samples were subjected to SDS-PAGE
using 4–20% precast gels followed by transfer onto nitrocellulose
membranes. The membranes were blocked with 1x PBS containing 5% milk
(PBS-milk) at room temperature for 20 min and then incubated overnight
at 4°C with PBS-milk containing 2 µg/ml phospho-ERK1/ERK2 antibody
(New England Biolabs). After washing for 20 min with 1x PBS containing
0.05% Tween 20 (PBS-Tween), membranes were incubated with horseradish
peroxidase-conjugated antirabbit antibody (Bio-Rad, Hercules, CA) for
1 h at room temperature (1:2000 dilution in PBS-milk). After
1 h washing in PBS-Tween, isolated proteins were detected using
SuperSignal West Pico chemiluminescence substrate (Pierce, Rockford,
IL). For subsequent probing with an antibody against total ERK1/ERK2
(New England Biolabs), membranes were reused after stripping the
phospho-ERK1/ERK2 antibody with the Western Blot Recycling kit (Alpha
Diagnostic Intl., San Antonio, TX).
Detection of RasGTP.
We measured activation of Ras by using the activation-specific probes,
as described elsewhere (19)
. The RBD of Raf1 in a pGEX
vector (pGEX-RasRBD construct) was generously provided by Douglas Lowy
(Laboratory of Cellular Oncology, National Cancer Institute, Bethesda,
MD). This construct was used for expression of the RBD as a fusion
protein with GST (GST-RBD) in E. coli. HEK-293 cells
transfected with either the RasGRP3-pQBI25 construct or the pQBI25
vector alone were grown in 60-mm dishes and serum-starved overnight.
After treatment with PMA or vehicle, cells were washed twice with 1x
Dulbecco’s PBS and resuspended in 300 µl of lysis buffer [50
mM Tris-HCl (pH 7.4), 25 mM
NaF, 1 mM sodium orthovanadate, 10
mM MgCl2, and protease
inhibitors]. After a 6-s sonication pulse, IGEPAL CA-630 (Sigma, St.
Louis, MI) was added at a 1% final concentration. Lysates were rotated
for 30 min at 4°C and then clarified by centrifugation. Aliquots of
the supernatant containing 300 µg of total protein were incubated
with 20 µg of GST-RasRBD. This GST-protein was precoupled to
gluthathione Sepharose 4B beads (Amersham Pharmacia Biotech). After 1-h
incubation at 4°C, the beads were washed 3 times with lysis buffer
and finally resuspended in 30 µl of 2x Laemmli buffer. Samples were
boiled for 5 min and a 20-µl sample was analyzed by SDS-PAGE using
4–20% precast gels (Novex-Invitrogen, Carlsbad, CA). Proteins were
transferred to nitrocellulose and then blocked with PBS-milk for 20 min
at room temperature. Immunostaining was performed as described above,
using 1 µg/ml Ras clone Ras10 antibody (Upstate Biotechnology,
Waltham, MA) and a horseradish peroxidase-conjugated antimouse antibody
(Bio-Rad). Aliquots of total lysates (20 µg) were also analyzed for
quantitation of total Ras. For the rat2 cells transfected with the
RasGRP3-Puro virus, cells were plated in a 10-cm dish overnight and
then treated with PMA or solvent. Lysates were incubated for 30 min
with GST-RasRBD coupled to gluthathione beads. Samples were run on
SDS-PAGE, blotted onto nitrocellulose membranes and probed with a
mixture of a panRas R02120 antibody 1:500 dilution (BD Biosciences, Los
Angeles, CA) and K-Ras antibody 1:100 dilution (Santa Cruz
Biotechnologies, Santa Cruz, CA) followed by horseradish peroxidase
antimouse antibody.
Confocal Microscopy Studies.
HEK-293 cells were seeded onto 40-mm circular glass coverslips at a
density of 5–10 x 104
cells/coverslip. Twenty-four h later, the cells were transfected with
either the RasGRP3-pQBI25 construct or the pQBI25 vector using
LipofectAMINE Plus. All of the experiments were performed 48–72 h
after transfection. Confocal images were collected with a Bio-Rad MRC
1024 confocal scan head (Bio-Rad) mounted on a Nikon Optiphot
microscope with a 60x planachromat lens. Excitation at 488 nm was
provided by a krypton-argon laser with a 522/32 emission filter for
green fluorescence. For live-cell imaging, a Biotechs Focht Chamber
System (Biotechs, Butler, PA) was inverted and attached to the
microscope stage with a custom stage adapter. The cells plated on a
40-mm coverslip were enclosed in the chamber, connected to a
temperature controller at 37°C, and media without phenol red were
perfused through the chamber with a Lambda microperfusion pump.
Sequential images of the same cell were collected at various time
points using LaserSharp software.
For the colocalization studies, labeling of the Golgi apparatus was done using 1 µM BODIPY TR C5-ceramide according to the manufacturer’s protocol (Molecular Probes Inc., Eugene, OR). The emission of the red fluorescence was detected using a 589/617 emission filter.
Localization of Ras by Immunocytochemistry.
HEK-293 cells were grown on glass coverslips. After 24–48 h, cells
were washed twice with 1x PBS and fixed in 1% paraformaldehyde for 10
min at room temperature. The cells were then washed twice with 1x PBS
for 5 min and incubated for 1 h with 8% BSA at room temperature.
The blocking solution was removed, and cells were washed with 1x PBS
for 5 min. Then, cells were incubated at 4°C overnight with 2.5
µg/ml Ras clone Ras10 antibody (Upstate Biotechnology) diluted in 1%
BSA. After washing twice in 1x PBS for 5 min, FITC-antimouse antibody
(Jackson ImunoResearch Laboratories, Inc., West Grove, PA) was added to
the coverslip for 2 h at room temperature in the dark. After a
final wash with 1x PBS, cells were mounted in VectaShield with
propidium iodide (Vector Laboratories, Inc., Burlingame, CA) and
examined by confocal microscopy.
Chromosomal Mapping.
To determine the chromosomal position of the human coding sequence for
RasGRP3, a BAC clone (H-NH0150E23) that contained the KIAA0846 sequence
was isolated and used as an in situ hybridization probe
labeled with FITC. Localization to normal lymphocyte chromosomes was
performed by the FISH Mapping Resource Center at the Hospital
for Sick Children (Toronto, Canada). To determine the chromosomal
location of the mouse homologue, we used Southern blotting to type a
panel of EcoRI-digested genomic DNA samples purchased from
The Backcross Mapping Resource (The Jackson Laboratory, Bar Harbor,
ME). A radiolabeled probe was generated using as template a 3.6-kb DNA
fragment corresponding to the 5' untranslated region of the mouse gene
encoding Rasgrp3.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The other two members of the family, RasGRP and RasGRP2,
are also included for comparison. All of the members have a RasGEF
domain belonging to the CDC25 family of GEFs and a RasGEF-associated
NH2-terminal Ras exchange motif (REM).
They also possess a pair of EF-hand calcium-binding domains, and a
potential diacylglycerol/phorbol ester binding motif C1. The presence
in RasGRP3 of a C1 domain that appears to have the structural features
appropriate for diacylglycerol/phorbol ester binding prompted us to
investigate the modulation of this protein by phorbol esters. Alignment
studies showed high sequence similarities between the RasGRP3 C1 domain
and those present in other diacylglycerol receptors, such as PKC
and
PKC
(Fig. 1B)
. As expected from this similarity, RasGRP3
bound phorbol esters with high affinity. Fig. 1C
shows the
saturation binding curve and Scatchard plot (inset) obtained
by using recombinant RasGRP3 protein and
[3
H]PDBu as a ligand. The dissociation constant
(Kd) for this binding was
1.53 ± 0.33 nM
(n = 4).
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was obtained in the presence of 100 µg/ml
phosphatidylserine. To determine the sensitivity of RasGRP3 binding to
the phospholipid composition, we examined two different vesicle
mixtures: POPA/POPC and POPS/POPC. Previous findings have shown
that these mixtures induced efficient phorbol ester binding for the
related family member RasGRP (20)
. The effect of
increasing concentrations of lipid vesicles (10–3000
µM) is shown in Fig. 2
. RasGRP3 displayed an almost identical pattern of phorbol ester binding
for both anionic phospholipids, with strong dependence on the mole
percentage content of anionic phospholipid. Weak binding levels were
observed at 5 mol % anionic phospholipid, with
EC50 values approximately 30-fold higher than the
EC50 values at 20 mol %
(EC50 POPA at 5 mol % = 6300 ± 1100 µM, POPA at 20 mol % = 235 ± 15 µM; POPS at 5 mol
% = 6100 ± 1000 µM,
POPS at 20 mol % = 273 ± 18
µM). In contrast to the previous results
observed for RasGRP (20)
, RasGRP3 required a greater mole
fraction of phosphatydylserine or phosphatidic acid to reach maximal
binding to phorbol esters. This dependence is more similar to what has
been found for some PKC isoforms, such as PKC (20)
.
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. The densitometric analysis revealed a maximal RasGRP3-dependent,
PKC-independent activation at 100 nM PMA, with a higher
concentration diminishing the response. The level of total ERK1/ERK2
remained constant for the time points and concentrations tested.
Neither the HEK-293 untransfected cells nor the empty vector
transfected pools (pQBI25) showed any PKC-independent ERK1/ERK2
phosphorylation induced by PMA treatment (Fig. 3, A and B)
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. This activation was further increased by treatment with the phorbol
ester PMA. The activation was independent of PKC because it was not
abolished or reduced by preincubation with 5 µM
GF109203X. Under the same concentrations, PMA did not significantly
affect the level of RasGTP in HEK-293 cells
transfected with the pQBI25 vector alone and assayed in the presence of
GF109203X (Fig. 4)
.
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. In an independent experiment, we found that treatment of these cells
with a calcium ionophore neither activated Ras nor augmented
PMA-induced Ras activation (data not shown).
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. There was also a punctate pattern of Ras distribution, possibly
indicative of localization in the Golgi apparatus and endosomes, which
has been described by others as part of the trafficking pool of Ras to
the plasma membrane (21)
. By the use of the GFP fusion
protein with RasGRP3 (RasGRP3-GFP), we looked at the subcellular
distribution of RasGRP3 in live HEK-293 cells. As shown on Fig. 7
, RasGRP3 localized mainly in the cytosol with some perinuclear
concentration. We investigated the ability of phorbol esters to recruit
RasGRP3 to the plasma membrane by following RasGRP3 redistribution in
individual cells. As a control, we performed translocation studies
using the GFP protein alone (pQBI25-transfected cells). The control GFP
protein distributed evenly throughout the cells, including the nucleus.
At the highest concentration of PMA tested (1 µM), no
changes in the subcellular localization of the GFP protein were
observed (Fig. 7A
, lower panel). In contrast, PMA
changed the distribution of RasGRP3-GFP in the HEK-293 cells in a
concentration-dependent manner. For concentrations up to 10
nM, no changes in localization were evident (Fig. 7A)
. PMA (100 nM) caused RasGRP3 to
localize preferentially to the plasma membrane after 15 min (Fig. 7A)
. At 1 µM, PMA induced not only
some plasma membrane localization but also perinuclear distribution and
nuclear membrane localization (Fig. 7A)
. To determine
whether the perinuclear distribution observed at 1
µM PMA corresponded to localization in the
Golgi apparatus, we visualized Golgi with Bodipy-Ceramide
(red) and followed the pattern of translocation of RasGRP3
(green) by confocal microscopy. After 15-min exposure to PMA
(1 µM), images showed colocalization of RasGRP3
in green and Bodipy-Ceramide in red, suggesting a redistribution of
RasGRP3 to the Golgi apparatus (Fig. 7C)
. We also looked at
the translocation pattern of RasGRP3 in response to the
membrane-permeable analogue of diacylglycerol, DOG. At the two
concentrations tested, 10 and 100 µM, DOG
induced redistribution of RasGRP3 to the perinuclear area and nuclear
membrane with some plasma membrane localization after 15-min treatment
(Fig. 7B)
.
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44 cM from the
centromere. Many mouse sequences in this region have homologous human
relatives in chromosome 2p2, as expected. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phosphatidylserine accounts for most of the anionic phospholipid present in the inner side of the plasma membrane and endosomes, and it is one of the main contributors to the electrostatic attraction of proteins to membrane compartments (24) . The requirement of RasGRP3 for anionic phospholipids suggested the possibility of RasGRP3 redistribution, or translocation, by phorbol ester treatment in intact cells. Our experiments confirmed that RasGRP3 did translocate in response to the phorbol ester PMA. We found two different patterns of translocation in live cells, depending on the concentration of PMA. At 100 nM PMA, we observed predominant plasma membrane localization of RasGRP3, whereas higher concentrations induced preferential perinuclear and nuclear membrane distribution. A membrane-permeable analogue of DAG, DOG, also induced RasGRP3 redistribution to the perinuclear region and, to a lesser extent, to the plasma membrane. The localization to the plasma membrane may be relevant for the activation of Ras, because it is in the plasma membrane where Ras localizes (25) . There are several examples of EFs for Ras that are recruited to the plasma membrane with activation, such as SOS (26) , RasGRF2 (27) , and RasGRP (5 , 6) . The significance of the perinuclear distribution of RasGRP3, however, remains to be investigated. Another target of RasGRP3 resides mainly in the Golgi apparatus and endosomes: the small G protein Rap1 (28 , 29) . Preliminary studies indicate that Rap1, like Ras, is activated by phorbol esters through RasGRP3.3 On the basis of the differential pattern of distribution, it is tempting to speculate that RasGRP3 would preferentially activate Rap1 over Ras at high concentrations of PMA. In a physiological context, this could provide a mechanism for differential modulation of Ras and Rap1 depending on the site and extend of the DAG generation in the cell. Additional studies are needed to examine this hypothesis.
One of the downstream targets of Ras is the Raf1//MEK//ERK cascade (30) . As expected from Ras stimulation, we found that RasGRP3 induced ERK activation in a PMA-dependent manner. It should be noted that the PMA-concentration profile for ERK activation did not correlate quantitatively with the translocation pattern of RasGRP3. Although 10 nM PMA induced almost maximal activation of ERK, redistribution of RasGRP3 was predominantly at high ligand concentrations. This shift in concentration-response curves would be consistent with the concept of "spare receptors" (31) , in which activation of a fraction of the receptors is sufficient for saturation of the downstream response.
In conclusion, our results demonstrate that RasGRP3 can serve as a phorbol ester-activated signaling pathway that independently of PKC modulates Ras. RasGRP3 and the other members of the GRP family represent particularly interesting targets of the phorbol ester tumor promoters. This is because the GRP proteins directly modulate Ras and related GTPases signaling molecules, the deregulation of which has been linked to carcinogenesis and tumor progression (32) . In addition, the chromosomal location for human RasGRP3 may be significant. Evidence that the RasGRP3 coding sequence is located in chromosome 2 was obtained from the UniGene database4 under UniGene Cluster Hs.24024 (see also Ref. 14 ), and by the use of FISH analysis, we mapped the gene in locus 2p23 (present results). Leukemias and lymphomas are frequently accompanied by chromosomal translocations and/or deletions, and in 50% of human primary acute myeloid leukemia (AML) cells constitutive activation of MAP kinase/ERK pathways has been observed (33) . Chromosomal rearrangements specifically affecting the 2p23 locus occur in a variety of leukemias, which suggests that one or more genes in that area can affect the proliferation and differentiation of the hematopoietic cells. Among other genes, the anaplastic lymphoma kinase (ALK) gene maps to chromosome 2p23 (34) , although in a proportion of the rearrangements, this gene is not affected (35) . The chromosomal localization of the RasGRP3 gene suggests that its potential role in human cancers of hematopoetic origin should be evaluated carefully.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at 37 Convent Drive MSC 4255, Building 37/Room 3A01,
Bethesda, MD 20892-4255. Phone: (301) 496-3189; Fax: (301) 496-8709;
E-mail: blumberp{at}dc37a.nci.nih.gov 
2 The abbreviations used are: PKC, protein
kinase C; GEF, guanine nucleotide exchange factor; MBP, maltose-binding
protein; GFP, green fluorescent protein; PDBu, phorbol 12,
13-dibutyrate; POPS, 1-palmitoyl-2-oleoyl-phosphatidylserine; POPA,
1-palmitoyl-2-oleoyl-phosphatidic acid; POPC,
1-palmitoyl-2-oleoyl-phosphatidylcholine; PMA, phorbol 12-myristate
13-acetate; RBD, Ras-binding domain; GST, glutathione
S-transferase; DOG, 1,2-dioctanoyl-sn-glycerol;
[14C]DPPC,
L-[1-14C]dipalmitoylphosphatidylcholine; ERK,
extracellular signal-regulated kinase; FISH, fluorescence in
situ hybridization; DAG, 1,2-diacyl-sn-glycerol;
GRP, guanyl nucleotide-releasing protein.
3 P. S. Lorenzo and P. M. Blumberg, unpublished
observations.
4 Internet address:
http://www.ncbi.nlm.nih.gov/UniGene.
Received 3/17/00. Accepted 11/20/00.
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REFERENCES |
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 D. M. Roberts, A. L. Anderson, M. Hidaka, R. L. Swetenburg, C. Patterson, W. L. Stanford, and V. L. Bautch A Vascular Gene Trap Screen Defines RasGRP3 as an Angiogenesis-Regulated Gene Required for the Endothelial Response to Phorbol Esters Mol. Cell. Biol., December 15, 2004; 24(24): 10515 - 10528. [Abstract] [Full Text] [PDF]
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Y. Aiba, M. Oh-hora, S. Kiyonaka, Y. Kimura, A. Hijikata, Y. Mori, and T. Kurosaki Activation of RasGRP3 by phosphorylation of Thr-133 is required for B cell receptor-mediated Ras activation PNAS, November 23, 2004; 101(47): 16612 - 16617. [Abstract] [Full Text] [PDF]
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M. Keiper, M. B. Stope, D. Szatkowski, A. Bohm, K. Tysack, F. vom Dorp, O. Saur, P. A. Oude Weernink, S. Evellin, K. H. Jakobs, et al. Epac- and Ca2+-controlled Activation of Ras and Extracellular Signal-regulated Kinases by Gs-coupled Receptors J. Biol. Chem., November 5, 2004; 279(45): 46497 - 46508. [Abstract] [Full Text] [PDF]
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C. Brodie, R. Steinhart, G. Kazimirsky, H. Rubinfeld, T. Hyman, J. N. Ayres, G. M. Hur, A. Toth, D. Yang, S. H. Garfield, et al. PKC{delta} Associates with and Is Involved in the Phosphorylation of RasGRP3 in Response to Phorbol Esters Mol. Pharmacol., July 1, 2004; 66(1): 76 - 84. [Abstract] [Full Text] [PDF]
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 S. A. Walker and P. J. Lockyer Visualizing Ras signalling in real-time J. Cell Sci., June 15, 2004; 117(14): 2879 - 2886. [Abstract] [Full Text] [PDF]
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M. B. Stope, F. vom Dorp, D. Szatkowski, A. Bohm, M. Keiper, J. Nolte, P. A. Oude Weernink, D. Rosskopf, S. Evellin, K. H. Jakobs, et al. Rap2B-Dependent Stimulation of Phospholipase C-{varepsilon} by Epidermal Growth Factor Receptor Mediated by c-Src Phosphorylation of RasGRP3 Mol. Cell. Biol., June 1, 2004; 24(11): 4664 - 4676. [Abstract] [Full Text] [PDF]
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B. Guilbault and R. J. Kay RasGRP1 Sensitizes an Immature B Cell Line to Antigen Receptor-induced Apoptosis J. Biol. Chem., May 7, 2004; 279(19): 19523 - 19530. [Abstract] [Full Text] [PDF]
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M. J. Caloca, J. L. Zugaza, M. Vicente-Manzanares, F. Sanchez-Madrid, and X. R. Bustelo F-actin-dependent Translocation of the Rap1 GDP/GTP Exchange Factor RasGRP2 J. Biol. Chem., May 7, 2004; 279(19): 20435 - 20446. [Abstract] [Full Text] [PDF]
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S. Madani, A. Hichami, M. Charkaoui-Malki, and N. A. Khan Diacylglycerols Containing Omega 3 and Omega 6 Fatty Acids Bind to RasGRP and Modulate MAP Kinase Activation J. Biol. Chem., January 9, 2004; 279(2): 1176 - 1183. [Abstract] [Full Text] [PDF]
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R. A. Rambaratsingh, J. C. Stone, P. M. Blumberg, and P. S. Lorenzo RasGRP1 Represents a Novel Non-protein Kinase C Phorbol Ester Signaling Pathway in Mouse Epidermal Keratinocytes J. Biol. Chem., December 26, 2003; 278(52): 52792 - 52801. [Abstract] [Full Text] [PDF]
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M. J. Caloca, J. L. Zugaza, and X. R. Bustelo Exchange Factors of the RasGRP Family Mediate Ras Activation in the Golgi J. Biol. Chem., August 29, 2003; 278(35): 33465 - 33473. [Abstract] [Full Text] [PDF]
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C. Teixeira, S. L. Stang, Y. Zheng, N. S. Beswick, and J. C. Stone Integration of DAG signaling systems mediated by PKC-dependent phosphorylation of RasGRP3 Blood, August 15, 2003; 102(4): 1414 - 1420. [Abstract] [Full Text] [PDF]
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L. Li, Y. Yang, G. W. Wong, and R. L. Stevens Mast Cells in Airway Hyporesponsive C3H/HeJ Mice Express a Unique Isoform of the Signaling Protein Ras Guanine Nucleotide Releasing Protein 4 That Is Unresponsive to Diacylglycerol and Phorbol Esters J. Immunol., July 1, 2003; 171(1): 390 - 397. [Abstract] [Full Text] [PDF]
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A. M. Norment, L. Y. Bogatzki, M. Klinger, E. W. Ojala, M. J. Bevan, and R. J. Kay Transgenic Expression of RasGRP1 Induces the Maturation of Double-Negative Thymocytes and Enhances the Production of CD8 Single-Positive Thymocytes J. Immunol., February 1, 2003; 170(3): 1141 - 1149. [Abstract] [Full Text] [PDF]
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G. W. Reuther, Q. T. Lambert, J. F. Rebhun, M. A. Caligiuri, L. A. Quilliam, and C. J. Der RasGRP4 Is a Novel Ras Activator Isolated from Acute Myeloid Leukemia J. Biol. Chem., August 16, 2002; 277(34): 30508 - 30514. [Abstract] [Full Text] [PDF]
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Y. Yang, L. Li, G. W. Wong, S. A. Krilis, M. S. Madhusudhan, A. Sali, and R. L. Stevens RasGRP4, a New Mast Cell-restricted Ras Guanine Nucleotide-releasing Protein with Calcium- and Diacylglycerol-binding Motifs. IDENTIFICATION OF DEFECTIVE VARIANTS OF THIS SIGNALING PROTEIN IN ASTHMA, MASTOCYTOSIS, AND MAST CELL LEUKEMIA PATIENTS AND DEMONSTRATION OF THE IMPORTANCE OF RasGRP4 IN MAST CELL DEVELOPMENT AND FUNCTION J. Biol. Chem., July 5, 2002; 277(28): 25756 - 25774. [Abstract] [Full Text] [PDF]
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M. G. Kazanietz Novel "Nonkinase" Phorbol Ester Receptors: The C1 Domain Connection Mol. Pharmacol., April 1, 2002; 61(4): 759 - 767. [Abstract] [Full Text] [PDF]
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N. Brose and C. Rosenmund Move over protein kinase C, you've got company: alternative cellular effectors of diacylglycerol and phorbol esters J. Cell Sci., January 12, 2002; 115(23): 4399 - 4411. [Abstract] [Full Text] [PDF]
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)
or 20 (•) mol % anionic phospholipid (POPA or
POPS) with POPC as the remainder. The results are
expressed as percentage of the binding at 3000 µM 20 mol
% anionic phospholipid. Values represent the mean ± SE
of three to four experiments per group.
