From the Departments of Pharmacology and
** Psychiatry and Behavioral Neurosciences and the
Program in Molecular Biology and Genetics,
Barbara Ann Karmanos Cancer Institute, Wayne State University,
Detroit, Michigan 48201 and ¶ Cell Signaling Technology Inc.,
Beverly, Massachusetts 01915
Received for publication, September 24, 2002, and in revised form, January 16, 2003
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ABSTRACT |
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The Ras-GRF1 exchange factor, which is regulated
by increases in intracellular calcium and the release of G The Ras GTPases are timed molecular switches that cycle between
GDP- and GTP-bound forms to control pathways of cellular growth and
differentiation (1). In addition to the well established roles for Ras
in the proliferation of normal and malignant cells (2), there is
increasing evidence that Ras also regulates critical functions in
terminally differentiated cells such as neurons (3). The GTPase cycle
is controlled by guanine nucleotide exchange factors
(GEFs)1 and GTPase-activating
proteins (GAPs) (4), with the balance between the effective GEF and GAP
activities determining the activation state of Ras because it is the
GTP-bound form that activates downstream effector pathways. The
principal control in many instances may be the activation process
through the activity or subcellular localization of the GEF, as there
is increasing evidence that these Ras activators are highly regulated
(5). In other cases, such as type 1 neurofibromatosis, where there is
loss of the Ras-GAP activity of the protein neurofibromin (6),
physiological control is shown to require the deactivation step.
Indeed, oncogenic mutations in Ras that block the GAP-catalyzed
deactivation of Ras from the GTP- to GDP-bound states (7), emphasize
the importance of this switch mechanism.
The Ras-GRF1 exchange factor (8), which is also termed
CDC25Mm (9, 10), is highly expressed in neurons of the
central nervous system (11-13). Ras-GRF1 is expressed predominantly at
synapses (14), a neuronal subcellular distribution it shares with the Ras-GAP termed SynGap (15). This colocalization suggests that the
proteins may reciprocally regulate Ras at synapses. Interestingly, Ras
(16) and the Ras effector mitogen-activated protein kinase (17, 18)
have been implicated in the control of synaptic plasticity, which is
the proposed cellular corollary of memory storage, whereas mice that
lack Ras-GRF1 have defects in memory (19, 20). Thus, Ras-GRF1 may
participate in the regulation of synaptic plasticity in the central
nervous system.
Ras-GRF1 couples heterotrimeric G proteins (21-24) and calcium signals
(8, 25) to the activation of Ras. The activation of Ras-GRF1 by G
protein-coupled receptors is closely associated with an increase in its
phosphorylation at certain serine residues (24), with the first of
these to be identified being Ser916 (in the mouse sequence,
equivalent to Ser898 in the rat sequence) (26).
Phosphorylation of Ser916/898, which is an in
vivo and in vitro substrate for protein kinase A (PKA),
is necessary for full activation of the Ras-GEF activity of Ras-GRF1
(26). The current study confirms that Ser916/898 is a
physiologically relevant site of regulated phosphorylation in the
endogenous Ras-GRF1 exchange factor that is expressed in rat forebrain.
Localization of this regulatory phosphorylation event to the apical
dendrites of prefrontal pyramidal cells suggests that Ras signaling
is activated at these loci.
Plasmids and Transfection--
Constructs in the pKH3 mammalian
expression vector (27) that encode murine Ras-GRF1, Ras-GRF1
Expression of recombinant wild-type Ras-GRF1-(900-983) and
Ras-GRF1-(900-983) with the S916A mutation as fusion proteins with glutathione S-transferase (GST) has been previously
described (26). GST-Ras-GRF1-(632-1262) (equivalent to GST-GRF1 Metabolic Labeling and Digestion of Ras-GRF1--
Forebrain
slices cut from postnatal day 15 rat brains were labeled in six-well
tissue culture plates in 1 ml/well phosphate-free Dulbecco's modified
Eagle's medium (Invitrogen) with 2 mCi of [32P]orthophosphate (ICN, Costa Mesa, CA) for 2 h in
a 37 °C incubator that was supplied with 95% O2 and 5%
CO2. The slices were lysed, and Ras-GRF1 was
immunoprecipitated using antibody sc-224 (Santa Cruz
Biotechnology, Santa Cruz, CA) as described (23). Immunoprecipitated labeled Ras-GRF1 was processed for Western blotting and digestion with
cyanogen bromide as described (26).
Development of Polyclonal Antibody 2152 against Ras-GRF1
Ser(P)916--
Antibodies were developed in rabbits
immunized with a 13-residue phosphopeptide that represents the region
flanking Ser916 of mouse Ras-GRF1. This sequence is highly
conserved (with only three conservative substitutions) in rat Ras-GRF1
(8). The specificity of the antibodies was tested, and
affinity-purified antibody 2152 was characterized as a selective agent
for recognition of both mouse and rat Ras-GRF1 only when phosphorylated
at the relevant serine (see below). Western blots were developed by
enhanced chemiluminescent detection (32).
Confocal Immunofluorescence--
Dual labeling indirect confocal
immunofluorescence of transfected PC12 cells was performed as
previously described (33). For neurite outgrowth experiments, the
primary antibodies used were anti-Myc monoclonal antibody 9e10 (1:1000
dilution; Sigma) for detection of cells expressing Myc-H-Ras and
anti-hemagglutinin-1 (HA1) polyclonal antibody Y-11 (1:150 dilution;
Santa Cruz Biotechnology) for detection of cells expressing
HA13-Ras-GRF1. The secondary antibodies used were
Cy3-coupled anti-mouse antibody (1:300 dilution; Jackson Laboratories,
Bar Harbor, ME) and Oregon Green-coupled anti-rabbit antibody (1:300
dilution; Molecular Probes, Inc., Eugene, OR). Pictures were taken with
a ×40 water immersion lens on a Zeiss LSM310 microscope. Neurite
outgrowth was quantified as previously described (34). To determine
phosphorylation of Ras-GRF1, the procedure was similar, except that the
primary antibodies used were anti-HA1 monoclonal antibody 12CA5 (1:500
dilution) for detection of HA13-Ras-GRF1 and polyclonal
antibody 2152 (1:300 dilution) for detection of Ras-GRF1 phosphorylated
at Ser916/898, and pictures were taken with a ×63 oil
immersion lens.
Forebrain slices of postnatal day 15 rat brain were fixed in 4%
paraformaldehyde in phosphate-buffered saline and incubated in 50%
sucrose/phosphate-buffered saline overnight at 4 °C. The slices were
then permeabilized for 10 min with methanol that had been precooled to
In Vitro Phosphorylation of Ras-GRF1
Constructs--
GST-GRF1-(900-983) (wild-type and mutant S916A) and
GST-GRF1-(632-1262) were reacted with PKA (Sigma) as described
(26).
Ras-GRF1 Is Highly Expressed in Rat Brain--
It is now clear
that there are multiple GEFs that can serve to activate Ras proteins,
including those in the Sos, Ras-GRF1/CDC25Mm,
Ras-guanyl nucleotide-releasing protein, and other families (5).
Much work has been performed on the ubiquitous Sos exchange factors
that activate Ras in response to stimulation of tyrosine kinase-mediated signals (28, 35, 36). In contrast, Ras-GRF1 is
expressed predominantly in the neurons of the central nervous system
(11-13) and activates Ras in response to G protein-coupled and calcium
signals (8, 21-24). To examine the relative expression levels of Sos
and Ras-GRF1 in rat brain, a quantitative Western blot protocol was
developed. Ras-GRF1 and Sos1 were expressed in COS-7 cells with
identical triple-HA1 epitope tags at their N termini. The lysates from
these transfections were standardized by Western blotting with anti-HA1
monoclonal antibody 12CA5. The standardized HA13-Ras-GRF1
and HA13-Sos1 lysates were then used to establish the
relative sensitivities of two polyclonal antibodies directed against
the Ras-GRF1 and Sos proteins (Fig.
1). The polyclonal antibodies detected
significantly more Ras-GRF1 than Sos in lysates of rat brain forebrain,
even though the sensitivity of the anti-Sos antibody is greater.
Correcting for the sensitivity of the antibodies, we found that there
was 11-fold more Ras-GRF1 than Sos in rat forebrain slices.
Ras-GRF1 Is Phosphorylated at Ser898 in
Forskolin-treated Slices of Rat Brain--
Ras-GRF1 is regulated by
a complex of mechanisms that include regulated phosphorylation
of serine residues (24). Phosphorylation of Ser916, which
is an in vivo substrate for PKA, has been shown to be necessary in model systems for full activation of the Ras-GEF activity
of Ras-GRF1 by muscarinic receptors (26). To test whether phosphorylation of this residue also occurs in endogenous Ras-GRF1, we
metabolically labeled rat forebrain slices with
[32P]orthophosphate, stimulated the slices with forskolin
to activate PKA, and immunoprecipitated Ras-GRF1. Ras-GRF1 was then
cleaved at methionine residues using cyanogen bromide, and the
fragments were separated by peptide PAGE (Fig.
2). Phosphorylation of mouse Ras-GRF1 at
Ser916 produces a 32P-labeled fragment with an
apparent mobility of ~6.5 kDa (26). The positions of the methionines
that flank this phosphorylation site are conserved between mouse and
rat. Transfection of COS-7 cells with rat Ras-GRF1 demonstrated that
the equivalent phosphorylation (at Ser898) was revealed in
a similarly sized cyanogen bromide cleavage product. Stimulation of
forebrain slices of rat brain with forskolin induced the appearance of
32P in the equivalent fragment of endogenous Ras-GRF1,
confirming that Ser898 is a site of regulated
phosphorylation in the brain.
Phosphorylation of Ras-GRF1 at Ser916 Promotes
Ras-dependent Outgrowth of Neurites in PC12 Cells--
We
have previously shown that phosphorylation of Ras-GRF1 at
Ser916 is necessary for maximal activation of Ras-GEF
activity in a biochemical assay with recombinant Ras substrate (26).
Because we have shown here that this phosphorylation event
occurred in the endogenous exchange factor in the brain, we
investigated whether this phosphorylation plays a critical role in Ras
activation in a neuronal context. We used the well established model of
neurite extension from PC12 cells in response to Ras activation (37). The results show that expression of either the C-terminal half of
Ras-GRF1 (Ras-GRF1 Development of an Antibody That Selectively Recognizes Ras-GRF1
Ser(P)916/898--
The appearance of the
32P-labeled fragment of Ras-GRF1 following cyanogen bromide
cleavage provides an assay for the regulated phosphorylation of
Ser916/898. This assay is time-consuming and provides poor
sensitivity, however. To develop a more sensitive and powerful assay
approach, antibodies that selectively recognize the
Ser(P)916/898 form of Ras-GRF1 were developed. To confirm
the selectivity of antibody 2152, it was tested against recombinant
Ras-GRF1 proteins that were subjected to phosphorylation by PKA (Fig.
4). The data show that antibody 2152 exhibited great selectivity for recognition of Ras-GRF1 only when it
was phosphorylated, with detectable signal produced from as little as
10 ng of Ras-GRF1 Ser(P)916, whereas unphosphorylated
Ras-GRF1 was only detected when microgram quantities were present. Note
that PKA treatment of the S916A mutant protein did not produce any
reactivity to antibody 2152.
Regulated Phosphorylation of Ras-GRF1 at
Ser916/898--
To test whether antibody 2152 recognizes both mouse and rat Ras-GRF1 expressed in mammalian cells, it
was tested against whole cell lysates of COS-7 cells that had been
transfected to express Ras-GRF1 and then treated with various agonists
(Fig. 5). The data show that antibody
2152 reacted with a single band that was present only in the cells
transfected to express Ras-GRF1 and that this band had the same
mobility as that recognized by an independent anti-Ras-GRF1 antibody
(sc-863). Furthermore, it is clear that the recognition of the mouse
and rat Ras-GRF1 proteins was similar and that all recognition was
absolutely dependent on phosphorylation at Ser916/898, as
no reactivity occurred in cells expressing Ras-GRF1(S916A). Both
forskolin and serum (the latter to a lesser extent, which is clearer
when the Western blots were given prolonged exposure) stimulated an
increase in the phosphorylation of Ras-GRF1 at Ser916/898.
Interestingly, treatment of the cells with thapsigargin, which increases cytosolic calcium levels (39), failed to increase phosphorylation of Ras-GRF1 at Ser916/898 above the basal
level. This result supports previous observations on transfected
fibroblasts that increases in calcium do not stimulate the
phosphorylation of Ras-GRF1 (40), although it has been reported that
Ras-GRF1 is an in vitro substrate for
calmodulin-dependent kinase II (14).
Multiple Agonists Stimulate the Phosphorylation of Ras-GRF1 at
Ser916/898 in PC12 Cells--
To investigate
the phosphorylation of Ras-GRF1 in a neuronal context, we coexpressed
Ras-GRF1 with muscarinic receptors in PC12 cells. Western blotting of
PC12 cell lysates (Fig. 6) demonstrated that activation of muscarinic receptors by carbachol, stimulation of
PKA by forskolin, and activation of endogenous Trk receptors by nerve
growth factor (NGF) all increased the phosphorylation of Ras-GRF1 at
Ser916. To extend these results, either
HA13-Ras-GRF1 or its S916A mutant was coexpressed in PC12
cells with muscarinic receptors, and the cells were stimulated and then
fixed and processed for indirect confocal immunofluorescence (Fig.
7). It is clear that antibody 2152 reactivity was again dependent on phosphorylation at
Ser916, as no reactivity was seen in untransfected cells,
in transfected cells that were not stimulated, or in stimulated cells
that were transfected with the Ras-GRF1(S916A) mutant. In agreement
with the Western blot results, carbachol, forskolin, and NGF all
induced the phosphorylation of Ras-GRF1 at Ser916.
Regulated Phosphorylation of Ras-GRF1 at Ser898 in Rat
Forebrain Slices--
To investigate whether endogenous Ras-GRF1 is
regulated by phosphorylation in a manner similar to that defined in the
model systems, antibody 2152 was used to assess phosphorylation of
Ras-GRF1 in rat forebrain slices. It was difficult to detect any
reactivity to antibody 2152 in lysates of brain slices by Western
blotting, perhaps suggesting that only a subset of Ras-GRF1
proteins (perhaps, in particular, neurons) is phosphorylated at
Ser898. When Ras-GRF1 was first immunoprecipitated from
the lysate using an antibody directed against the C terminus (sc-224),
the regulated phosphorylation at Ser898 became apparent by
Western blotting (Fig. 8). There was
little reactivity in Ras-GRF1 from untreated slices, but there were
clear increases following stimulation with carbachol, forskolin, or NGF. Thus, just as in the expression systems constructed in PC12 cells,
endogenous Ras-GRF1 is phosphorylated at Ser898 following
activation of muscarinic or Trk receptors or PKA.
To investigate the distribution of Ras-GRF1 Ser(P)898 in
the cortex, brain slices were fixed and processed for indirect confocal immunofluorescence with antibodies 2152 (anti-Ras-GRF1
Ser(P)916/898) and sc-224 (anti-Ras-GRF1). Prefrontal
cortex includes two classes of neurons, GABAergic interneurons
and glutamate-secreting pyramidal cells. The latter class is easily
recognizable by its distinctive morphology, including a prominent
single apical dendrite that branches in the upper layers. In these
immunolocalization studies, we observed expression of Ras-GRF1
throughout pyramidal neurons, including the cell bodies (Fig.
9), whereas phosphorylated Ras-GRF1 was strikingly localized to the apical dendrites of pyramidal cells (Fig. 9).
This study identifies Ser916/898 of the Ras-GRF1
exchange factor as a target for regulated in vivo
phosphorylation in both transfected model systems of COS-7 and PC12
cells and endogenously in rat brain slices. The role of Ras-GRF1 in
central neurons is intriguing (41, 42), but confusing, as the
phenotypes of mice deficient in Ras-GRF1 are not in complete agreement.
Brambilla et al. (19) described Ras-GRF1-deficient mice that
were impaired in amygdala-dependent learning and memory
tests such as emotional conditioning of memory consolidation. The
authors stated that there were no deficits in the Morris water maze
test, suggesting that hippocampal function was normal, but did find
larger field excitatory postsynaptic potentials upon
electrophysiological recording from both the amygdala and hippocampus
(19). Subsequent work has detailed the hyperexcitability of hippocampal
neurons from these mice (43). Giese et al. (20) described
smaller-than-control mice with defects in
hippocampus-dependent learning and memory as revealed in
contextual discrimination, social transmission of food preference, and
Morris water maze tests. A third group of Ras-GRF1-deficient mice,
studied by Itier et al. (44), were also found to be small,
with evidence that this characteristic is due to a decrease in
postnatal insulin-like growth factor I production that is secondary to
a reduction in pituitary growth hormone levels. Because pituitary
growth hormone is under the control of hypothalamic neurotransmission
and Ras-GRF1 is normally expressed in the postnatal hypothalamus, the
authors suggested that hypothalamic function may be reduced in their
Ras-GRF1-deficient mice, but did not report results from any other
tests of neuronal function (44). Nevertheless, there is strong support
for the concept that Ras-GRF1 is highly expressed in certain central
neurons and has the potential to play critical roles in processes (such as memory) to which Ras signaling has previously been linked (3, 45,
46). In this regard, it is interesting to note that Ras-GRF1 is
regulated by cAMP/PKA phosphorylation (26, 47), signaling elements that
are thought to play a key role in synaptic plasticity and hence memory
storage in the brain (17). The identification of activated Ras-GRF1 in
the apical dendrites of prefrontal pyramidal neurons in this study is
completely consistent with its postulated role as an integrator of
neuronal signal transduction (41, 42).
One possibility that might have resolved the differences in the
phenotypes of mice that are deficient in Ras-GRF1 would be variable
compensation from the highly homologous Ras-GRF2 protein (48).
Recently, however, mice deficient in Ras-GRF2 have been described to
have neither an apparent phenotype nor any compensatory changes in the
expression of Ras-GRF1 (49). Conversely, Ras-GRF1-deficient mice have
no alteration in Ras-GRF2 expression. Furthermore, results from mice
doubly deficient in both Ras-GRF1 and Ras-GRF2 were also reported.
Remarkably, there is no further phenotypic effect beyond that produced
by loss of Ras-GRF1 alone, suggesting that there is no overlap of
function between these highly similar GEFs (49). In this regard, it is
important to note that the Ser916/898 phosphorylation site,
although conserved in Ras-GRF1 proteins from rodents and humans, is
absent in Ras-GRF2. Thus, it is plausible that this regulatory
mechanism is part of the unique function of Ras-GRF1 that cannot be
replaced by Ras-GRF2.
Phosphorylation of Ras-GRF1 at Ser916 has previously been
shown to be essential for maximal activation of Ras-GRF1 in a
biochemical assay with recombinant Ras substrate (26). In the current
study, we have shown that there is a regulatory function for residues 900-975 of Ras-GRF1 to increase Ras activation in the PC12 system and
that this function is also lost by point mutation of the
Ser916 phosphorylation site. Although Ras-GRF1 is
phosphorylated at multiple sites in response to agonist stimulation,
Ser916 is the only detectable site of phosphorylation
in this region (26). These results therefore support the significance
of Ras-GRF1 phosphorylation at Ser916/898 both as
a marker for increased Ras activation within a neuronal context and in
the physiological control of neuronal Ras activation.
Muscarinic stimulation was previously demonstrated to increase the
phosphorylation and activity of Ras-GRF1 in mouse brain explants (23);
thus, stimulation of rat brain Ras-GRF1 Ser898
phosphorylation by carbachol treatment was expected. Activation of
muscarinic receptor signaling in the cortex is also correlated with
memory function in humans (50), with broad pharmacological significance
(51). Whether Ras-GRF1 phosphorylation and activation of Ras in
response to muscarinic agonists are a physiological part of this
process remains to be demonstrated.
The Ser916/898 context fits a consensus PKA substrate
sequence (52) and does indeed serve as a PKA site both in
vitro and in vivo in model systems (26).
Phosphorylation at this site was not, however, recorded in in
vitro experiments with PKA and recombinant Ras-GRF1 by Baouz
et al. (47). The basis for this difference is unclear, but
the current study provides further in vivo support for this
PKA-dependent phosphorylation through demonstration of stimulation of Ser916/898 phosphorylation by forskolin
treatment. More surprising was the robust ability of NGF to stimulate
Ras-GRF1 phosphorylation at Ser916/898 in both the
transfected PC12 model and the endogenous protein in rat brain slices.
Previously, Ras-GRF1 has generally been found to be regulated
independently from growth factor/tyrosine kinase pathways (21,
23), although Ras-GRF1 was reported to associate with the NGF-activated
TrkA receptor (53). The current result suggests that the interaction of
the TrkA and Ras-GRF1 signaling pathways may be functionally important
in those neurons that express both proteins.
In addition to Ras-GEF activity that is stimulated by a G protein
Ras-GRF1 provides an example for other GEFs that are also
subject to complex regulation through phosphorylation (60, 61), allosteric interactions (62-64), and subcellular redistribution (65). The activation of Ras-GRF1 is also complicated by the potential for dimerization and for effects that may be mediated through
the activation of Raf, the Ras effector, even in the absence of
apparent further activation of Ras (66). It is likely that these
complexities reflect the underlying function of Ras-GRF1 and other GEFs
to serve as key integrators and controllers of signaling pathways with
activities beyond the simple control of GTP binding to their
substrates. For example, an emerging model suggests that the GEFs
provide a scaffold or framework function that can select the downstream
targets of their substrate GTPases (67, 68). Thus, it is essential to
consider the cellular and subcellular context within which the GEF acts
to activate Ras signal transduction. In this regard, the selectivity
and sensitivity of anti-Ras-GRF1 Ser(P)916/898 antibody
2152 will provide a powerful approach toward the elucidation of Ras
activation pathways in central neurons.
subunits from heterotrimeric G proteins, plays a critical role in the
activation of neuronal Ras. Activation of G protein-coupled receptors
stimulates an increase in the phosphorylation of Ras-GRF1 at certain
serine residues. The first of these sites to be identified,
Ser916 in the mouse sequence (equivalent to
Ser898 in the rat sequence), is required for full
activation of the Ras exchange factor activity of Ras-GRF1 by
muscarinic receptors. We demonstrate here that Ras-GRF1 is highly
expressed in rat brain compared with the Sos exchange factor and
that there is an increase in incorporation of 32P into
Ser898 of brain Ras-GRF1 following activation of protein
kinase A. Phosphorylation of Ras-GRF1 at Ser916 is also
required for maximal induction of Ras-dependent neurite outgrowth in PC12 cells. A novel antibody (termed 2152) that
selectively recognizes Ras-GRF1 when it is phosphorylated at
Ser916/898 confirmed the regulated phosphorylation of
Ras-GRF1 by Western blotting in both model systems of transfected COS-7
and PC12 cells and also of the endogenous protein in rat forebrain
slices. Indirect confocal immunofluorescence of transfected PC12 cells
using antibody 2152 demonstrated reactivity only under conditions in
which Ras-GRF1 was phosphorylated at Ser916/898. Confocal
immunofluorescence of cortical slices of rat brain revealed widespread
and selective phosphorylation of Ras-GRF1 at Ser898. In the
prefrontal cortex, there was striking phosphorylation of Ras-GRF1 in
the dendritic tree, supporting a role for Ras activation and signal
transduction in neurotransmission in this area.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
N,
Ras-GRF1
900, and the S916A mutant have previously been
described (23, 26). Ras-GRF1
976 was prepared by PCR using a
5'-primer with a BamHI restriction site from the template
pKH3Ras-GRF1
N and verified by sequencing. Mammalian expression
vectors for rat Ras-GRF1 (8), Sos1 (28), Myc-tagged H-Ras (29), and
muscarinic receptor subtypes 1 and 2 (30) were generously provided by
Profs. L .A. Feig, M. Czech, J. Jackson, and M. R. Brann, respectively. COS-7 cells were transfected by calcium phosphate
coprecipitation (27), and PC12 cells by electroporation (31).
N)
was expressed from the vector pGEX.GRF1-(632-1262), which was
constructed by subcloning the BamHI/EcoRI insert
from pKH3GRF1
N (23) into pGEX-2T.
20 °C; rinsed with phosphate-buffered saline; and blocked with
phosphate-buffered saline containing 2% bovine serum albumen, 5% goat
serum, and 0.25% Triton X-100 for 4 h at room temperature.
Indirect confocal immunofluorescence was carried out using the
following primary polyclonal antibodies: antibody 2152 (anti-Ras-GRF1
Ser(P)916/898; 1:200 dilution); antibody sc-224
(anti-Ras-GRF1; 1:200 dilution); or, as a negative control, antibody
sc-863 (anti-Ras-GRF1 antibody that is competent for Western blotting,
but does not recognize the protein in immunoprecipitation or
immunofluorescence protocols; 1:200 dilution). The secondary antibody
used was Oregon Green-coupled anti-rabbit immunoglobulin (1:150
dilution). Pictures were taken with an Olympus Fluoview laser scanning
confocal microscope using a ×20 objective.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Ras-GRF1 is expressed at a higher level
compared with Sos in rat forebrain slices. Lysates of COS-7 cells
expressing HA13-Ras-GRF1 and HA13-Sos1 were
subjected to Western blotting with the anti-HA1 monoclonal antibody
(left panels) and standardized by dilution such that
equivalent levels of the expressed proteins were present. These amounts
were then run in parallel with lysates of rat brain slices (right
panels) and subjected to Western blotting with polyclonal
antibodies to either Ras-GRF1 (upper right panel) or Sos
(lower right panel). In all cases, a graded scale of four
3-fold dilutions of the lysates was loaded to ensure that the signals
could be measured in a range where the signals were not saturated. Data
shown are representative of four independent experiments and were
collected digitally using a Fuji Film LAS-1000 Plus imaging system.
Quantification was performed with ImageGauge Version 3.3 software.
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Fig. 2.
Cyanogen bromide digests of Ras-GRF1 reveal
the phosphopeptide that reflects Ser898 phosphorylation in
forskolin-treated COS-7 cells and rat forebrain slices. COS-7
cells transfected with pMT3gluGRF1 (8) and rat forebrain slices were
labeled with 32Pi and then treated with
dimethyl sulfoxide vehicle (-) or 100 µM
isobutylmethylxanthine (IBMX) plus 20 µM forskolin
(FSK) (+). The Ras-GRF1 proteins were immunoprecipitated and
digested with cyanogen bromide, and the fragments were separated by
SDS-PAGE using a Tricine cathode buffer to sharpen resolution of the
phosphopeptides (26). The results shown are autoradiographs of a region
of the dried gels, with molecular mass markers (in kilodaltons) shown
to the left. Exposure times were overnight for the COS-7 cell-derived
samples and 2 weeks with intensifying screens for the brain-derived
samples. The arrow indicates a phosphopeptide fragment that
was stimulated by the activation of PKA and represents phosphorylation
of Ser898 (26). The result is representative of three
independent experiments.
N) or a Myc-tagged H-Ras protein alone did not
induce neurite outgrowth (Fig. 3). It has
previously been demonstrated that wild-type Ras proteins (those without
a constitutively activating, oncogenic mutation) are not sufficient to
induce neurite outgrowth (37), and this result further suggests that
the Ras-GRF1
N protein is not able to activate the endogenous Ras
protein in PC12 cells to induce neurite extension. Because H-Ras is the
preferred in situ substrate for Ras-GRF1 (29), we assayed
for neurite extension in cotransfections of Ras-GRF1
N and wild-type
H-Ras and found robust outgrowth. Deletion of the Ras-GRF1 construct to
leave the C-terminal third (Ras-GRF1
900), although removing the Ras
exchange motif sequence that is common to Ras-GEFs (5), did not reduce
the H-Ras-dependent induction of neurite outgrowth. Further
deletion to leave the C-terminal quarter of Ras-GRF1 (Ras-GRF1
976) did, however, significantly reduce neurite extension. This deletion leaves intact the CDC25 Ras-GEF domain that is necessary for
biochemical activity (38) and so reveals a stimulatory function within
residues 900-975 of Ras-GRF1. The same reduction in neurite extension
was produced by point mutation of the Ser916
phosphorylation site to alanine (Fig. 3).
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Fig. 3.
Phosphorylation of Ras-GRF1 at
Ser916 promotes Ras-dependent outgrowth of
neurites in PC12 cells. PC12 cells were transfected by
electroporation and then grown for 48 h before processing for
indirect confocal immunofluorescence using red detection for the Myc
epitope tags at the N termini of the wild-type H-Ras constructs and
green detection for the HA1 epitope tags at the N termini of the
Ras-GRF1 constructs. Results shown in A-F are
representative red fluorescence results (except for E, where
green fluorescence is shown) from transfections with GRF1 N plus
H-Ras (A), GRF1
900 plus H-Ras (B),
GRF1
900(S916A) (GRF1
900,916A) plus H-Ras
(C), GRF1
976 plus H-Ras (D), GRF1
N alone
(E), and H-Ras alone (F). Scale
bar = 20 µm. Neurite extension was quantified in the red
fluorescence channel by measuring the projections that were longer than
the diameter of the cell body. In all cases where cotransfections were
performed, clear signals in both the red and green channels were
required for a cell to be scored. Four independent experiments were
performed, with 25 positive cells measured from each trial. Results for
the longest neurite produced and for the sum of all neurites are
presented in G and H as means ± S.D.
Mutation of Ser916 or deletion of residues 900-975 from
Ras-GRF1 significantly reduced both parameters of neurite outgrowth
(p < 0.001).
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Fig. 4.
Antibody 2152 selectively recognizes Ras-GRF1
Ser(P)916. Recombinant GST fusion proteins of
the indicated regions of Ras-GRF1 were incubated with (+) or without
(-) PKA in the presence of ATP/Mg2+ and then separated by
SDS-PAGE on two gels run in parallel. A, one gel was
transferred to nitrocellulose and subjected to Western blotting with
antibody 2152. Note that reactivity was seen only when the GST-Ras-GRF1
constructs were phosphorylated by PKA and was not seen when
Ser916 was mutated to alanine (916A).
B, the other gel was stained with Coomassie Blue to confirm
the relative loading of the recombinant proteins. C,
experiments were repeated with graded loading of the unphosphorylated
(open symbols) and phosphorylated (closed
symbols) substrates to establish the degree to which antibody 2152 recognizes Ras-GRF1 only when it is phosphorylated at
Ser916. Data shown were collected digitally from Western
blots using the Fuji Film LAS-1000 Plus imaging system and analyzed
with ImageGauge Version 3.3. software. wt, wild-type.
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Fig. 5.
Regulated phosphorylation of Ras-GRF1 at
Ser916/898 in COS-7 cells. COS-7 cells were
transfected with pKH3Ras-GRF1 (to express wild-type mouse
Ras-GRF1 (mGRF1 wt)), pKH3Ras-GRF1(S916A) (to express mouse
Ras-GRF1(S916A) (mGRF1 916A)), pMT3gluRas-GRF1 (to express
wild-type rat Ras-GRF1 (rGRF1 wt)), or an empty vector
control ( ). The cells were deprived of serum overnight and then
stimulated with 100 µM IBMX plus 20 µM
forskolin, with 10% fetal calf serum, or with 200 nM
thapsigargin. Whole cell extracts were prepared for Western blotting
with a 1:2000 dilution of affinity-purified antibody 2152 (upper
panel), which showed a strong increase in phosphorylation upon
forskolin treatment. Prolonged exposure (middle panel)
revealed a low level of basal phosphorylation that was increased by
serum stimulation, but not by thapsigargin. The blot was then stripped
(69) and reprobed for total Ras-GRF1 proteins using antibody sc-863
(lower panel) to demonstrate the reproducibility of the
transfection and loading. Data shown are representative of three
independent experiments. anti-GRF1[916phospho],
anti-Ras-GRF1 Ser(P)916.
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Fig. 6.
Regulated phosphorylation of Ras-GRF1 at
Ser916 in PC12 cells as revealed by Western blotting.
PC12 cells were cotransfected with pKH3Ras-GRF1 plus expression vectors
for human muscarinic receptor subtypes 1 and 2. The cells were deprived
of serum overnight and then stimulated with 100 µM
carbachol, with 100 µM IBMX plus 20 µM
forskolin, or with 10 ng/ml NGF. Whole cell extracts were prepared for
Western blotting (32) with antibody 2152 (raised against Ras-GRF1
Ser(P)916 (GRF1(phospho916)); right
panel) and with antibody sc-863 (raised against total Ras-GRF1;
left panel). Data shown are representative of two
independent experiments.
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Fig. 7.
Regulated phosphorylation of Ras-GRF1 at
Ser916 in PC12 cells as revealed by indirect confocal
immunofluorescence. PC12 cells were cotransfected with
pKH3Ras-GRF1 (left panels) or pKH3Ras-GRF1(S916A)
(right panels) plus expression vectors for human muscarinic
receptor subtypes 1 and 2. The cells were deprived of serum overnight
and then stimulated with 100 µM carbachol, with 100 µM IBMX plus 20 µM forskolin, or with 10 ng/ml NGF. The cells were fixed and processed for indirect confocal
immunofluorescence using red detection for the HA1 epitope tags at the
N termini of the Ras-GRF1 constructs and green detection for antibody
2152 (raised against Ras-GRF1 Ser(P)916
(Ras-GRF1phospho916)). The fluorescence images are presented
overlaid with a phase-contrast image of the cells that reveals the
presence of untransfected control cells. Note that green fluorescence
due to antibody 2152 reactivity was found only when wild-type Ras-GRF1
(Ras-GRF1,wt) was present (thus colocalized with red
fluorescence and appears yellow) and only in cells that had
been stimulated with agonists that induce Ser916
phosphorylation. Data shown are representative of five independent
experiments. Ras-GRF1,916A, Ras-GRF1(S916A).
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Fig. 8.
Regulated phosphorylation of endogenous
Ras-GRF1 at Ser898 in rat forebrain slices as revealed by
Western blotting. Rat forebrain slices were stimulated with
100 µM carbachol, with 100 µM IBMX plus 20 µM forskolin, or with 10 ng/ml NGF for 20 min at room
temperature. Lysates were prepared, with 2.5% reserved for direct
analysis. Ras-GRF1 was immunoprecipitated from 97.5% of the lysate
using antibody sc-224; the immunoprecipitates were then split, with
10% subjected to Western blotting for total Ras-GRF1 using antibody
sc-863 (left panel) and 90% subjected to Western blotting
with antibody 2152 (raised against Ras-GRF1 Ser(P)898
(GRF1(phospho898)); right panel). Negative
control immunoprecipitations ((-ve) i.p.) were performed
with rabbit anti-mouse immunoglobulin (Jackson Laboratories). Data
shown are representative of four independent experiments.
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Fig. 9.
Regulated phosphorylation of endogenous
Ras-GRF1 at Ser898 in rat cerebral cortex as revealed by
indirect confocal immunofluorescence. Forebrain slices of
postnatal day 15 rat brains were fixed and processed for indirect
confocal immunofluorescence. The primary antibodies used were as
follows: antibody 2152, raised against Ras-GRF1 Ser(P)898
(A, the yellow arrows indicate apical dendrites
of pyramidal cells in the medial prefrontal cerebral cortex, and the
inset shows a lower magnification view); antibody sc-224,
raised against total Ras-GRF1 (B1, the yellow
arrows indicate apical dendrites of pyramidal cells in the medial
prefrontal cerebral cortex, and the red arrows indicate cell
bodies); and antibody sc-863, which recognizes Ras-GRF1 only in Western
blot protocols, but not in immunofluorescence and so was used as a
staining control (B2).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit-dependent pathway and is associated with
increased serine phosphorylation (23), Ras-GRF1 has also been
demonstrated to exhibit Rac-GEF activity that can be induced by the
coexpression of G protein
subunits and is associated with
increased tyrosine phosphorylation (54). Both of these pathways can be
induced by the same agonist, lysophosphatidic acid, at least in NIH-3T3 fibroblast model systems (24, 55). Tyrosine phosphorylation of Ras-GRF1
that is induced by v-Src increases Rac-GEF activity, but does
not increase Ras-GEF activity (56). In contrast,
ACK1-dependent tyrosine phosphorylation of Ras-GRF1
increases Ras-GEF activity, but does not induce Rac-GEF activity (57).
These results suggest that there may be multiple sites of tyrosine
phosphorylation on Ras-GRF1 with distinct effects. Because ACK1 can
couple to both growth factor pathways and the CDC42 small
GTPase, it is possible that ACK1 may participate in the interactions
between the NGF/TrkA pathway and Ras-GRF1 or between CDC42 and Ras-GRF1
(58, 59). In view of the close parallels between the control of the
Ras-GEF and Rac-GEF activities of Ras-GRF1, it is possible that
phosphorylation of Ser916/898 may also participate in the
control of Rac exchange activity, but this remains unclear.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. D. R. Lowy for the original gift of the plasmid encoding CDC25Mm and Drs. M. R. Brann, M. Czech, J. Jackson, and L. A. Feig for gifts of plasmids encoding human muscarinic receptors, Sos1, Myc-tagged H-Ras, and rat Ras-GRF1, respectively. NGF was generously provided by Genentech, Inc. Initial work on the relative expression of Ras-GRF1 and Sos was performed by C. L. Smith in the laboratory of Dr. I. G. Macara. The production of the anti-Ras-GRF1 Ser(P)916 antibody was carried out by Cell Signaling Technology, Inc.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants R01 CA381150 (to R. R. M.) and R01 MH43985 (to R. A.). Work performed at the Cell Imaging and Cytometry Facility Core was supported by NIEHS Center Grants P30 ES06639 and NCI Grant P30 CA22453 from the National Institutes of Health.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.
§ Supported by Initiative for Minority Student Development Grant GM-58905 from the National Institutes of Health.
Supported by Cell Signaling Technology, Inc.
§§ To whom correspondence should be addressed: Dept. of Pharmacology, Wayne State University, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-6022; Fax: 313-577-6739; E-mail: r.mattingly@wayne.edu.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M209805200
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ABBREVIATIONS |
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The abbreviations used are: GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; PKA, protein kinase A; GST, glutathione S-transferase; HA1, hemagglutinin-1; NGF, nerve growth factor; IBMX, isobutylmethylxanthine; Tricine, N-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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