T-type calcium channel trigger p21ras signaling pathway to ERK in Cav3.1-expressed HEK293 cells.
ABSTRACT We constructed a new cell line which stably expressed Cav3.1 and Kir2.1 subunits in HEK293 cells (HEK293/Cav3.1/Kir2.1) in order to investigate the unknown cellular signaling pathways of T-type voltage-dependent calcium channels. The new cell line has a stable resting membrane potential and can activate T-type Ca(2+) channels by KCl-mediated depolarization. We showed that Cav3.1 activation resulted in the level of p21(ras)-GTP in the cells being rapidly decreased during the first 2 min, and then recovering between 2 min and 15 min. The kinetics of ERK activation following Cav3.1 stimulation was also investigated. ERK activation was decreased from 2 min to 5 min after KCl stimulation, which means that Cav3.1 activation reduced ERK activity in the very early stages of activation. In addition, similar results for Cav3.1 activation were also shown in the case of Sos1, Grb2, and Shc, which means that Cav3.1 activation triggers p21(ras) and that this signal is transferred to ERK by Sos1, Grb2, and Shc.
- SourceAvailable from: J David Spafford[Show abstract] [Hide abstract]
ABSTRACT: Cav3 T-type channels are low-voltage-gated channels with rapid kinetics that are classified among the calcium-selective Cav1 and Cav2 type channels. Here, we outline the fundamental and unique regulators of T-type channels. An ubiquitous and proximally located "gating brake" works in concert with the voltage-sensor domain and S6 alpha-helical segment from domain II to set the canonical low-threshold and transient gating features of T-type channels. Gene splicing of optional exon 25c (and/or exon 26) in the short III-IV linker provides a developmental switch between modes of activity, such as activating in response to membrane depolarization, to channels requiring hyperpolarization input before being available to activate. Downstream of the gating brake in the I-II linker is a key region for regulating channel expression where alternative splicing patterns correlate with functional diversity of spike patterns, pacemaking rate (especially in the heart), stage of development, and animal size. A small but persistent window conductance depolarizes cells and boosts excitability at rest. T-type channels possess an ion selectivity that can resemble not only the calcium ion exclusive Cav1 and Cav2 channels but also the sodium ion selectivity of Nav1 sodium channels too. Alternative splicing in the extracellular turret of domain II generates highly sodium-permeable channels, which contribute to low-threshold sodium spikes. Cav3 channels are more ubiquitous among multicellular animals and more widespread in tissues than the more brain centric Nav1 sodium channels in invertebrates. Highly sodium-permeant Cav3 channels can functionally replace Nav1 channels in species where they are lacking, such as in Caenorhabditis elegans.Pflügers Archiv - European Journal of Physiology 02/2014; · 4.87 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Intracellular Ca(2+) transient is crucial in initiating the differentiation of mesenchymal cells into chondrocytes, but whether voltage-gated Ca(2+) channels are involved remains uncertain. Here, we show that the T-type voltage-gated Ca(2+) channel Cav3.2 is essential for tracheal chondrogenesis. Mice lacking this channel (Cav3.2(-/-)) show congenital tracheal stenosis because of incomplete formation of cartilaginous tracheal support. Conversely, Cav3.2 overexpression in ATDC5 cells enhances chondrogenesis, which could be blunted by both blocking T-type Ca(2+) channels and inhibiting calcineurin and suggests that Cav3.2 is responsible for Ca(2+) influx during chondrogenesis. Finally, the expression of sex determination region of Y chromosome (SRY)-related high-mobility group-Box gene 9 (Sox9), one of the earliest markers of committed chondrogenic cells, is reduced in Cav3.2(-/-) tracheas. Mechanistically, Ca(2+) influx via Cav3.2 activates the calcineurin/nuclear factor of the activated T-cell (NFAT) signaling pathway, and a previously unidentified NFAT binding site is identified within the mouse Sox9 promoter using a luciferase reporter assay and gel shift and ChIP studies. Our findings define a previously unidentified mechanism that Ca(2+) influx via the Cav3.2 T-type Ca(2+) channel regulates Sox9 expression through the calcineurin/NFAT signaling pathway during tracheal chondrogenesis.Proceedings of the National Academy of Sciences 04/2014; · 9.81 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: CD13 is a large cell surface peptidase expressed on the monocytes and activated endothelial cells that is important for homing to and resolving the damaged tissue at sites of injury. We showed previously that cross-linking of human monocytic CD13 with activating Abs induces strong adhesion to endothelial cells in a tyrosine kinase- and microtubule-dependent manner. In the current study, we examined the molecular mechanisms underlying these observations in vitro and in vivo. We found that cross-linking of CD13 on U937 monocytic cells induced phosphorylation of a number of proteins, including Src, FAK, and ERK, and inhibition of these abrogated CD13-dependent adhesion. We found that CD13 itself was phosphorylated in a Src-dependent manner, which was an unexpected finding because its 7-aa cytoplasmic tail was assumed to be inert. Furthermore, CD13 was constitutively associated with the scaffolding protein IQGAP1, and CD13 cross-linking induced complex formation with the actin-binding protein α-actinin, linking membrane-bound CD13 to the cytoskeleton, further supporting CD13 as an inflammatory adhesion molecule. Mechanistically, mutation of the conserved CD13 cytoplasmic tyrosine to phenylalanine abrogated adhesion; Src, FAK, and ERK phosphorylation; and cytoskeletal alterations upon Ab cross-linking. Finally, CD13 was phosphorylated in isolated murine inflammatory peritoneal exudate cells, and adoptive transfer of monocytic cell lines engineered to express the mutant CD13 were severely impaired in their ability to migrate into the inflamed peritoneum, confirming that CD13 phosphorylation is relevant to inflammatory cell trafficking in vivo. Therefore, this study identifies CD13 as a novel, direct activator of intracellular signaling pathways in pathophysiological conditions.The Journal of Immunology 08/2013; · 5.52 Impact Factor
T-type calcium channel trigger p21rassignaling pathway to ERK in
Cav3.1-expressed HEK293 cells
Juhyun Choia,1, Jong-Hwa Parkb,1, Oh Yeun Kwona, Sunoh Kima, Ji Hyung Chungc,
Dae Sik Limd, Key Sun Kima, Hyewhon Rhima,2, Ye Sun Hanb,*,2
aBiomedical Research Center, Korea Institute of Science and Technology, Seoul, Korea
bDepartment of Advanced Technology Fusion, Konkuk University, 1, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Korea
cResearch Institute of Aging Science, and Cardiovascular Genome Center, Yonsei University, Seoul 120-752, Korea
dDepartment of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, Korea
Accepted 3 May 2005
Available online 27 July 2005
We constructed a new cell line which stably expressed Cav3.1 and Kir2.1 subunits in HEK293 cells (HEK293/Cav3.1/Kir2.1) in order to
investigate the unknown cellular signaling pathways of T-type voltage-dependent calcium channels. The new cell line has a stable resting
membrane potential and can activate T-type Ca2+channels by KCl-mediated depolarization. We showed that Cav3.1 activation resulted in the
level of p21ras-GTP in the cells being rapidly decreased during the first 2 min, and then recovering between 2 min and 15 min. The kinetics of
ERK activation following Cav3.1 stimulation was also investigated. ERK activation was decreased from 2 min to 5 min after KCl stimulation,
which means that Cav3.1 activation reduced ERK activity in the very early stages of activation. In addition, similar results for Cav3.1
activation were also shown in the case of Sos1, Grb2, and Shc, which means that Cav3.1 activation triggers p21rasand that this signal is
transferred to ERK by Sos1, Grb2, and Shc.
D 2005 Elsevier B.V. All rights reserved.
Theme: Cellular and molecular biology
Topic: Calcium channel structure, function, and expression
Keywords: Cav3.1 T-type calcium channel; HEK293/Cav3.1/Kir2.1 cells; p21ras; ERK
Voltage-dependent calcium channels (VDCC) in neurons
and other cells have been divided into high and low
voltage-activated (HVA and LVA or T-type) classes
[7,8,26]. T-type VDCCs were found to mediate the low-
threshold spike and to be involved in rebound burst firing,
oscillations, and resonance . T-type VDCCs character-
istically begin to activate at about ?70 mV under
physiological conditions, producing a maximum current at
between ?40 and ?20 mV [7,20]. The current waveform is
transient with a division into rapidly and more slowly
inactivating subtypes [9,21]. Although a class of LVA T-
type calcium channel has been cloned and its native role has
been identified, the molecular function of T-type VDCCs
has so far eluded investigation [12,14,23]. T-type VDCCs
have properties which differ from those of HVA VDCCs,
such as a more negative voltage range for their activation
and inactivation and more rapid gating kinetics . It has
been reported that T-type VDCCs participate in cardiac
pacemaking , as well as the regulation of vascular tone
and hormone secretion [24,37].
In sensory neurones, endogenous Ras is involved in the
tonic upregulation of VDCC , and the activation of the
0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
* Corresponding author. Fax: +82 2 452 5558.
E-mail address: firstname.lastname@example.org (Y.S. Han).
1These authors contributed equally to this work.
2These co-corresponding authors contributed equally to this work.
Brain Research 1054 (2005) 22 – 29
Ras-MAPK pathway was also shown to suppress endoge-
nous T-type VDCC in Swiss 3T3 cells . It remains to be
elucidated how activation of the MAPK pathway inhibits T-
type Ca2+channels. To investigate the unknown cellular
signaling pathways of T-type Ca2+channels, we have
reached the need of making a useful cell line which has a
stable resting membrane potential and can activate T-type
Ca2+channels by biochemical tools such as KCl or channel
activators. Among the inwardly rectifying family of potas-
sium channel subunits (Kir), Kir2 family members show
strong rectification, preferentially passing potassium ions
into the cell. It has been demonstrated that Kir2.1 is one of
major determinants of resting membrane potential in many
cell types . We therefore devised human embryonic
kidney (HEK293) cells stably transfected with Kir2.1
subunit (HEK293/Cav3.1/Kir2.1) from the cells stably
express Cav3.1 subunit, a subset of T-type a1 subunits
We also performed yeast two hybrid assay using intra-
cellular domains of Cav3.1 as baits. In this assay we found
several binding candidates in human brain library (manu-
script in preparation). These included RanBPM, a novel
Met-interacting protein which stimulates Ras activation by
recruiting Sos . Therefore, we investigated a link
between T-type VDCCs and Ras activity.
In the present study, we showed that Cav3.1 activation
reduces the activation of p21rasin HEK293/Cav3.1/Kir2.1
cells. After the activation of Cav3.1 by KCl, the level of
p21ras-GTP rapidly decreased for the first 2 min, and then
recovered between 2 min and 15 min. We also found the
Cav3.1 activation was correlated with ERK activity, as well
as with other signaling molecules, such as Sos1, Grb2, and
2. Materials and methods
2.1. Cloning of human Kir2.1 cDNA
The full-length gene for human Kir2.1 cDNA was
amplified from a human brain cDNA library in plasmid
(Takara, Kyoto) with cKir2.1 forward (5V -CCGCT-
CGAGGCCGCCATGGGCAGTGTGAG-3V ) and cKir2.1
reverse (5V -CCGGAATTGTCATATCTCCGATTCTCGCC-
3V ) as primers. The PCR product was cloned into the XhoI/
EcoRI site of pCMVpuro (Clontech).
2.2. Generation of stably transfected
HEK293 cells stably expressing human Cav3.1 subunit
of T-type Ca2+channels were kindly provided from Dr.
Perez-Reyes (University of Virginia) and grown in Dulbec-
co’s modified Eagle’s medium (DMEM) containing 10%
fetal bovine serum, penicillin (100 units/ml), streptomycin
(100 Ag/ml), and geneticin (500 Ag/ml). For the generation
of HEK293/Cav3.1/Kir2.1 cell line, Kir2.1 (cloned into
pCMVpuro) was transfected with HEK293/Cav3.1 cells
using pEQPAM3 and pVSV-G (Clontech) for the production
of a retrovirus including the Kir2.1 gene. A standard
calcium phosphate transfection procedure was performed.
These retrovirus-infected HEK293/Cav3.1 cells positively
selected with puromycin (1 Ag/ml) in DMEM medium to
generate HEK293/Cav3.1/Kir2.1 cell line which stably
expressed both Cav3.1 and Kir2.1 subunits. Cells were
incubated in a humid atmosphere of 5% CO2and 95% air at
2.3. Intracellular Ca2+imaging
The acetoxymethyl-ester form of fura-2 (fura-2/AM;
Molecular probes, Eugene, OR) was used as the fluorescent
Ca2+indicator. Cells were incubated for 40–60 min at room
temperature with 5 AM fura-2/AM and 0.001% Pluronic F-
127 in a HEPES-buffered solution composed of the fol-
lowing (in mM): 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10
HEPES, 10 glucose, whose pH was adjusted to 7.4 with
NaOH. The cells were then washed with HEPES-buffered
solution and placed on an inverted microscope (Olympus,
Japan). The cells were illuminated using a xenon arc lamp,
and the required excitation wavelengths (340 and 380 nm)
were selected by means of a computer-controlled filter
wheel (Sutter Instruments, CA). Data were acquired every 2
s and a shutter installed in the light path was closed between
exposures to protect the cells from phototoxicity. Emitter
fluorescence light was reflected through a 515-nm long-pass
filter to a frame transfer cooled CCD camera, and the ratios
of emitted fluorescence were calculated using a digital
fluorescence analyzer and converted to intracellular-free
Ca2+concentration ([Ca2+]i). All imaging data were
collected and analyzed using Universal Imaging software
(West Chester, PA).
2.4. Immunoprecipitation and Western blot analysis
Stimulation of cells was terminated by washing twice
with ice-cold phosphate-buffered saline (PBS). The cells
were then lysed with 50 mM HEPES buffer, at pH 7.5,
containing 150 mM NaCl, 10% glycerol, 1% Triton X-100,
1.5 mM MgCl2, 1 mM EGTA, 10 Ag/ml leupeptin, 10 Ag/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 0.2
mM sodium orthovanadate for 20 min at 4 -C. The cells
were scraped from the dishes and centrifuged at 15,000?g
for 20 min at 4 -C. The resulting supernatant was mixed
with the antibodies which had been bound to protein A/G
plus agarose (Santa Cruz Biotechnology, Santa Cruz, CA),
rotated for 1 h at 4 -C, and then the beads were spun down
and washed with lysis buffer. The resulting immunopreci-
pitates were boiled in sample buffer, electrophoresed in SDS
polyacrylamide gel, transferred to a PVDF membrane
(Millipore, Bedford, MA), and then blotted with the
J. Choi et al. / Brain Research 1054 (2005) 22–29
2.5. In-vivo Ras activation assay
HEK293/Cav3.1/Kir2.1 cells were treated with or with-
out 150 mM KCl and lysed for a subsequent in vivo Ras
activation assay. Cell lysates (1 mg) prepared from
HEK293/Cav3.1/Kir2.1 cells were incubated with 40 Ag
of Raf-1 RBD agarose (Upstate Biotech) at 4 -C for 2 h.
The agarose beads were collected, washed, resuspended in
the sample buffer (Upstate Biotech), and resolved on an
SDS–PAGE gel, followed by Western blotting with anti-
Ras monoclonal antibody (Upstate Biotech). The cell
lysates, incubated with GDP and GTPgS (Upstate Bio-
tech), were served as negative and positive controls,
respectively. The cell lysates of each sample were used
for Western blotting with anti-Ras monoclonal antibody as
a loading control.
2.6. ERK activity assay
This assay was performed according to a previously
described method . Cells were stimulated with 150
mM KCl and harvested as described above. Lysates,
containing 500 Ag protein, were immunoprecipitated with
2 Ag of anti-p42ERKantibody (Santa Cruz Biotechnology).
Immunoprecipitation was carried out at 4 -C for 2 h
before adding protein A/G plus agarose (Santa Cruz
Biotechnology) and incubating for an additional 1 h. The
immunoprecipitates were washed twice in lysis buffer and
twice in 20 mM HEPES buffer, at pH 7.4, containing 10
mM MgCl2 and 0.2 mM sodium orthovanadate and
incubated in 0.25 mg/ml myelin basic protein (MBP),
50 AM ATP, and 5 ACi [g-32P]ATP for 15 min at 30 -C.
The reaction was terminated by the addition of 2?
sample buffer and the protein was subjected to electro-
phoresis in a 12% SDS–PAGE gel and visualized by
3.1. High KCl-mediated [Ca2+]iincrease in
In order to investigate the unknown cellular signaling
pathways of T-type Ca2+channels, we constructed a new cell
line which stably expressed Cav3.1 and Kir2.1 subunits in
HEK293 cells (HEK293/Cav3.1/Kir2.1) . To confirm the
activation of Cav3.1 T-type Ca2+channels directly in
HEK293/Cav3.1/Kir2.1 cells, we measured KCl-mediated
changes in intracellular Ca2+concentration ([Ca2+]i) using
fura-2-based digital imaging techniques. In HEK293/Cav3.1
cells, there was no detectable change of [Ca2+]iby 150 mM
treatment of KCl (data not shown). However, application of
high concentration of KCl (150 mM, 30 s) produced a
transient increase of [Ca2+]iin HEK293/Cav3.1/Kir2.1 cells.
Treatment of 150 mM KCl induced the increase of [Ca2+]i
from 75 to 120 nM in a regular HEPES-buffered solution
(Fig. 1). Furthermore, this increase was totally blocked by
co-treatment of 10 AM mibefradil, the potent T-type Ca2+
channel blocker, with partial recovery. These results indicate
that the increase of [Ca2+]iby KCl-induced depolarization is
fully responsible for Cav3.1 channel activated in HEK293/
Cav3.1/Kir2.1 cells. Altogether, the detailed functional anal-
ysis performed in this study by comparing HEK293/Cav3.1
and HEK293/Cav3.1/Kir2.1 cells provides a compelling
evidence that the stable expression of Kir2.1 channels only
made it possible to construct a cell line which could activate
T-type Ca2+channels by KCl-mediated depolarization .
3.2. Cav3.1 activation rapidly reduces p21ras-GTP in
The activity of p21rasis regulated through its binding of
guanine nucleotides, the GTP-bound active form of the
Fig. 1. High KCl-mediated [Ca2+]iincrease in HEK293/Cav3.1/Kir2.1 cells. [Ca2+]irecording in HEK293/Cav3.1/Kir2.1 cells by membrane depolarization
using 150 mM KCl. Application of T-type calcium channel blocker, mibefradil (1 AM, 1 min), reduced the 150 mM KCl (30 s)-induced [Ca2+]iincrease by
93.6% in this cell line.
J. Choi et al. / Brain Research 1054 (2005) 22–29
protein being able to interact with effector proteins such as
Raf-1 and phosphatidylinositol 3-kinase (PI-3-K) . The
kinetics of Cav3.1 stimulated p21rasactivation has, as yet,
not been studied.
In the present study, p21rasactivation was assessed by
measuring the amount of p21ras-GTP, using Raf1-RBD and
the p21rasmonoclonal antibody described in the Materials
and methods section. Firstly, we investigated whether the
activation of Cav3.1 reduces the activation of p21ras. The
initial decrease in p21ras-bound GTP, which peaks at 2 min,
is followed by a subsequent increase in p21Ras-bound GTP
which returns to its initial value at 15 min and thereafter
decreases again to attain the zero level at 120 min post-
stimulation (Fig. 2A). The application of 150 mM KCl over
a period of 2 h triggers a transient increase in [Ca2+]i(about
130 s, peak level = 133 nM) (Fig. 2C). This increase of
[Ca2+]iwas completely inactivated ([Ca2+]ilevel = 56.7 T
0.6 nM) after 15 min. These data provide evidence that the
early period of stimulation of Cav3.1 results in a decrease in
the amount of p21ras-GTP in HEK293/Cav3.1/Kir2.1 cells.
This sudden decrease of p21ras-GTP is recovered at 15 min
after stimulation, and then gradually decreases again up
until 2 h. Compared to the HEK293/Cav3.1/Kir2.1 cells, in
which KCl stimulation altered the level of p21ras-GTP, no
such change was observed in the HEK293/Cav3.1 cells (Fig.
2B). The application of 150 mM KCl over a period of 1 h
did not alter the response to Cav3.1 in any of the HEK293/
Cav3.1 cells ([Ca2+]ilevel = 58.8 T 1.2 nM, n = 11) (Fig.
2D). These data showed that the very early inactivation of
p21rasis solely dependent on the stimulation of Cav3.1 by
3.3. The effect of Cav3.1 activation on ERK
The p21raseffectors, which are known to predominantly
mediate growth stimulatory signals, are the serine/threonine
Raf kinases . To date, p21rasis known to interact,
through the effector region, with the N-terminal domain of
Raf, regulating its kinase activity [25,36]. Raf activates
MEK by phosphorylating its two regulatory serine residues
. MEK1 and MEK2 are dual specific kinases capable of
phosphorylating regulatory serine/threonine and tyrosine
residues on ERK (also referred to as p44 and p42 MAP
kinase) . Thus, when activated, ERK is itself able to
phosphorylate the serines and threonines of cytoplasmic and
nuclear proteins, such as transcription factors, thereby
transducing proliferative and differentiation signals to the
nucleus . ERK can also phosphorylate membrane
proteins including ion channels .
In this study, we investigated the kinetics of ERK
activation following Cav3.1 stimulation. ERK activation
was assessed both by the delayed mobility of the phos-
phorylated form on SDS–PAGE and the ability of
immunoprecipitated p42ERKto phosphorylate myelin basic
Fig. 2. Activation of Cav3.1 briefly reduces p21rasin HEK293/Cav3.1/Kir2.1 cells. (A) HEK293/Cav3.1/Kir2.1 cells or (B) HEK293/Cav3.1 cells which do not
express Kir2.1 were exposed to 150 mM KCl and incubated for different periods of time. Cleared cell lysates were incubated with Raf-1 RBD. The beads were
then washed, and the proteins were resolved by SDS–PAGE. The amount of activated Ras bound to Raf-1 RBD beads was determined by anti-p21ras
immunoblotting. Activation of Cav3.1 did not affect p21rasin HEK293/Cav3.1 cells (B). Cell lysates were also directly subjected to anti-Ras immunoblotting to
determine the levels of Ras in each sample. (C) [Ca2+]irecording of 150 mM KCl responses in HEK293/Cav3.1/Kir2.1 cells. Application of 150 mM KCl (over
2 h) triggers a transient increase in [Ca2+]i(about 130 s, peak level = 133 nM). This increase of [Ca2+]iwas completely inactivated ([Ca2+]ilevel = 56.7 T 0.6
nM) after 15 min. (D) [Ca2+]irecording of 150 mM KCl responses in HEK293/Cav3.1 cells. The application of 150 mM KCl (over 1 h) did not alter the
responses to Cav3.1 in any of the HEK293/Cav3.1 cells ([Ca2+]ilevel = 58.8 T 1.2 nM, n = 11).
J. Choi et al. / Brain Research 1054 (2005) 22–29
protein (MBP) (Figs. 3A and B). As shown in Figs. 3A and
B, the data obtained by these methods are consistent with
the KCl-induced peak of ERK activation which occurred at
15 min. However, ERK activation decreased from 2 min to
5 min after KCl stimulation, which means that Cav3.1
activation reduced ERK activity in the very early stages of
activation. According to our p21rasactivation study, these
variations in ERK activity in stimulated HEK293/Cav3.1/
Kir2.1 cells are correlated with the amount of p21ras-GTP.
The time course for ERK activation appears to be consistent
with that of Ras activity and Cav3.1 activation suggesting
that Cav3.1 activation regulates Ras/ERK activity.
3.4. Cav3.1 activation stimulates tyrosine phosphorylation
of Shc and its association with Grb2 and Sos1
It has been shown that the Shc proteins are involved in
activation of Ras via receptor tyrosine kinases [10,30]. In
this study we investigated whether Cav3.1 activation could
bring about Shc phosphorylation in HEK293/Cav3.1/Kir2.1
cells. As shown in Fig. 3C, a KCl-induced peak of
phosphorylated Shc occurred at 15 min. However, phos-
phorylated Shc activation was decreased from 2 min to 5
min, i.e., in the very early period following KCl stimulation,
which means that Cav3.1 activation reduced Shc phosphor-
ylation in the very early stages. This process of phosphor-
ylation persisted for at least 120 min, with a peak occurring
at 15 min. The anti-Shc antibody was able to immunopre-
cipitate all three isoforms of Shc, as determined by
immunoprecipitation and immunoblotting with the same
antibody (Fig. 3C). Although the variation of phospho-
p52Shcwithin each sample were not distinguishable, this
phenomenon can be explained in light of a previous study
. Several agonists have been shown to induce Shc
phosphorylation, not only at Tyr317 but also at Tyr239 and
Tyr240 [16,17,35]. It has been reported that phosphorylation
at positions Tyr239 and Tyr240 does not contribute to
p21ras/ERK activation and has little involvement with Grb2
association . Shc phosphorylation creates a binding site
for the SH2 domain of the SH2/SH3 domain-containing
adaptor protein, Grb2 . The SH3 domains of Grb2 have
been shown to interact with Sos1 [6,13]. This event leads to
the recruitment of Sos1 to the membrane and enables it to
activate p21ras. To verify whether, and with what kinetics,
Grb2 associates with Shc in HEK293/Cav3.1/Kir2.1 cells
stimulated with KCl, we performed immunoprecipitations
with anti-Shc antibody at different time points post Cav3.1
activation, followed by immunoblotting with an anti-Grb2
antibody. As shown in Fig. 4A, Grb2/Shc association
suddenly increased between 5 and 15 min following the
activation of Cav3.1. Moreover, to confirm the Grb2 to
associate with the phosphorylated form of p52Shc, we
precipitated phospho-p52Shcwith Grb2. As shown in Fig.
4B, Grb2 can precipitate phospho-p52Shcbetween 5 and 30
min and peak was shown at 15 min after the activation of
Cav3.1. Immunoblotting analysis showed that the p52Shc
isoform, which is the most prevalent isoform in HEK293/
Cav3.1/Kir2.1, plays a major role in the association of Shc
3.5. Peak activation of p21rasand ERK in 15 min after
Cav3.1 activation is correlated with Grb2/Shc interaction
To verify the association of Grb2 with Sos1, Sos1
immunoprecipitates were immunoblotted with anti-Grb2
antibody. As shown in Fig. 4C, Sos1 was able to associate
with Grb2 in HEK293/Cav3.1/Kir2.1 cells and this associ-
ation rapidly decreased at 2 min after KCl treatment and
then recovered its initial level at 15 min. After about 30 min,
the association began to decrease. All Sos1 immunoblotting
experiments were generated by over-exposure. These results
indicate that KCl stimulation increases the Grb2/Sos1
complex at 15 min, which correlates temporally with
p21rasactivation. Finally, we verified whether Sos1 is
associated with Shc, as a result of the association of Grb2
with both Sos1 and Shc, after KCl stimulation, and whether
Fig. 3. Monophasic activation of ERK by KCl and Shc phosphorylation during the time period of KCl stimulation of HEK293/Cav3.1/Kir2.1 cells. HEK293/
Cav3.1/Kir2.1 cells were stimulated with KCl (150 mM) for the times indicated. (A) Immunoblot detection of the two isoforms of ERK. Activation is verified
by the appearance of bands with delayed mobility related to the phosphorylated form of the protein. (B) ERK activity assayed by the ability of the
immunoprecipitated p42ERKto phosphorylate MBP as substrate. (C) Cell lysates prepared from HEK293/Cav3.1/Kir2.1 cells were immunoprecipitated with
anti-Shc polyclonal antibody. Immunoprecipitates were analyzed by SDS–PAGE and immunoblotted with anti-phosphotyrosine (anti-PY) antibody and anti-
Shc antibody as indicated. Shown here is a representative result; the experiment was repeated three times.
J. Choi et al. / Brain Research 1054 (2005) 22–29
the association of Sos1/Shc follows the kinetics of the
association of Shc with Grb2. Sos1 was immunoprecipitated
from KCl treated or untreated HEK293/Cav3.1/Kir2.1 cells
and immunoblotted with anti-Shc antibody. In can be seen
in Fig. 5A that Shc can precipitate Sos1 between 5 and 30
min, and that this precipitation peaks at 15 min after the
activation of Cav3.1 by 150 mM KCl treatment. This result
was also correlated with the p21rasactivation event.
However, we could not clearly verify the immunoprecipi-
tation and immunoblotting of Sos1 in detail (Fig. 5B). This
phenomenon can be explained by the relative excess of
cytosolic Grb2 and Shc compared to the small amount of
Sos1 under the experimental conditions used in this study.
For three decades Ca2+has been known to affect cell
proliferation and differentiation [3,5]. This provided the
initial evidence of the potential link between Ca2+and Ras
signaling. Recently, increased intracellular Ca2+resulting
from Ca2+influx through voltage-dependent ion channels
was found to activate Ras in rat pheochromocytoma (PC12)
cells and primary cultures of rat cortical neurons [11,29]. In
this study, we showed that the activation of Cav3.1, a T-type
voltage-dependent calcium channel, results in a rapid
reduction of the p21rassignaling pathway, which peaks at
15 min, and that this signal was transferred to ERK. The
construction of HEK293/Cav3.1/Kir2.1 cells specifically
activating Cav3.1 was very useful in this assay. Cav3.1
activation resulted in the level of p21ras-GTP in the cells
being rapidly decreased during the first 2 min and the level
of p21ras-GTP gradually increased up to 15 min, then
rapidly decreased again (Fig. 2A).
The initial decrease at 2 min was different with general
signaling processes. Most signaling induced by stimulation
is gradually increased or decreased by the time. It is possible
that an initial decrease of p21ras signaling might be the
resultant of using stably transformed HEK293/Cav3.1/
Kir2.1 cells. The experiments for the identification of this
behavior are now investigating.
ERK activation was observed at 15 min, as shown in Fig.
3B, and then gradually decreased. This phenomenon was
also mentioned in another recent report . From this data,
it was concluded that the role of Cav3.1 in HEK293/Cav3.1/
Kir2.1 activation is to trigger Ras signaling, and that this
signal is transferred to ERK with a unique pattern. Recent
Fig. 5. Grb2 mediates the association of Sos1 and Shc. Lysates from
HEK293/Cav3.1/Kir2.1 cells stimulated with KCl (150 mM) for the times
indicated were immunoprecipitated with anti-Shc (A) or anti-Sos1 anti-
bodies (B). The resultant immunoprecipitates were resolved by SDS–
PAGE and then immunoblotted with anti-Shc and anti-Sos1 antibodies.
Shown here is a representative result; the experiment was repeated three
Fig. 4. Activation of Cav3.1 induces association of Shc/Grb2 and Sos1/Grb2. Lysates from HEK293/Cav3.1/Kir2.1 cells stimulated with KCl (150 mM) for the
times indicated were immunoprecipitated with anti-Shc (A) or anti-Grb2 antibodies (B). The resultant immunoprecipitates were resolved by SDS–PAGE and
then immunoblotted with anti-Grb2 and anti-PY antibodies, respectively. (C) HEK293/Cav3.1/Kir2.1 cells stimulated with KCl (150 mM) for the indicated
times. Cell lysates were immunoprecipitated with anti-Sos1 antibody. Shown here is a representative result; the experiment was repeated three times.
J. Choi et al. / Brain Research 1054 (2005) 22–29
evidence suggests that p21rasreduces the l-type calcium
channel current in cardiac Myocytes . In this report,
p21raswas found to diminish l-channel promoter activity
through the Raf-MEK-ERK pathway. It was reported that
p21rasreduces the T-type calcium channel density in Swiss
3T3 fibroblasts [19,33]. Similar results for Cav3.1 activation
were also shown in the case of Sos1, Grb2, and Shc. Sos1 is
normally localized to the cell cytosol, however, in response
to growth factor stimulation, it is recruited to the plasma
membrane. p21rasis localized to the interior of the plasma
membrane, so simply targeting Sos to this membrane
efficiently regulates p21rasactivation. This is a key concept
in the activation–deactivation cycle of p21ras. Sos is
recruited to the plasma membrane in complexes with the
adaptor proteins, growth-factor receptor-bound protein 2
(Grb2) and Src-homology-2 domains containing Shc, which
bind to phosphotyrosine-binding domains. In our results,
Sos1, Grb2, and Shc were correlated with p21raskinetics
after Cav3.1 activation, which means that Shc, Grb2, and
Sos are involved in the activation pathways upstream of Ras
and it is activation of Ras that triggers ERK activation via
Raf and MEK. The data presented here will likely improve
our understanding of T-type calcium channel signaling in
the brain and may provide a basic understanding of how an
ion channel activates transcriptional regulation and cell
This work was supported by the intramural program
of KIST, the National Research Laboratory Program
(M10400000046-04J0000-04610), Real Time Molecular
Imaging Project, the Brain Research Center of the 21st
Century Frontier Research Program (M103KV010004-
03K2201-00420), and the 21C Frontier Functional Proteo-
mics Project from the Korean Ministry of Science and
Technology. The authors extend their appreciation to Dr.
Perez-Reyes for providing HEK293 cells stably expressing
human Cav3.1 T-type Ca2+channels.
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