Activation of P2Y2 Receptors by UTP and ATP
Stimulates Mitogen-activated Kinase Activity through a Pathway That
Involves Related Adhesion Focal Tyrosine Kinase and Protein Kinase
C*
Stephen P.
Soltoff
§,
Hava
Avraham¶,
Shalom
Avraham¶, and
Lewis C.
Cantley
From the
Divisions of Signal Transduction and
¶ Hematology/Oncology, Department of Medicine, Beth Israel
Deaconess Medical Center and the
Department of Cell Biology,
Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
We examined downstream signaling events that
followed the exposure of PC12 cells to extracellular ATP and UTP, and
we compared the effects of these P2 receptor agonists
with those of growth factors and other stimuli. Based on early
findings, we focused particular attention on the mitogen-activated
protein (MAP) kinase pathway. ATP and/or UTP produced increases in
tyrosine phosphorylation of multiple proteins, including p42 MAP (ERK2)
kinase, related adhesion focal tyrosine kinase (RAFTK) (PYK2, CAK
),
focal adhesion kinase (FAK), Shc, and protein kinase C
(PKC
). MAP
(ERK2) kinase activity (quantified by substrate phosphorylation) was
increased by UTP, ATP, phorbol 12-myristate 13-acetate, ionomycin, and
growth factors. UTP and ATP were equipotent (EC50 ~25
µM) in stimulating MAP kinase activity, suggesting that
these effects were mediated via the Gi-linked
P2Y2 (P2U) receptor. Consistent with this, the UTP- and ATP-promoted activation of MAP kinase was diminished in
pertussis toxin-treated cells. Treatment of cells with pertussis toxin
also reduced both the UTP-dependent increases in
intracellular calcium ion concentration
([Ca2+]i) and the tyrosine
phosphorylation of RAFTK. Similarly, when
[Ca2+]i elevation was prevented
using BAPTA and EGTA, the activation of MAP kinase by UTP and ionomycin
was blocked, and the tyrosine phosphorylation of RAFTK was reduced. The
UTP-promoted increase in MAP kinase activity was partially reduced in
cells in which PKC was down-regulated, suggesting that both
PKC-dependent and PKC-independent pathways were involved.
PKC
, which increases MAP kinase activity in some systems, became
tyrosine-phosphorylated within 15 s of exposure of cells to ATP or
UTP; but epidermal growth factor, nerve growth factor, and insulin had
little effect. UTP also promoted the association of Shc with Grb2.
These results suggest that the P2Y2 receptor-initiated
activation of MAP kinase was dependent on the elevation of
[Ca2+]i, involved the recruitment
of Shc and Grb2, and was mediated by RAFTK and PKC.
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INTRODUCTION |
P2-type purinoceptors constitute a diverse set of
proteins that are linked by their common ability to bind extracellular
ATP and elicit an increase in intracellular Ca2+ and other
ions. ATP is a neurotransmitter or co-transmitter in some systems
(1-3), and this has been the proposed rationale for the existence of
P2 receptors on some cells. Previously, classifications of
receptor subtypes were based on pharmacological hierarchies of the
responses to ATP analogs (4, 5). The P2 subclassifications have been reorganized based on recent molecular biology approaches and
now enfold more than a dozen types of P2 receptors into a family of two main receptor types: P2X and P2Y
purinoceptors. P2X receptors are ligand-gated ion channels,
and P2Y receptors are seven transmembrane proteins that are
GTP-dependent protein (G-protein)1-coupled
receptors (6, 7).
In preliminary studies we observed that ATP and UTP produced
alterations in the tyrosine phosphorylation of multiple proteins in
PC12 cells, including one similar in mass to mitogen-activated protein
(MAP) (p42 ERK) kinase, suggesting the involvement of the
P2Y2 receptor in various signal transduction events. This receptor is a 53-kDa protein (8) that is linked by a heterotrimeric G-protein to phospholipase C, and UTP promotes the hydrolysis of
phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol
1,4,5-trisphosphate, which activate protein kinase C (PKC) and elevate
the intracellular calcium ion concentration ([Ca2+]i), respectively. The
P2Y2 receptor has been postulated to be activated by the
physiological release of intracellular ATP from cells, and thus ATP may
act as an autocrine or paracrine factor (9-11). However, signaling
molecules that are downstream of the P2Y2 and other
P2 receptors have not been characterized as well as those
activated by growth factors.
In the present study we focused attention on several proteins,
particularly those involved in the MAP kinase cascade, to determine which signaling molecules were stimulated by activation of the P2Y2 receptor by ATP and UTP. In addition, we compared the
effects of extracellular nucleotides and growth factors on PKC
tyrosine phosphorylation. PKC
, a Ca2+-insensitive member
of the PKC family of proteins, is phosphorylated on tyrosine residues
in response to various stimuli. These include the activation of
platelet-derived growth factor receptors in NIH3T3 and 32D cells (12),
muscarinic and substance P receptors in freshly isolated parotid acinar
cells (13), and the transformation of cells with the oncogenic Ras
protein (14). In 32D cells, a murine myeloid progenitor cell line, the
tyrosine phosphorylation of PKC
was associated with cell
differentiation (13). However, as reported here, we find that nerve
growth factor (NGF), which promotes differentiation (neurite outgrowth)
in PC12 cells, did not promote the tyrosine phosphorylation of PKC
;
in contrast, the activation of the P2Y2 receptor was much
more effective in promoting this phosphorylation.
Our results indicate that MAP (p42 ERK) kinase activity was increased
by UTP by a mechanism distinct from that of growth factors. MAP kinase
activation by UTP was correlated with the tyrosine phosphorylation of
related adhesion focal tyrosine kinase (RAFTK), a protein that is
upstream of MAP kinase, and conditions that reduced the tyrosine
phosphorylation of RAFTK also reduced the activation of MAP kinase by
UTP. The tyrosine phosphorylation of p42 MAP kinase, PKC
, RAFTK, and
other proteins in cells treated with ATP and UTP indicates that
stimulation of the P2Y2 receptor promotes the activation of
multiple signaling molecules and demonstrates that tyrosine
phosphorylation is involved in mediating signal transduction events
initiated by the activation of this G-protein-coupled receptor.
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MATERIALS AND METHODS |
Reagents--
All chemicals were reagent grade or better.
Dulbecco's modified Eagle's medium and phorbol 12-myristate
13-acetate (PMA) were obtained from Life Technologies, Inc. Calf serum,
horse serum, UTP, and ATP were purchased from Sigma. EGF was from
Upstate Biotechnology Co. (Lake Placid, NY), insulin from Collaborative
Biomedical Products (Bedford, MA), and NGF (2.5 S) from Boehringer
Mannheim. [32P]ATP (specific activity, 3,000 Ci/mmol) was
purchased from NEN Life Science Products. Anti-phosphotyrosine
(anti-P-Tyr) antibody was a generous gift of Dr. Tom Roberts (Dana
Farber, Boston). Polyclonal anti-RAFTK antibody was produced as
described previously (15). Polyclonal anti-Shc (S14630) and monoclonal
anti-Shc (S14620) antibodies were purchased from Transduction
Laboratories (Lexington, KY). Anti-PKC
(SC-213) and anti-ERK2
(SC-154) antibodies were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Pertussis toxin (PTX; product 180) was purchased from
List Biological Laboratories, Inc. (Campbell, CA).
Cell Culture--
PC12 cells were grown in 100-mm-diameter
dishes at 37 °C in a mixture of 95% air and 5% CO2 in
Dulbecco's modified Eagle's medium with 5% calf serum and 5% horse
serum. Cells were used at or near confluence. In all experiments, cells
were maintained overnight in low serum medium (Dulbecco's modified
Eagle's medium plus 0.05% calf serum, 0.05% horse serum). Cells were
exposed to stimuli by exchanging this medium with medium containing
stimuli or vehicle. Cells in which PKC was down-regulated were exposed to 1 µM PMA or vehicle (0.06% dimethyl sulfoxide)
overnight (~16 h). In some experiments cells were exposed to PTX (100 ng/ml) or vehicle (0.1 mM sodium phosphate, pH 7.0; NaCl,
0.5 mM) overnight (~16 h). In experiments in which
elevations in intracellular calcium elevation were blocked,
serum-starved cells were exposed to 10 µM BAPTA or
vehicle (0.1% dimethyl sulfoxide) for 1 h and then switched to
low serum medium ± 5 mM EGTA ± stimuli or
vehicle for 5 min.
Immunoprecipitation and Western Blotting--
After cells were
exposed to a stimulatory agent for the designated time, the cells were
washed twice with an ice-cold buffered saline solution (137 mM NaCl, 20 mM Tris base, 1 mM
EGTA, 1 mM EDTA, 0.2 mM vanadate, pH 7.5) and
were lysed in lysis buffer (137 mM NaCl, 20 mM
Tris base, pH 7.5, 1 mM EGTA, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40) containing the following phosphatase and protease inhibitors: 1 mM vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM
ZnCl2, 4.5 mM sodium pyrophosphate, 2 mg/ml
NaF, 2 mg/ml
-glycerophosphate, 1 µg/ml leupeptin, and 1 µg/ml
pepstatin. The lysates were vortexed thoroughly and centrifuged at
16,000 × g (Eppendorf 5414 microcentrifuge). The
cleared supernatants were transferred to fresh microcentrifuge tubes. A
portion (5-10% of the volume) of the lysate was removed and combined
with an equal volume of 2 × sample buffer (62.5 mM
Tris, pH 6.8, 10% (v/v) glycerol, 6.25% (v/v) SDS, 0.72 N
-mercaptoethanol, and bromphenol blue for color). The remainder was
incubated at 4 °C for 3 h or overnight with anti-PKC
(1-2
µg/ml), anti-ERK2 (1 µg/ml), polyclonal anti-Shc (1 µg/ml),
anti-RAFTK (5 µl), or anti-P-Tyr antibody (~6 µg/ml), plus
protein G-Sepharose (4 mg/ml) with the anti-RAFTK antibody and protein
A-Sepharose (4 mg/ml) with the other antibodies. At the end of the
incubation, the immunoprecipitates were collected by centrifugation.
The immunoprecipitates were washed three times in ice-cold
phosphate-buffered saline (137 mM NaCl, 15.7 mM
NaH2PO4, 1.47 mM
KH2PO4, 2.68 mM KCl, 1% Nonidet
P-40, pH 7.4), twice in 0.1 M Tris, pH 7.5, and 0.5 M LiCl, and two times in TNE (10 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.5). All wash solutions
contained 0.2 mM vanadate. The majority of the TNE was
removed, the remaining volume was diluted with an equal volume of
2 × sample buffer, and the samples were boiled for 5-10 min. The
immunoprecipitated proteins and the lysate fractions were subjected to
electrophoresis or stored at
80 °C before electrophoresis.
Samples were subjected to electrophoresis on an SDS-polyacrylamide
separating gel with a 3% stacking gel. The separating gel was 12% for
anti-Shc immunoprecipitates, and 7% for all others. Proteins were
transferred to 0.2-µm pore size nitrocellulose filters, and the
filters were blocked with TBS (20 mM Tris, pH 7.6, 137 mM NaCl) and 2% (w/v) bovine serum albumin for 1 h.
The filters were washed in TTBS (TBS and 0.2% (v/v) Tween 20) three
times. The nitrocellulose filters were exposed to blotting antibodies in TTBS and 1% bovine serum albumin for ~16 h at 4 °C. The
filters were washed three times in TTBS and exposed to anti-rabbit or anti-mouse horseradish peroxidase (Boehringer Mannheim) at a 1:10,000 dilution in TTBS and 1% bovine serum albumin for 1 h. All washes and exposure to the secondary antibody were performed at room temperature. Filters were washed three times with TTBS and twice with
TBS and were visualized on x-ray film (Kodak) using a chemiluminescence system (Amersham or NEN Life Science Products). In some experiments, the filters were stripped of antibodies by exposing them to 62.5 mM Tris, pH 6.8, 0.1 M
-mercaptoethanol, and
2% (w/v) SDS at 70 °C for 40 min. The stripped filters were washed
several times in TTBS, once in TBS, blocked with TBS and 2% bovine
serum albumin for 1 h, and reprobed with antibody overnight. Blots
were then treated as described above.
ERK2/MAP Kinase Activity--
ERK2 was immunoprecipitated from
PC12 cells by incubating cleared lysates with anti-ERK2 antibody and
protein A-Sepharose beads for 3-4 h (see above). The
immunoprecipitates were washed two times with RIPA buffer containing 1 mM vanadate and two times with kinase buffer (50 mM Tris, pH 7.5, 5 mM MgCl2).
Immunoprecipitates were resuspended in a final volume of 50 µl of
kinase buffer containing myelin basic protein (MBP) (200 µg/ml kinase
buffer), and the kinase assay was initiated with the addition of 40 µM ATP plus [32P]ATP (1 µCi). After 30 min, in some experiments 20 µl of the supernatant was removed and
spotted onto p81 Whatman paper. An identical volume of supernatant was
collected and spotted from a kinase mixture prepared in the absence of
immunoprecipitated proteins as a control, and this background value of
radioactivity was subtracted from all samples. Unreacted
[32P]ATP was removed by rinsing the p81 papers five times
with 0.22 M phosphoric acid, and the phosphorylated MBP on
each p81 paper was quantified by scintillation counting. In other
experiments the supernatant was added to an equal volume of 2 × sample buffer, boiled, and subjected to SDS-PAGE using a 15%
separating gel. The phosphorylated MBP was quantified using a molecular
imager system (Bio-Rad GS-363). Similar results were obtained using the two methods of MAP kinase quantification.
[Ca2+]i--
PC12 cells were suspended
in a physiological salt solution containing 2 µM fura-2
acetoxymethylester and 10 µM probenecid in a
physiological salt solution for 50 min, as reported previously for
other cells (16). The cells were washed twice, and
[Ca2+]i was monitored at room
temperature in a SPEX FluoroMax-2 spectrofluorometer.
[Ca2+]i was quantified from the
fluorometric ratio emitted at 510 nm from dual wavelength excitation at
340 and 380 nm.
Data--
Immunoblots similar to those shown in the figures were
obtained in two or more independent experiments. The numbers
(n) of independent assays of ERK2 activity were as noted on
the figures or in the text.
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RESULTS |
UTP, ATP, and PMA Promote the Tyrosine Phosphorylation of Multiple
Proteins, Including MAP Kinase--
Analysis of the tyrosine
phosphorylation pattern of cells treated with UTP, ATP, and PMA
indicated that multiple proteins were phosphorylated after various
lengths of exposure to ATP and UTP (Fig.
1). These included proteins at the
following apparent molecular masses (and the time of peak tyrosine
phosphorylation): ~159 kDa (UTP or ATP for 1 min), ~102 kDa and
~90 kDa (ATP or UTP for 1 or 5 min), ~76 kDa (UTP or PMA for 5 min), ~63 and ~67 kDa (UTP or ATP for 1 min), ~49 kDa (UTP or ATP
for 1 min), ~40 kDa and ~44 kDa (UTP and PMA for 5 min). Thus,
there were distinct proteins that exhibited different peak times in
tyrosine phosphorylation when exposed to P2Y2 agonists or
PMA. Several proteins were identified by reprobing the blots with
antibodies to known proteins of similar size. The ~40- and ~44-kDa
proteins co-localized with the MAP kinases ERK2 and ERK1 (not shown),
respectively, which are proteins of 42 and 44 kDa. This suggested that
activation of the P2Y2 (P2U) receptor activated
MAP kinase and affected other signaling proteins in these cells.

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Fig. 1.
UTP, ATP, and PMA increase the tyrosine
phosphorylation of multiple proteins in PC12 cells. Cells were
exposed to UTP (100 µM) or ATP (100 µM) for
0.2-15 min or to PMA (200 µM) for 5 min. Cells were
lysed, and a fraction of the lysate was subjected to SDS-PAGE and was
immunoblotted using anti-P-Tyr antibody (IB Ab; 1 µg/ml).
Molecular mass markers are shown on the left, and the
arrows on the right indicate the location of some
of the proteins that displayed an increase in tyrosine phosphorylation.
For additional information, see "Results."
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PTX Reduces the P2Y2 Receptor-mediated Increase in MAP
Kinase Activity--
Additional experiments were performed to confirm
that MAP kinase activity was increased by activation of the
P2Y2 receptor. In vitro phosphorylation assays
were performed on ERK2 kinase immunoprecipitates using MBP as a
substrate (see "Materials and Methods"). MAP kinase activity was
increased in cells exposed to UTP (100 µM) or ATP (100 µM) for 5 min (Fig.
2A). This period of time (5 min) was one at which there appeared to be a readily detectable
increase in tyrosine phosphorylation of MAP kinase promoted by these
stimuli (Fig. 1). In experiments in which the effects of UTP (100 µM) and ATP (100 µM) were both measured
after a 5-min exposure, the activation of MAP kinase by UTP was
67.0 ± 9.0% (10) as large as that produced by ATP, consistent
with the ability of either UTP or ATP to bind to and activate the
P2Y2 receptor. There were negligible increases in MAP
kinase activity in cells treated with UTP (100 µM) for 1 min. PMA, EGF, NGF, and the calcium ionophore ionomycin also produced
large increases in MAP kinase activity after a 5-min exposure (Fig.
2A), as reported by others.

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Fig. 2.
Extracellular nucleotides, growth factors,
and calcium ionophore increase MAP kinase activity in PC12 cells.
Panel A, cells were exposed to ATP (100 µM),
UTP (100 µM), PMA (200 nM), NGF (100 ng/ml),
EGF (100 ng/ml), or ionomycin (10 6 M) for the
indicated periods of time (in min). Cells were lysed, and p42 ERK2 was
immunoprecipitated using anti-p42 ERK2 antibody. MAP (ERK2) kinase
activity was assayed by measuring the phosphorylation of MBP and is
shown relative to the basal (untreated) level. The values are
means ± S.E., and the numbers in
parentheses at the bottom of each bar
indicate the number of individual experiments. For additional
information, see "Results." Panel B, concentration dependence of ATP and UTP on MAP kinase activation. Cells were treated
with 3, 10, 100, or 1,000 µM nucleotide for 5 min, and MAP kinase activity was measured as in panel A. For each
experiment, MAP kinase activation was calculated by subtracting the
basal rate of MAP kinase activity from that obtained in the presence of
the different concentrations of ATP or UTP and normalizing the
increases in MAP kinase activity to that obtained using 1000 µM ATP or UTP, respectively. Data represent the mean ± S.E. of three (ATP) or four (UTP) experiments, except 3 µM UTP (n = 1). The absolute value of MAP
kinase activity at 1,000 µM UTP was 0.45 ± 0.04 (n = 3) times that at 1,000 µM ATP.
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The concentration dependences of ATP and UTP on MAP kinase activity
also were examined. Although the absolute magnitudes of MAP kinase
activities were different for the two nucleotides at each
concentration, the relative effects of different concentrations of each
nucleotide were very similar, and the EC50 value was ~25 µM for both ATP and UTP (Fig. 2B). This is
consistent with the stimulatory effects of ATP and UTP on MAP kinase
being caused by the activation of a P2Y2 receptor.
The activation of MAP kinase by UTP and ATP was reduced by about 40%
in PTX-treated cells (Fig. 3). In
contrast, the basal MAP kinase activity and the activation of MAP
kinase by EGF and ionomycin were not significantly affected by PTX
treatment. These results are consistent with the P2Y2
receptor being coupled to Gi, a PTX-sensitive G-protein,
and suggest that the activation of MAP kinase by UTP and ATP is
initiated by the activation of this receptor.

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Fig. 3.
PTX reduces the activation of MAP kinase by
UTP and ATP. PC12 cells were treated with PTX (100 ng/ml) or
vehicle overnight and subsequently exposed to vehicle (water), UTP (100 µM), ATP (100 µM), ionomycin
(10 6 M), or EGF (100 ng/ml) for 5 min. Cells
were lysed, and p42 ERK2 was immunoprecipitated using anti-ERK2
antibody. The immunoprecipitates were used to assay MAP (ERK2) kinase
activity by measuring the phosphorylation of MBP (see "Materials and
Methods"). For each treatment, the MAP kinase activity is presented
relative to the activity measured in the absence of PTX. The values are
means ± S.E. The number of experiments is shown in
parentheses at the bottom of each bar.
PTX produced a significant (p 0.05) decrease in the
activation of MAP kinase in UTP- and ATP-treated cells but not in
basal, EGF-, or ionomycin-treated cells.
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Activation of MAP Kinase by UTP Requires the Elevation of
[Ca2+]i--
Because UTP elevates
[Ca2+]i in PC12 (17) and other
cells (for review, see Ref. 5) and calcium ionophores activate MAP
kinase in PC12 (18) and other cells, we examined whether the
UTP-dependent activation of MAP kinase was dependent on an elevation of [Ca2+]i. To block the
elevation of [Ca2+]i, PC12 cells
were loaded with the calcium chelator BAPTA, and extracellular calcium
was depleted by the addition of EGTA to the medium. These conditions
reduced the activation of MAP kinase by UTP and ionomycin to near basal
levels, but the activation of MAP kinase by EGF was not affected
significantly (Fig. 4). Treatment of
cells with BAPTA/EGTA also blocked the tyrosine phosphorylation of ERK2
and ERK1 by UTP and ionomycin but not by EGF (not shown). These results
indicated that the elevation of
[Ca2+]i mediated the activation of
MAP kinase by UTP and ionomycin but not by EGF.

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Fig. 4.
UTP- and ionomycin-dependent
increases in MAP kinase activity are promoted by increases in
[Ca2+]i. PC12 cells were
pretreated with either dimethyl sulfoxide (open) or 10 µM BAPTA-AM (hatched) for 1 h and then
exposed to UTP (100 µM), ionomycin (10 6
M), or EGF (100 ng/ml) for 5 min. Cells that were
pretreated with BAPTA-AM were also exposed to EGTA (5 mM)
during the 5-min exposure to stimuli. Cells were lysed, and p42 ERK2
was immunoprecipitated using anti-ERK2 antibody. MAP (ERK2) kinase
activity was quantified by measuring the phosphorylation of MBP (see
"Materials and Methods"). The UTP- and ionomycin-promoted
increases, but not the EGF-promoted increases, in MAP kinase were
reduced in BAPTA/EGTA-treated cells. The values are means ± S.E.
from three separate experiments.
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We also examined the effects of PTX on alterations of
[Ca2+]i by different stimuli. The
elevation of [Ca2+]i by 100 µM UTP was reduced by 50% in PTX-treated PC12 cells
compared with control cells (not shown), consistent with the coupling
of Gi between the P2Y2 receptor and
phospholipase C. In contrast, PTX did not affect the increase in
[Ca2+]i which was produced by
10
8 M bradykinin (not shown). Along with the
BAPTA/EGTA data presented in Fig. 4, these results suggest that an
increase in [Ca2+]i is upstream of
the the P2Y2 receptor-initiated activation of MAP
kinase.
RAFTK Is Involved in Mediating the Activation of MAP Kinase by
UTP--
RAFTK (also called PYK2 and CAK
), is a protein tyrosine
kinase that can couple G-protein-linked receptors to the MAP kinase activation cascade (19-21). PYK2/RAFTK is phosphorylated on tyrosine residues when [Ca2+]i is elevated
or PKC is activated (20). Because the effects of UTP on MAP kinase
activation require elevations in [Ca2+]i (Fig. 4), we examined the
tyrosine phosphorylation status of RAFTK to determine if it might be
involved in mediating the MAP kinase activation by UTP. In anti-RAFTK
immunoprecipitations, RAFTK was phosphorylated on tyrosine residues in
cells exposed to UTP, ionomycin, and EGF (Fig.
5A), indicating that all three stimuli activated this kinase. In most experiments, the tyrosine phosphorylations initiated by UTP and ionomycin were much greater than
those produced by EGF. The tyrosine phosphorylation of focal adhesion
kinase (FAK), a tyrosine kinase that has substantial sequence homology
to RAFTK (hence the name, Related Adhesion
Focal Tyrosine Kinase) was also
increased by UTP and EGF (Fig. 5B). EGF produced a greater
increase in FAK tyrosine phosphorylation than did UTP, and ionomycin
decreased the phosphorylation to levels below that found under basal
conditions.

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Fig. 5.
The UTP-dependent increase in the
tyrosine phosphorylation of RAFTK is sensitive to PTX and increases in
[Ca2+]i. PC12 cells were
treated as indicated with vehicle (dimethyl sulfoxide or water), UTP
(100 µM), ionomycin (10 6 M), or
EGF (100 ng/ml) for 1 min and lysed. Proteins were immunoprecipitated (IP) using anti-RAFTK or anti-FAK antibody. The
immunoprecipitated proteins were subjected to SDS-PAGE, transferred to
nitrocellulose, and immunoblotted (IB) using anti-P-Tyr
antibody (1 µg/ml). Panel A, the arrow on the
right indicates the location of RAFTK. The tyrosine
phosphorylation of RAFTK was increased more by UTP and ionomycin than
by EGF. Panel B, the arrow on the
right indicates the location of FAK. The tyrosine
phosphorylation of FAK was increased more by EGF than by UTP.
Panel C, cells were pretreated with dimethyl sulfoxide or
BAPTA-AM (10 µM), followed by stimuli ± EGTA (5 mM) as described in Fig. 4. The arrow on the
right indicates the location of RAFTK. Exposure of the cells
to BAPTA plus EGTA (B/E) reduced the increase in RAFTK
tyrosine phosphorylation by UTP and ionomycin but not by EGF.
Panel D, cells were pretreated overnight with PTX (100 ng/ml) or vehicle. The UTP-promoted increase in the tyrosine phosphorylation of RAFTK was reduced in PTX-treated cells (upper panel). The blots were stripped and reprobed using anti-RAFTK antibody (lower panel) to demonstrate the level of RAFTK in
the immunoprecipitations.
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In cells loaded with BAPTA and exposed to EGTA, the tyrosine
phosphorylation of RAFTK was decreased significantly in UTP- and
ionomycin-treated cells but not in EGF-treated cells (Fig. 5C). In multiple experiments, exposure of cells to
BAPTA/EGTA blocked the ionomycin-promoted phosphorylation of RAFTK more
completely than this treatment blocked the UTP-promoted
phosphorylation. This was probably because both the elevation of
[Ca2+]i and the activation of PKC
contributed to the UTP-promoted phosphorylation of RAFTK, and
BAPTA/EGTA blocked the former. These results are consistent with the
involvement of RAFTK in the activation of MAP kinase by UTP and suggest
that at least part of the response is caused by the UTP-promoted
increase in [Ca2+]i.
The UTP-promoted tyrosine phosphorylation of RAFTK was also decreased
substantially in PTX-treated cells (see Fig. 7D), consistent with the inhibitory effect of PTX on the elevation of
[Ca2+]i (see above) and a role for
[Ca2+]i upstream of RAFTK
activation. These results suggest the involvement of RAFTK in the
activation of MAP kinase by UTP and indicate that a large part of the
MAP kinase activation is caused by the UTP-promoted increase in
[Ca2+]i.
MAP Kinase Activation by UTP Involves PKC--
To determine
whether PKC was involved in the activation of MAP kinase by UTP, the
response of cells to UTP was measured in cells in which PKC was
down-regulated by treating the cells overnight with PMA. The exposure
of PKC-down-regulated cells to UTP for 5 min produced a reduced but
significant increase in activity (Fig.
6A). In contrast, the basal
MAP kinase activity was not affected significantly in
PKC-down-regulated cells, and the effect of an acute addition of PMA
was completely ineffective in stimulating MAP kinase activity. These
findings suggest that MAP kinase activation via stimulation of the
P2Y2 receptor involves both PKC-dependent and
PKC-independent pathways.

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Fig. 6.
PKC down-regulation blocks the activation of
MAP kinase by UTP, ATP, and PMA, and reduces the shift to a
hyperphosphorylated form of p42 ERK. PC12 cells were treated
overnight with PMA (1 µM) or dimethyl sulfoxide
(control). Cells were exposed to UTP (100 µM), ATP (100 µM), PMA (200 nM), or vehicle (dimethyl sulfoxide or water) for 5 min, lysed, and p42 ERK2 was
immunoprecipitated. Panel A, anti-ERK2 immunoprecipitates
were used to assay MAP (ERK2) kinase activity by measuring the
phosphorylation of MBP (see "Materials and Methods"). All
measurements of MAP kinase activity were normalized to that measured
under control basal conditions (no agonist, no PMA overnight).
Pretreatment with PMA completely blocked the response to the acute
addition of PMA and blocked the majority of the response to UTP. The
values are means ± S.E. from four separate experiments. Panel B, the anti-ERK2 immunoprecipitates were subjected to
SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted
using anti-ERK2 antibody (0.2 µg/ml) and subsequently reprobed with anti-P-Tyr antibody (1 µg/ml). The arrows on the
right designate the upper form of p42 ERK2, which is
phosphorylated on multiple amino acid residues, including tyrosine. In
cells pretreated with PMA overnight, the shift to a hyperphosphorylated
form was blocked completely in cells treated acutely (5 min) with PMA
but was present at a reduced level in cells treated with UTP (5 min).
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The effects of PKC down-regulation on the activation of MAP kinase were
also observable by Western blotting the immunoprecipitated ERK2
proteins. In anti-ERK2 blots, ERK2 was identified as a doublet in cells
treated with UTP, ATP, or PMA for 5 min (Fig. 6B). In cells
in which PKC was down-regulated by overnight treatment of the cells
with PMA, the upper protein was observed in UTP-treated cells but not
in cells acutely exposed to PMA. When these blots were stripped and
reblotted using anti-P-Tyr antibody, a tyrosine-phosphorylated band
co-migrated with the upper form of the doublet under all conditions in
which the doublet was observed. The upper band was present in
PKC-down-regulated cells that were subsequently treated with UTP,
although this band was of lesser intensity than that seen in cells in
which PKC was not down-regulated (Fig. 6B, upper panel). These results were parallel to those obtained in the MAP kinase in vitro substrate phosphorylation assay (Fig.
6A). Thus, UTP, ATP, and PMA all promote the tyrosine
phosphorylation of MAP kinase which is dependent on the activation of
PKC.
UTP and ATP Promote the Tyrosine Phosphorylation of
PKC
--
Preliminary studies indicated that exposure of cells to
ATP initiated the tyrosine phosphorylation of PKC
in PC12 cells. Because activation of the P2Y2 receptor by UTP and ATP
increased MAP kinase activity in PC12 cells in a
PKC-dependent manner (see above) and the overexpression of
PKC
increased MAP kinase activity (22) and was associated with cell
differentiation (12) in other cells, we examined further the tyrosine
phosphorylation of PKC
to (a) determine whether the
effects on PKC
were mediated by the activation of a P2Y2
receptor and (b) compare the effects of ATP with various
growth factors including NGF, which causes PC12 cells to differentiate.
Among a selection of P2 purinoceptor agonists, ATP and UTP
promoted the largest increase in phosphorylation, and BzATP produced a
much smaller increase, consistent with the nucleotide-dependent activation of the P2Y2
receptor (Fig. 7A). All three
of these compounds will bind to P2Y2 receptors, although BzATP is not a very effective or potent agonist (23).
,
-methylene ATP and 2-methyl-thio-ATP, agonists of P2X receptors and
other P2Y receptors, produced little alteration in tyrosine
phosphorylation. Thus, although P2X receptors have been
found on PC12 cells (24), activation of the P2Y2 receptor
appeared to be selective for promoting the tyrosine phosphorylation of
PKC
. Activation of the EGF and insulin receptors, two receptor
tyrosine kinases that activate pathways involving the tyrosine
phosphorylation of other proteins in these cells, did not produce
significant changes in tyrosine phosphorylation of PKC
(Fig.
7A).

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Fig. 7.
PKC is phosphosphorylated on tyrosine
residues in cells exposed to UTP, ATP, and PMA. PC12 cells were
exposed to stimuli, lysed, and PKC was immunoprecipitated using
anti-PKC antibody. Proteins were separated by SDS-PAGE, transferred
to nitrocellulose filters, and probed with anti-P-Tyr antibody (1 µg/ml) unless otherwise indicated. Proteins were visualized on x-ray
film using enhanced chemiluminescence techniques. Panel A,
cells were treated with PMA (200 nM, 5 min), EGF (100 ng/ml, 1 min), insulin (100 nM, 1 min), nucleotide analogs
(100 µM, 1 min), or vehicle (dimethyl sulfoxide or water,
1 min). A molecular mass marker is shown on the left, and
the arrow on the right indicates the location of the tyrosine-phosphorylated form of PKC . , -ATP,
, -methylene ATP; 2-M-SATP, 2-methyl-thio-ATP. ATP,
UTP, and PMA increased the tyrosine phosphorylation of PKC .
Panel B, cells were exposed to ATP (100 µM) or
UTP(100 µM) for 0.2-15 min or PMA (200 nM) for 5 min. A molecular mass marker is shown on the left, and
the arrow on the right indicates the location of
the tyrosine-phosphorylated form of PKC . ATP and UTP produced rapid
increases in the tyrosine phosphorylation of PKC . Panel
C, cells were exposed to UTP (100 µM), NGF (100 ng/ml), or EGF (100 ng/ml) for 0.2-5 min or to PMA (200 nM) for 5 min. A molecular mass marker is shown on the
left, and the arrow on the right
indicates the location of the tyrosine phosphorylated form of PKC .
UTP and PMA, but not EGF and NGF, produced large increases in the
tyrosine phosphorylation of PKC . Panel D, cells were
treated with vehicle ( ) or 1 µM PMA (+PMA) overnight to down-regulate PKC, and subsequently they were exposed to
an acute addition of PMA (200 nM) for 5 min. Anti-PKC
immunoprecipitates were immunoblotted using anti-PKC antibody (0.2 µg/ml) and were reprobed subsequently with anti-P-Tyr antibody (1 µg/ml). The arrows on the right designate the
location of the tyrosine-phosphorylated form of PKC . The amount of
immunoprecipitable PKC was reduced significantly in cells pretreated
with PMA (upper panel). Control cells, but not cells in
which PKC was down-regulated, responded to the acute addition of PMA
with an increase in PKC tyrosine phosphorylation (lower
panel).
|
|
The time course of PKC
tyrosine phosphorylation was examined using
ATP and UTP. The largest increases in tyrosine phosphorylation occurred
in cells exposed to ATP or UTP for ~15 s or 1 min, and the
phosphorylation returned to near basal levels after 5 min or more (Fig.
7B). These results indicate that activation of the P2Y2 receptor produces very rapid increases in the tyrosine
phosphorylation of PKC
. This event temporally precedes the peak
activation of MAP kinase (Fig. 2A) by UTP, consistent with
the possibility that the stimulatory effect of UTP on MAP kinase could
involve PKC
(see "Discussion").
The effects of UTP, NGF, and EGF on PKC
tyrosine phosphorylation
were compared over an expanded time course. At times of 0.2, 1, and 5 min of exposure, neither NGF nor EGF produced increases in PKC
tyrosine phosphorylation which were of a magnitude similar to those
produced by a 0.2- or 1-min exposure to UTP or by a 5-min exposure to
PMA (Fig. 7C). Slight increases in PKC
tyrosine
phosphorylation were sometimes evident for EGF- or NGF-treated cells
during longer exposures of the immunoblot using enhanced
chemiluminescence techniques. In contrast, NGF and EGF produced large
increases in tyrosine phosphorylation of other proteins, observed on
anti-P-Tyr immunoblots of cell lysates (not shown). There was no
detectable increase in the tyrosine phosphorylation of PKC
produced
by the exposure of PC12 cells to the calcium ionophore ionomycin
(10
6 M) (not shown). These results indicate
that the phosphorylation of PKC
in response to UTP at these early
times (
1 min) is not mimicked by the activation of growth factor
receptor tyrosine kinases or the elevation of
[Ca2+]i.
The amount of PKC
in anti-PKC
immunoprecipitates from PC12 cells
treated overnight with PMA (1 µM) was reduced greatly
compared with cells in which PKC was not down-regulated (Fig.
7D, upper panel). Unlike the control cells, the
cells in which PKC was down-regulated did not respond to the acute
addition of PMA (200 nM, 5 min) with a detectable increase
in tyrosine phosphorylation (Fig. 7D, lower panel). These data parallel the diminished effect of PMA and UTP on the activation of MAP kinase in PKC-down-regulated cells (Fig. 6A) and are consistent with the potential involvement of
PKC
in the activation of MAP kinase (see "Discussion").
Activation of the P2Y2 Receptor Affects Shc and
Grb2--
Because RAFTK/PYK2 appears to activate MAP kinase by
engaging Shc and Grb2 (20), we also examined whether UTP promotes the formation of a Grb2·Shc complex. There was an increase in the tyrosine phosphorylation of two forms of Shc (46 and 52 kDa) after treatment of PC12 cells with UTP for 1 and 5 min (Fig.
8A). EGF, NGF, ionomycin, and
PMA also produced substantial increases in the tyrosine phosphorylation
of these forms of Shc. EGF and NGF also increased the tyrosine
phosphorylation of the 66-kDa form of Shc (Fig. 8A). Grb2
was co-immunoprecipitated with Shc in cells treated with all of these
stimuli, and the amount of immunoprecipitated Grb2 (Fig. 8B)
was directly proportional to the tyrosine phosphorylation of Shc (Fig.
8A), consistent with the recruitment of Grb2 to Shc via its
SH2 domain. In cells treated with UTP for various times, the peak
increase in the tyrosine phosphorylation of Shc (Fig. 8A)
and the co-immunoprecipitation of Grb2 (Fig. 8B) was at 5 min, similar to the time of the peak increase in MAP kinase activity (Figs. 1 and 2A). These results suggest that Shc and Grb2
are signaling molecules involved in the downstream effects of the activation of the P2Y2 receptor in PC12 cells, and they
place RAFTK between the P2Y2 receptor and these molecules
in the signaling cascade involved in the activation of MAP kinase by
UTP.

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Fig. 8.
UTP, ionomycin, and other stimuli increase
the tyrosine phosphorylation of Shc and its association with Grb2.
PC12 cells were exposed to UTP (100 µM), EGF (100 ng/ml),
NGF (100 ng/ml), ionomycin (10 6 M), and PMA
(200 nM) for 0.2-5 min as indicated. Cells were lysed, and
proteins were immunoprecipitated using anti-Shc antibody. The
immunoprecipitated proteins were subjected to SDS-PAGE, transferred to
nitrocellulose, and immunoblotted using anti-P-Tyr antibody (1 µg/ml)
or anti-Grb2 antibody (0.2 µg/ml). Upper panel, the arrows designate the 46-, 52-, and 66-kDa forms of Shc.
There is a time-dependent increase in the tyrosine
phosphorylation of Shc in cells exposed to the various stimuli.
Lower panel, the association of Grb2 with Shc is related
directly to the amount of the tyrosine phosphorylation of Shc.
|
|
 |
DISCUSSION |
In this study it is demonstrated that multiple signaling proteins,
including p42 ERK2/MAP kinase, PKC
, Shc, FAK, and RAFTK are
phosphorylated on tyrosine residues in PC12 cells exposed to ATP and/or
UTP. UTP and ATP had similar potencies in activating MAP kinase (Fig.
2B), and PTX blocked the activation of MAP kinase by UTP and
ATP to a similar degree (Fig. 3), indicating that the effects were
mediated by a G-protein-coupled P2Y2/P2U
receptor. The involvement of RAFTK/PYK2/CAK
in the activation of MAP
kinase by some stimuli was indicated by previous findings demonstrating that the overexpression of PYK2/RAFTK activated MAP kinase in a
PYK2/RAFTK concentration-dependent manner, and the
expression of kinase-dead PYK2/RAFTK reduced the activation of MAP
kinase (20). PYK2/RAFTK is activated by multiple stimuli, including the
elevation of [Ca2+]i and the
activation of PKC. In the present studies of PC12 cells, the
involvement of RAFTK in the activation of MAP kinase by UTP was
suggested by the following: (a) the similar [Ca2+]i dependence of
UTP-stimulated RAFTK tyrosine phosphorylation (Fig. 5C) and
MAP kinase activation (Fig. 4); (b) the similar PTX
sensitivity of the UTP-stimulated RAFTK tyrosine phosphorylation (Fig.
5D) and MAP kinase activation (Fig. 3); and (c)
the ability of the calcium ionophore ionomycin to mimic the effects of
UTP on both RAFTK tyrosine phosphorylation (Fig. 5A) and MAP
kinase activation (Fig. 2A). The PTX sensitivity of the
effects of UTP on [Ca2+]i, RAFTK
tyrosine phosphorylation, and MAP kinase activation are consistent with
the coupling of the P2Y2 receptor to a Gi-type G-protein, and the Ca2+ sensitivities of RAFTK and MAK
kinase indicate that the UTP-promoted elevation of
[Ca2+]i is an important signal
between P2Y2 receptor activation and the activation of MAP
kinase. Other P2Y2 receptor-promoted effects were also
blocked by PTX in different cells, including HL-60 (25), smooth muscle
(26), and human airway epithelial (27) cells.
UTP, ionomycin, PMA, EGF, and NGF all stimulated the activation of MAP
kinase (Fig. 2A), the tyrosine phosphorylation of Shc (Fig.
8A), and the association of Grb2 and Shc (Fig.
8B). Thus, these results are consistent with the convergence
of serpentine receptor-mediated and growth factor receptor-mediated
activation of MAP kinase occurring at the level of Shc/Grb2 in the MAP
kinase activation cascade (28, 29). However, because our results indicate that EGF also stimulated the tyrosine phosphorylation of
RAFTK, albeit to a much less degree than UTP or ionomycin, and because
neither this phosphorylation nor the activation of MAP kinase by EGF
was sensitive to decreases in calcium, we cannot rule out a
contribution of RAFTK in the EGF-dependent activation of
MAP kinase. The activation of MAP kinase by EGF may involve the
stimulation of RAFTK/PYK2/CAK
primarily via a
PKC-dependent pathway.
A number of studies have characterized effects of ATP and other
P2 analogs on PC12 cells (30-35). The results of these and other studies indicated that PC12 cells have multiple P2
subtypes. A P2X receptor was cloned from PC12 cells (24).
Various studies observed effects of UTP on PC12 cells. ATP and UTP had
similar potencies in promoting intracellular calcium mobilization,
indicating the presence of a P2Y2 receptor (17). However,
other studies reported that UTP was ineffective in elevating
[Ca2+]i (36), and one study
concluded that the UTP-sensitive nucleotide receptor in PC12 cells was
not the 53-kDa UTP receptor (P2Y2 receptor). Part of the
reason for the differences in reports from various laboratories
using PC12 cells may result from variations in PC12 cell lines in
different laboratories.
In the present studies we show that UTP is more effective than the
growth factors NGF, EGF, or insulin at stimulating the tyrosine
phosphorylation of PKC
, although these growth factors promote the
tyrosine phosphorylation of a number of other signaling molecules. The
maximum increase in the UTP-dependent tyrosine phosphorylation of PKC
occurred in cells exposed to UTP for
1 min (Fig. 7B), a time earlier than the
UTP-dependent increase in MAP kinase activity, which did
not occur until well after 1 min of UTP exposure (Fig. 2A).
Overnight treatment of PC12 cells with PMA resulted in the
down-regulation of PKC
(Fig. 5D) as well as a substantial
decrease in the PMA- and UTP-dependent activation of MAP
kinase (Fig. 6A). However, these studies alone are
insufficient to support a conclusion that PKC
is involved in the
activation of MAP kinase by extracellular nucleotides because PC12
cells express other members of the PKC family of proteins, including the isoforms
,
,
,
, and
(37, 38). In addition, MAP kinase can be activated by both PKC-dependent and
PKC-independent pathways (Fig. 6, A and B). The
transfection of constitutively active PKC
into COS1 cells resulted
in the activation of MAP/ERK kinase (MEK) and MAP kinase in a
Raf-dependent fashion, and this was not produced by transfection of
constitutively active PKC
and PKC
, suggesting that activation of
PKC
does lead to MAP kinase activation in some systems (22). It
remains to be determined whether PKC
and/or other PKC family members
are involved in the UTP-promoted activation of MAP kinase in PC12
cells. In addition, there are contrasting reports of whether the
tyrosine phosphorylation of PKC
increases (12, 39) or reduces (14,
40) the activation of the enzyme. However, a more important
consideration may be that PKC
translocates from a cytosolic location
to a cellular membrane. This may be more relevant than measurements
demonstrating a fractional reduction in its activity because the
translocation of PKC presumably will be to the sites of their
physiological substrates.
The P2Y2 receptor is found on a wide variety of cell types,
including freshly isolated cells as well as multiple cell lines (41;
for review, see Ref. 5). In some cells extracellular ATP has mitogenic
effects (42, 43), but in others ATP acts as an antiproliferative agent
(44). In primary cultures of dog tracheal epithelial cells, UTP
promoted the activation and phosphorylation of the Na-K-Cl cotransport
protein (45). UTP and/or ATP activates Cl
secretion in
many epithelial cells. Recently it was suggested that the
P2Y2/P2U receptor is involved in modulating the
cystic fibrosis transmembrane conductance regulator (CFTR). This model suggests that intracellular ATP released through the CFTR activates P2Y2 receptors, which, in turn, stimulate chloride
secretion via the activation of outwardly rectifying chloride channels
by a G-protein-coupled mechanism (9, 43). The precise mechanism of
activation was not defined, but other channels, notably the cardiac
K+ channel, are activated by G
.
A number of studies have shown that growth factors and
G-protein-coupled receptors activate certain signaling proteins in common and that tyrosine phosphorylation also plays a role in signaling
pathways activated by G-proteins (for review, see Ref. 46). G
subunits also can activate MAP kinase in a Ras-dependent manner in response to activation of the tyrosine kinase insulin-like growth factorI receptor (29). This activation was sensitive to PTX, and
the effect of G-protein-linked receptors on MAP kinase activation was
dependent on Ras activation and was blocked by tyrosine kinase
inhibitors, indicating that tyrosine kinase receptors and
G-protein-linked receptors both activate MAP kinase through both
tyrosine kinase activation and G-proteins (29). Subsequently, the
Gi-protein-coupled activation of MAP kinase by G
was
found to be mediated by the tyrosine phosphorylation of Shc (28), an
event that is shared with the activation of receptor protein tyrosine
kinase receptors. Our results are consistent with this model.
The ATP- and UTP-promoted increases in MAP kinase activity and the
increases in tyrosine phosphorylation of multiple proteins by ATP and
UTP demonstrate that G-protein-coupled P2Y2 receptors and
growth factor receptors, including those for EGF and NGF, activate some
intracellular signaling proteins and pathways in common. Other
signaling proteins, notably PKC
, were uniquely activated by UTP and
ATP. These results add to the expanding list of the involvement of
tyrosine phosphorylation in events promoted by G-protein-linked
receptors.
 |
ACKNOWLEDGEMENTS |
We thank Michelle Bradford and Margaret
Lubkin for excellent technical assistance and Dr. Zvi Greenberg for use
of the SPEX fluorometer.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants DE10877 and HL55445.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.
§
To whom correspondence should be addressed: Division of Signal
Transduction, Beth Israel Deaconess Medical Center, Harvard Institutes
of Medicine, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-0949;
Fax: 617-667-0957; E-mail: ssoltoff{at}bidmc.harvard.edu.
1
The abbreviations used are: G-protein,
GTP-dependent protein; MAP, mitogen-activated protein;
[Ca2+]i, intracellular calcium ion
concentration; PKC, protein kinase C; NGF, nerve growth factor; RAFTK,
related adhesion focal tyrosine kinase; PMA, phorbol 12-myristate
13-acetate; EGF, epidermal growth factor; P-Tyr, phosphotyrosine; PTX,
pertussis toxin; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N
,N
-tetraacetic
acid; MPB, myelin basic protein; PAGE, polyacrylamide gel
electrophoresis; FAK, focal adhesion kinase; BzATP,
3-O-(4
- benzoyl)-benzoyl-ATP.
 |
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