Activation of P 2Y2 Receptors by UTP and ATP Stimulates Mitogen-activated Kinase Activity through a Pathway That Involves Related Adhesion Focal Tyrosine Kinase and Protein Kinase C*

We examined downstream signaling events that followed the exposure of PC12 cells to extracellular ATP and UTP, and we compared the effects of these P 2 recep- tor 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 b ), focal adhesion kinase (FAK), Shc, and protein kinase C d (PKC d ). 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 (EC 50 ; 25 m M ) in stimulating MAP ki- nase activity, suggesting that these effects were mediated via the G i -linked P 2Y2 (P 2U ) 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 ([Ca 2 1 ] i ) and the tyrosine phosphorylation of RAFTK. Similarly, when [Ca 2

P 2 -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 Ca 2ϩ and other ions. ATP is a neurotransmitter or co-transmitter in some systems (1)(2)(3), and this has been the proposed rationale for the existence of P 2 receptors on some cells. Previously, classifications of receptor subtypes were based on pharmacological hierarchies of the responses to ATP analogs (4,5). The P 2 subclassifications have been reorganized based on recent molecular biology approaches and now enfold more than a dozen types of P 2 receptors into a family of two main receptor types: P 2X and P 2Y purinoceptors. P 2X receptors are ligand-gated ion channels, and P 2Y receptors are seven transmembrane proteins that are GTPdependent 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 P 2Y2 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 ([Ca 2ϩ ] i ), respectively. The P 2Y2 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 P 2Y2 and other P 2 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 P 2Y2 receptor by ATP and UTP. In addition, we compared the effects of extracellular nucleotides and growth factors on PKC␦ tyrosine phosphorylation. PKC␦, a Ca 2ϩ -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 differentia-tion (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 P 2Y2 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 P 2Y2 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.
Cell Culture-PC12 cells were grown in 100-mm-diameter dishes at 37°C in a mixture of 95% air and 5% CO 2 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 M PMA or vehicle (0.06% dimethyl sulfoxide) overnight (ϳ16 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 (ϳ16 h). In experiments in which elevations in intracellular calcium elevation were blocked, serumstarved cells were exposed to 10 M BAPTA or vehicle (0.1% dimethyl sulfoxide) for 1 h and then switched to low serum medium Ϯ 5 mM EGTA Ϯ stimuli or vehicle for 5 min.
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-m 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 ϳ16 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 MgCl 2 ). Immunoprecipitates were resuspended in a final volume of 50 l of kinase buffer containing myelin basic protein (MBP) (200 g/ml kinase buffer), and the kinase assay was initiated with the addition of 40 M ATP plus [ 32 P]ATP (1 Ci). After 30 min, in some experiments 20 l 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 [ 32 P]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 ϫ 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.
[Ca 2ϩ ] i -PC12 cells were suspended in a physiological salt solution containing 2 M fura-2 acetoxymethylester and 10 M probenecid in a physiological salt solution for 50 min, as reported previously for other cells (16). The cells were washed twice, and [Ca 2ϩ ] i was monitored at room temperature in a SPEX FluoroMax-2 spectrofluorometer. [Ca 2ϩ ] 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.

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): ϳ159 kDa (UTP or ATP for 1 min), ϳ102 kDa and ϳ90 kDa (ATP or UTP for 1 or 5 min), ϳ76 kDa (UTP or PMA for 5 min), ϳ63 and ϳ67 kDa (UTP or ATP for 1 min), ϳ49 kDa (UTP or ATP for 1 min), ϳ40 kDa and ϳ44 kDa (UTP and PMA for 5 min). Thus, there were distinct proteins that exhibited different peak times in tyrosine phosphorylation when exposed to P 2Y2 agonists or PMA. Several proteins were identified by reprobing the blots with antibodies to known proteins of similar size. The ϳ40and ϳ44-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 P 2Y2 (P 2U ) receptor activated MAP kinase and affected other signaling proteins in these cells.
PTX Reduces the P 2Y2 Receptor-mediated Increase in MAP Kinase Activity-Additional experiments were performed to confirm that MAP kinase activity was increased by activation of the P 2Y2 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 M) or ATP (100 M) 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 M) and ATP (100 M) were both measured after a 5-min exposure, the activation of MAP kinase by UTP was 67.0 Ϯ 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 P 2Y2 receptor. There were negligible increases in MAP kinase activity in cells treated with UTP (100 M) 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.
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 EC 50 value was ϳ25 M 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 P 2Y2 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 P 2Y2 receptor being coupled to G i , 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.  (18) and other cells, we examined whether the UTP-dependent activation of MAP kinase was dependent on an elevation of [Ca 2ϩ ] i . To block the elevation of [Ca 2ϩ ] 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 [Ca 2ϩ ] i mediated the activation of MAP kinase by UTP and ionomycin but not by EGF.
We also examined the effects of PTX on alterations of [Ca 2ϩ ] i by different stimuli. The elevation of [Ca 2ϩ ] i by 100 M UTP was reduced by 50% in PTX-treated PC12 cells compared with control cells (not shown), consistent with the coupling of G i between the P 2Y2 receptor and phospholipase C. In contrast, PTX did not affect the increase in [Ca 2ϩ ] i which was produced by 10 Ϫ8 M bradykinin (not shown). Along with the BAPTA/ EGTA data presented in Fig. 4, these results suggest that an increase in [Ca 2ϩ ] i is upstream of the the P 2Y2 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 [Ca 2ϩ ] i is elevated or PKC is activated (20). Because the effects of UTP on MAP kinase activation require elevations in [Ca 2ϩ ] 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.
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 UTPpromoted phosphorylation. This was probably because both the elevation of [Ca 2ϩ ] 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 [Ca 2ϩ ] i .
The UTP-promoted tyrosine phosphorylation of RAFTK was also decreased substantially in PTX-treated cells (see Fig. 7D 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 P 2Y2 receptor involves both PKC-dependent and PKC-independent pathways.
The effects of PKC down-regulation on the activation of MAP  2؉ ] i . PC12 cells were pretreated with either dimethyl sulfoxide (open) or 10 M BAPTA-AM (hatched) for 1 h and then exposed to UTP (100 M), ionomycin (10 Ϫ6 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 Ϯ S.E. from three separate experiments. 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 comigrated 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.

FIG. 4. UTP-and ionomycin-dependent increases in MAP kinase activity are promoted by increases in [Ca
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 P 2Y2 receptor by UTP and ATP increased MAP kinase activity in PC12 cells in a PKC-dependent manner (see above) and the overexpression of PKC␦ in-creased 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 P 2Y2 receptor and (b) compare the effects of ATP with various growth factors including NGF, which causes PC12 cells to differentiate. Among a selection of P 2 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 P 2Y2 receptor (Fig. 7A). All three of these compounds will bind to P 2Y2 receptors, although BzATP is not a very effective or potent agonist (23). ␣,␤-methylene ATP and 2-methyl-thio-ATP, agonists of P 2X receptors and other P 2Y receptors, produced little alteration in tyrosine phosphorylation. Thus, although P 2X receptors have been found on PC12 cells (24), activation of the P 2Y2 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 . Cells were exposed to UTP (100 M), ATP (100 M), 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 Ϯ 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 g/ml) and subsequently reprobed with anti-P-Tyr antibody (1 g/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). these cells, did not produce significant changes in tyrosine phosphorylation of PKC␦ (Fig. 7A).
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 ϳ15 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 P 2Y2 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 Ϫ6 M) (not shown). These results indicate that the phosphorylation of PKC␦ in response to UTP at these early times (Յ1 min) is not mimicked by the activation of growth factor receptor tyrosine kinases or the elevation of [Ca 2ϩ ] i .
The amount of PKC␦ in anti-PKC␦ immunoprecipitates from PC12 cells treated overnight with PMA (1 M) 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 P 2Y2 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 Grb2⅐Shc 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 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 g/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 M, 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 M) or UTP(100 M) 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 M), 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 (Ϫ) or 1 M PMA (ϩPMA) 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 g/ml) and were reprobed subsequently with anti-P-Tyr antibody (1 g/ml). The arrows on the right designate the location of the tyrosinephosphorylated 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) .   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 M), EGF (100 ng/ml), NGF (100 ng/ml), ionomycin (10 Ϫ6 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 g/ml) or anti-Grb2 antibody (0.2 g/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.
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 P 2Y2 receptor in PC12 cells, and they place RAFTK between the P 2Y2 receptor and these molecules in the signaling cascade involved in the activation of MAP kinase by UTP. 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 Gprotein-coupled P 2Y2 /P 2U 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 [Ca 2ϩ ] 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 [Ca 2ϩ ] i dependence of UTPstimulated 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 [Ca 2ϩ ] i , RAFTK tyrosine phosphorylation, and MAP kinase activation are consistent with the coupling of the P 2Y2 receptor to a G i -type G-protein, and the Ca 2ϩ sensitivities of RAFTK and MAK kinase indicate that the UTP-promoted elevation of [Ca 2ϩ ] i is an important signal between P 2Y2 receptor activation and the activation of MAP kinase. Other P 2Y2 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-de-pendent pathway.
A number of studies have characterized effects of ATP and other P 2 analogs on PC12 cells (30 -35). The results of these and other studies indicated that PC12 cells have multiple P 2 subtypes. A P 2X 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 P 2Y2 receptor (17). However, other studies reported that UTP was ineffective in elevating [Ca 2ϩ ] i (36), and one study concluded that the UTPsensitive nucleotide receptor in PC12 cells was not the 53-kDa UTP receptor (P 2Y2 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 UTPdependent tyrosine phosphorylation of PKC␦ occurred in cells exposed to UTP for Յ 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 downregulation 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 P 2Y2 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 Ϫ secretion in many epithelial cells. Recently it was suggested that the P 2Y2 /P 2U receptor is involved in modulating the cystic fibrosis transmembrane conductance regulator (CFTR). This model suggests that intracellular ATP released through the CFTR activates P 2Y2 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 ϩ channel, are acti-vated 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 G i -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 P 2Y2 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.