ATP-stimulated Activation of the Mitogen-activated Protein Kinases through Ionotrophic P2X2 Purinoreceptors in PC12 Cells

Extracellular purine nucleotides elicit a diverse range of biological responses through binding to specific cell surface receptors. The ionotrophic P2X subclass of purinoreceptors respond to ATP by stimulation of calcium ion permeability; however, it is unknown how P2X purinoreceptor activation is linked to intracellular signaling pathways. We report that stimulation of PC12 cells with ATP results in the activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 and was wholly dependent upon extracellular calcium ions. Treatment of the cells with adenosine, AMP, ADP, UTP, or α,β-methylene ATP was without effect; however, MAP kinase activation was abolished by pretreatment with suramin and reactive blue 2. The calcium-activated tyrosine kinase, Pyk2, acts as an upstream regulator of the MAP kinases and became tyrosine phosphorylated following treatment of the cells with ATP. We have ruled out the involvement of depolarization-mediated calcium influx because specific blockers of voltage-gated calcium channels did not affect MAP kinase activation. These data provide direct evidence that calcium influx through P2X2 receptors results in the activation of the MAP kinase cascade. Finally, we demonstrate that a different line of PC12 cells respond to ATP through P2Y2 purinoreceptors, providing an explanation for the conflicting findings of purine nucleotide responsiveness in PC12 cells.

Adenine nucleotides act through specific cell surface receptors to provoke a variety of biological responses (1,2). In the nervous system, extracellular ATP has been postulated to serve as a classical neurotransmitter and to also act as a co-transmitter through its coordinate packaging and release with norepinephrine and acetylcholine (3). The analysis of these roles of ATP has been complicated by its catabolism into other biologically active species and the unexpected diversity of cell surface purinoreceptors. Purinoreceptors have been classified into two primary classes, the P1 receptors are responsive to adenosine, whereas the P2 class receptors respond to a variety of purine nucleotides, including ATP. Presently, twelve P2 subtypes have been identified and assigned to two mechanistically distinct subclasses of purinoreceptors (1). The metabotrophic purinoreceptors of the P2Y subclass (formerly P2u, P2t, and P2y) initiate their biological actions by G-protein-dependent activation of phospholipase C and subsequent elevation of intracellular calcium levels through liberation of calcium from internal stores (4). The P2X purinoreceptors comprise a distinct subclass of receptors that are ligand-gated calcium channels functionally related to glutamate and nicotinic acetylcholine receptors. The ionotrophic P2X receptors regulate intracellular calcium levels through the ligand-stimulated increase in calcium permeability; thus their actions are dependent upon extracellular calcium ions. Although the ability of the P2X receptors to evoke a rise in intracellular calcium levels is well documented, it is entirely unclear how these receptors are linked to intracellular signaling pathways subserving their biological actions. In PC12 cells, activation of P2X receptors has been shown to be responsible for stimulation of neurotransmitter release (5-9). A nervous system-specific P2X receptor subtype, P2X2, was initially cloned from PC12 cells and exhibits a distinctive pharmacological profile (10) and has been implicated in mediating many of the biological actions of ATP. Michel et al. (11) have recently demonstrated that the P2X2 receptor appears to be principally responsible for the ATPstimulated calcium influx in PC12 cells, extending and confirming a number of earlier reports of the involvement of the P2X subclass in mediating the action of ATP.
PC12 cells have also been reported to respond to purinergic agonist through other classes of purinoreceptors, giving rise to an extensive and controversial literature (1,4). Recently, Soltoff and colleagues (12) have demonstrated that a line of PC12 cells respond to purinergic ligands through P2Y receptors. A direct comparison of these two PC12 cell lines has revealed that they exhibit distinctly different responses to ATP mediated through either P2X2 or P2Y receptors, demonstrating diversity in purinoreceptor sensitivity within PC12 cell lines.
Calcium plays well defined and critical roles in the regulation of a diverse range of intracellular events. Elevation of intracellular calcium levels provokes the activation of a number of protein serine/threonine kinases and signaling cascades (13,14). The recent discovery of a calcium-stimulated tyrosine kinase, Pyk2, has provided a mechanistic linkage between calcium and well described signaling systems dependent upon protein tyrosine phosphorylation, most prominently, the MAP 1 kinase cascade (15).
The MAP kinases ERK1 and ERK2 are components of a critical signaling pathway linking membrane receptors to cytoplasmic and nuclear effectors (16). Signaling through the MAP kinase cascade is initiated through activation of p21 ras that results in the serial phosphorylation and activation of Raf family members, MAP/ERK kinase and the MAP kinases. Once activated, the MAP kinases phosphorylate cytoplasmic effectors and are translocated to the nucleus where they act on transcription factors and mediate changes in gene expression. The goal of the present study was to establish the linkage of the ionotrophic calcium influx through P2X2 purinoreceptors to activation of intracellular signaling cascades and the stimulation of the MAP kinases.

Materials
Nerve growth factor (NGF) was purchased from Austral Biological (San Ramon, CA). All other reagents were from Sigma unless otherwise specified. Reactive blue 2 was purchased from Polysciences (Warrington, PA). Antibodies to the EGF receptor, ERKs, and Pyk2 were from Santa Cruz (Santa Cruz, CA). The anti-phosphotyrosine antibody, 4G10, was from Upstate Biotechnology Inc. (Lake Placid, NY). The anti-phospho-MAP kinase antibody was purchased from New England Biolabs (Beverly, MA).

Methods
Cell Culture-The PC12 cells used in this study were obtained from Dr. Eric Shooter (Stanford University) and have been maintained in this laboratory since 1980 under conditions similar to those described originally by Greene and Tischler (17). This line of PC12 cells was cultured in DMEM containing 10% horse serum and 5% fetal bovine serum in an atmosphere of 10% CO 2 . Cells were serum-starved for 16 h prior to harvest in DMEM containing 0.5% fetal bovine serum. A second line of PC12 cells was obtained from Dr. Stephen Soltoff (Harvard University) and cultured in DMEM containing 5% horse serum and 5% fetal bovine serum in an atmosphere of 5% CO 2 . The cells were harvested by trituration in phosphate-buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.1% dextrose. The cells were collected by centrifugation, resuspended in PBS (2 ϫ 10 6 cells/ml), and treated as indicated. Following treatment, cells were collected by centrifugation and resuspended in TEV buffer (20 mM Tris, pH 7.4, 1 mM EGTA, 100 mM sodium orthovanadate) containing 20 mM p-nitrophenyl phosphate, 1 mg/ml aprotinin, and 1 mg/ml bacitracin. Lysis was performed by sonication. Lysates were clarified by centrifugation at 13,000 rpm for 15 min at 4°C. Protein concentration was determined by the method of Bradford (18) using BSA as standard.
Phospho-MAP Kinase and Phosphotyrosine Detection-The antiphospho-MAP kinase antibody specifically detects only the activated, dually phosphorylated forms of the MAP kinases ERK1 and ERK2. Aliquots of the cellular lysates (50 g of protein) were resolved using 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked overnight at 4°C in Tris-buffered saline containing 0.1% Tween 20 (TBS-Tween) and 5% BSA. Membranes were then incubated at 25°C for 1 h in the presence of antiphospho-MAP kinase antibody (1:1000 dilution into TBS-Tween containing 5% BSA), followed by three washes in TBS-Tween for 15 min each. The membranes were then incubated in horseradish peroxidaseconjugated goat anti-rabbit antibody for 1 h at 25°C (1:4000 dilution into 5% milk in TBS-Tween). The membranes were then washed three times in TBS-Tween for 15 min, and the MAP kinases were visualized by chemiluminesence. Tyrosine phosphoproteins were detected by Western blot analysis using the anti-phosphotyrosine antibody, 4G10 (Upstate Biotechnology Inc.). Aliquots of cellular lysates (50 g of protein) were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes, blocked in 5% BSA, and probed with the indicated antibody.
Immunoprecipitation of Pyk2 and EGF Receptor-PC12 cells were collected and resuspended in PBS or calcium-free PBS containing 1 mM EGTA and then stimulated for 2 min with 100 M ATP or 20 ng/ml EGF or stimulated for 5 min with 75 mM KCl. The cells were lysed in RIPA buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% SDS, and 0.5% deoxycholate) containing 1 mM phenylmethylsulfonyl fluoride, 1 g/ml aprotinin, and 1 g/ml leupeptin. The lysates were cleared by centrifugation, and the cleared lysates were preincubated with protein A-agarose beads for 30 min on ice. Aliquots contain-ing 1.5 mg of protein of the resulting supernatants were then incubated for 30 min at 4°C in the presence of either a Pyk2 antibody or an EGF receptor antibody. Protein A-agarose (20 l) was added, and the tubes were rocked at 4°C for an additional 4 h. The beads were washed three times in RIPA buffer and twice in TEV buffer. Proteins bound to the beads were resolved by SDS-PAGE and blotted to polyvinylidene difluoride membranes as described above. The resulting Western blots were probed with the antiphosphotyrosine antibody, 4G10, as described above, stripped, and reprobed with the antibody used for immunoprecipitation.

RESULTS
Extracellular ATP Activates the MAP Kinases ERK1 and ERK2 in PC12 Cells-The ability of purinoreceptors to activate intracellular signaling events was investigated by incubating the cells with ATP and evaluating the activation of the MAP kinases ERK1 and ERK2 by their reactivity with a phosphospecific ERK antibody. Extracellular ATP stimulated ERK phosphorylation within PC12 cells following a 5-min incubation with 100 M ATP (Fig. 1A). The level of activation of the MAP kinases by ATP was routinely not as great as that elicited by treatment with NGF. Extracellular ATP activated both ERK1 and ERK2 in a dose-dependent manner, reaching maximal levels upon treatment with 100 M ATP (Fig. 1B). The EC 50 of ATP-stimulated MAP kinase activity was estimated to be 25 M.
Kinetic experiments revealed that ATP stimulated the rapid and transient activation of the MAP kinases. Maximal activation of ERK1 and ERK2 was observed within 2 min of ATP addition and returned to near basal levels within 60 min (Fig.  2). The ATP-induced activation of the MAP kinases was considerably more rapid than that produced by growth factor treatment, which typically does not reach maximal levels until 5-10 min of stimulation of PC12 cells (5).
Extracellular Calcium Is Required for the Activation of MAP Kinases ERK1 and ERK2-The mechanisms by which metabotrophic and ionotrophic purinoreceptors mediate an elevation of intracellular calcium levels are distinct. The former is dependent upon phosphoinositide-dependent release from intracellular stores, whereas the latter is because of opening of a plasma membrane ligand-gated calcium channel. We tested the dependence of ATP-stimulated MAP kinase activation on extracellular calcium by incubating PC12 cells in calcium-containing and calcium-free PBS (Fig. 3). The activation of the MAP kinases was completely dependent upon the presence of extracellular calcium, whereas the response to NGF was not. Analysis of tyrosine phosphoproteins revealed that ATP treatment of the cells did not have significant effect on protein tyrosine phosphorylation, with the exception of the 42-44-kDa MAP kinases. These data document the requirement for extracellular calcium in the response of the MAP kinases for the effect of ATP but not NGF and indicate that the activation of FIG. 1. ERK1 and ERK2 are activated in PC12 cells following stimulation by extracellular ATP. A, PC12 cells were serum starved for 16 h prior to stimulation with either 100 M ATP or 50 ng/ml NGF for 5 min at 37°C. B, the dose dependence of MAP kinase activation was evaluated by treating the cells for 5 min with the indicated amounts of extracellular ATP. ERK activation was evaluated by Western blot analysis using an activation state-specific phospho-ERK antibody. The blots were stripped and reprobed with an anti-ERK antibody to verify uniform protein levels.
the MAP kinases was because of the action of ionotrophic P2X receptors. Moreover, treatment of the cells with dantrolene, which blocks the release of intracellular calcium by acting as an antagonist to the inositol triphosphate receptors, had no effect on the activation ATP-stimulated activation of the MAP kinases (data not shown). This finding verifies that extracellular calcium and not calcium from internal stores was responsible for the activation of the ERKs. One possible consequence of elevated calcium levels is the activation of cPKC isoforms; however, down-regulation of PKC by chronic phorbol ester treatment of the cells had no effect upon the activation of the MAP kinases by extracellular ATP, suggesting that MAP kinase activation occurs independently of PKC action (data not shown).
Ligand-specific Activation of the MAP Kinases ERK1 and ERK2 in PC12 Cells-To investigate both the specificity of the response and the identity of the receptors involved in the activation of the MAP kinase cascade, a battery of purinoreceptor ligands were tested for their ability to activate the MAP ki-nases in PC12 cells (Fig. 4). Each P2 receptor subtype is defined by its relative response to different purinergic ligands (1,4). UTP has been demonstrated to be a selective agonist of the P2Y2 and P2Y4 subclasses of purinergic receptors but showed no ability to activate the MAP kinases in our PC12 cells at a concentration of 100 M or greater (Figs. 4 and 8). Because the metabolic breakdown products of ATP stimulate other purinoreceptor subtypes, we tested their possible participation in the response. However, ADP, AMP, and adenosine were without effect. These data rule out the involvement of P1 and metabotrophic P2Y receptors.
The P2X receptors comprise the ionotrophic subclass of purinoreceptors, of which seven subtypes have been identified (1). We observed a highly selective effect of ATP on the activation of the MAP kinases and an insensitivity of the response to a,␤methylene ATP. This latter finding is of some importance because it also clearly discriminates between the response of P2X1 and P2X2 receptor subtypes, both of which have been reported to be expressed on PC12 cells (19). P2X1 receptors are activated by a,␤-methylene ATP, whereas P2X2 receptors are not. These data indicate that ATP-mediated activation of the MAP kinases was because of the action of P2X2 receptors. Moreover, the EC 50 for ATP of the P2X1 receptor is approximately 1 mM (1), which differs from the EC 50 of P2X2 receptors, consistent with the dose response observed here (Fig. 1).
The effect of ATP on P2X purinoreceptors is selectively antagonized by suramin and reactive blue 2 (8,9,11,20). Pretreatment of PC12 cells with either of these agents at concentrations that block ATP-driven calcium influx (11) for 30 min prior to stimulation with ATP abolished the ability of ATP to stimulate the MAP kinases (Fig. 4). The sensitivity of the response to these antagonists is consistent with the identification of a P2X2 receptor linked to activation of the MAP kinases and rules out the involvement of P2X4 and P2X6 purinoreceptor. P2X3 and P2X5 receptors are not known to be expressed in PC12 cells. Reverse transcription-polymerase chain reaction analysis of the PC12 cell line used in this study was found not to express the P2X7 (P2Z) purinergic receptor. 2 L-type Calcium Channels Are Not Linked to MAP Kinase Activation-ATP has been reported to result in the depolarization of PC12 and other cells (6). One consequence of membrane depolarization is to open voltage-sensitive calcium channels. We tested whether the ATP-stimulated activation of the MAP kinases was because of movement of calcium ions through these channels. PC12 cells were treated with the selective L-type calcium channel blockers, diltiazem, nifedipine, and verapamil. Preincubation of the cells with each of these drugs for 30 min at a concentration of 100 M was followed by ATP stimulation for 5 min. All of the channel blockers applied failed to inhibit the activation of ERK1 and ERK2 by ATP, demonstrating that L-type channels are not involved in this response (Fig. 5).
ATP Treatment Induces the Tyrosine Phosphorylation of Pyk2-Exposure of PC12 cells to ATP resulted in the induction of the tyrosine phosphorylation of Pyk2 (Fig. 6), which is correlated with its enzymatic activation (15). Treatment of the cells with KCl also stimulated Pyk2 tyrosine phosphorylation because of depolarization of the cells (15). EGF treatment did not change Pyk2 tyrosine phosphorylation levels. The ability of ATP or KCl to stimulate Pyk2 phosphorylation was abolished when the incubations were performed in the absence of calcium in the medium. ATP stimulated the rapid and evanescent activation of PYK2, reaching maximal levels within 2 min and returning to basal levels within 5 min (Fig. 6). The kinetics of PYK2 activation was similar to that observed with the MAP kinases.
The EGF receptor has been shown to become tyrosine phosphorylated in response to elevation of intracellular calcium levels following depolarization (21) or ligand-stimulated G␣ q and G␣ 11/12 activation (22) and acts as a surrogate signaling intermediate mediating the activation of the MAP kinase cascade. We tested whether ATP treatment of PC12 cells would enhance EGF receptor tyrosine phosphorylation. We were unable to detect any change in phosphotyrosine levels in response to either ATP or KCl. These data suggest that the ATP-stimulated activation of the MAP kinase cascade does not employ the EGF receptor as an intermediate in the activation of this signaling pathway (Fig. 7).
Clonal Differences in PC12 Cell Response to Purinergic Stimulation-Recently, Soltoff and colleagues (12) reported that PC12 cells utilized P2Y receptors in their response to ATP stimulation. We directly compared the PC12 cell line main-tained in this laboratory with those provided by Dr. Soltoff. The PC12 cell line used in this study were uniform, round, and refractile when viewed by phase microscopy, similar to the original clonal PC12 line described by Greene and Tischler (17). In contrast, the PC12 cell line obtained from Dr. Soltoff was morphologically heterogeneous with the majority of the cells exhibiting a pleiomorphic appearance and were phase gray (Fig. 8A).
The response of the two cell lines to purinergic ligands and NGF was compared (Fig. 8B). The PC12 cell line maintained in this laboratory exhibited a robust activation of the MAP kinases in response to NGF and also responded to ATP, albeit less robustly. This cell line did not respond to UTP, as described above (Fig. 4). The PC12 cell line used in the study by Soltoff et al. (12) responded to treatment of ATP and UTP by activation of the MAP kinases, a response consistent with their conclusion that this response was mediated by P2Y receptors.  Aliquots of these lysates were subjected to Western blot analysis using the phospho-specific ERK antibody to determine levels of ERK activation (C). Blots were stripped and reprobed with an anti-ERK antibody to confirm uniform loading (D).
Significantly, the response of the latter cells to NGF was of approximately the same magnitude as that produced by ATP. These results demonstrate that clonal variation accounts for the differences seen in these two studies. DISCUSSION Extracellular ATP and its catabolites have a diverse range of biological effects that are mediated by purinoreceptors (2,3). ATP can stimulate the rapid depolarization of cells as well as mediate long-lived changes in cellular metabolism. This range of biological actions reflects the involvement of multiple classes of purinoreceptors. These receptors are ubiquitously expressed and comprise several distinct subclasses. There has been substantial confusion over the identity and actions of the purinoreceptors, owing largely to the lack of specific agonists or antagonists. Molecular analysis of purinoreceptors and their effectors has now established that ATP acts principally through ionotrophic P2X and metabotrophic P2Y receptor subclasses. The linkage of the P2Y receptors to intracellular signaling events occurs through G-protein-coupled pathways, most notably those activating phospholipase C (4). Interestingly, both P2X and P2Y receptor subclasses stimulate an increase in intracellular calcium levels but accomplish this through mechanistically distinct pathways. It is of particular interest that the biological effects elicited by the two receptor subtypes are also distinct and provide direct evidence that the cell must possess sophisticated mechanisms to selectively deploy calcium (and its effectors) to drive specific intracellular signaling systems (1,2,4,23). One of the major unresolved issues surrounding the action of P2X receptors is how they are linked to intracellular signaling systems that subserve their specific biological effects. The present study provides direct evidence that calcium flux through the P2X2 receptors results in the activation of the MAP kinases.
PC12 cells have been extensively used as a model system to investigate both growth factor and purinoreceptor action. ATP has been demonstrated to depolarize PC12 cells (6), raise intracellular calcium levels (7,11,24), and elevate phosphoinositide levels (25). The principal biological effect of ATP is its action as a secretogogue, causing the release of catecholamines (5-7, 8, 9). However, the cloning and functional characterization of purinoreceptor subtypes has provided insight into the biological actions of these molecules (1). Considerable controversy exists over the involvement of the various purinoreceptors in the response of PC12 cells to purine agonists. This study demonstrates that much of the confusion over the action of purinergic ligands can be accounted for by the use of PC12 cell lines expressing different classes of purinergic receptors. The recent report of Soltoff and colleagues that PC12 cells responded to ATP by activation of the MAP kinases through P2Y receptors lead us to directly compare the cell line used in that study with those maintained in this laboratory. It is significant that we found dramatic differences in both the morphology of the two cell lines and in their sensitivity to purinergic ligands, reflective of their expression of different purinoreceptor classes. The two populations of PC12 cells were maintained under different culture conditions that may have presented different selection pressures on the cells and may account for the loss of the original morphological phenotype described by Greene and Tischler (17). The results presented here are an illustration of the inherent problem with the use of this cell type, and they provide a clear demonstration of the phenotypic instability of PC12 cells.
We have shown that exposure of PC12 cells to ATP results in the rapid and transient activation of the MAP kinases. The data strongly suggest that this is because of activation of the P2X2 receptor. We have ruled out the involvement of P1 purinergic receptors on the basis of the absence of an effect of adenosine or AMP, which act as agonists at these receptors. The ATP-mediated activation of the MAP kinases in PC12 cells is not because of metabotrophic P2Y purinoreceptors as evidenced by the insensitivity of this response to UTP, ADP, and the absolute dependence of the effect on extracellular calcium. The metabotrophic, G-protein-linked P2Y receptors have been shown to mediate the activation of the MAP kinases in cortical astrocytes (23,26,27) and another line of PC12 cells (12). Michel et al. (11) have convincingly argued that P2Z receptors are not responsible for ATP-stimulated calcium permeability in PC12 cells. Further, we have demonstrated that this receptor subclass is not expressed in our PC12 cells.
The ionotrophic P2X subclass of receptors comprise a structurally related family of ligand-gated ion channels with two transmembrane domains that are likely to assemble in homomeric and heteromeric oligomers, forming a calcium-specific channel (1). The ATP-stimulated calcium influx (11) and MAP kinase activation in PC12 cells is a consequence of the activa-  ). B, the two PC12 cell lines, the line used in this study (left panels), and the line obtained from Dr. S. Soltoff (right panels) were deprived of serum for 16 h and then treated with 100 mM ATP, UTP for 2 min or 50 ng/ml NGF for 5 min. Aliquots of these lysates were subjected to Western blot analysis using the phospho-specific ERK antibody to determine levels of ERK activation (top panels). Blots were stripped and reprobed with an anti-ERK antibody to confirm uniform loading (bottom panels). Cont., control. tion of the P2X2 subtype. The ATP EC 50 of approximately 25 M was observed for both MAP kinase activation and calcium influx (11), distinguishing the P2X2 from the P2X1 receptor, because the latter exhibits a much lower sensitivity (EC 50 ϭ 1 M) to ATP. A distinctive feature of the P2X2 subtype is its insensitivity to a,␤-methylene ATP (1,11). a,␤-Methylene ATP was unable to activate the MAP kinases; similarly, ATP-stimulated calcium influx was not inhibited by this agent (11). These data suggest that neither the P2X1 nor the P2X3 subtype is activated, because both are sensitive to the action of this ligand. This finding has been confirmed using an electrophysiological approach (28). Moreover, we found that the ATP stimulation of the MAP kinases was blocked by suramin and reactive blue 2. These findings are consistent with the observation that ATP-stimulated calcium influx (7,11,25) and dopamine release (20) were inhibited by these agents. P2X4 and P2X6 subtypes are not sensitive to purinergic inhibitor, suramin (1). This observation provided further support for the identification of the P2X2 subtype being responsible for both MAP kinase activation and calcium influx (11) in PC12 cells.
Calcium ions are able to provoke a diverse range of intracellular events. There is ample evidence that the cell possesses sophisticated mechanisms to compartmentalize and selectively deploy calcium and that its effectors elicit specific cellular effects. The biological effects of changes in intracellular calcium concentration are dependent upon the mechanisms through which calcium levels are elevated (14), as evidenced by the ability of activation of L-type calcium channels to promote cellular survival, whereas ligand-gated calcium influx through N-methyl-D-aspartic acid receptors is linked to excitotoxic cell death. It is presently not understood how these specific biological effects of calcium are achieved. Depolarization of neurons results in elevation of intracellular calcium levels through the opening of voltage-gated calcium channels (14). Extracellular ATP elicits the depolarization of PC12 cells (20). We have provided data indicating that agents that block L-type calcium channels have no effect on the ability of ATP to activate the MAP kinases, indicating that the calcium permeability is mediated principally by the P2X receptor. There is presently no evidence indicating the opening of voltage-sensitive calcium channels subsequent to ATP-mediated depolarization (7,11,25). Paradoxically, K ϩ -stimulated depolarization of these cells results in opening of the voltage-sensitive calcium channels and activation of the MAP kinases ( Fig. 6 and Refs. 21 and 29).
The MAP kinases ERK1 and ERK2 are responsible for propagation of mitogenic signals in response to growth factor stimulation, resulting in changes in cellular morphology, metabolism, and gene expression. In PC12 cells, the ERKs have been demonstrated to be activated by growth factor stimulation with NGF and EGF. The receptors for these factors are themselves tyrosine kinases that undergo autophosphorylation, with the resulting phosphotyrosine residues catalyzing the formation of a signaling complex leading to the activation of p21 ras . p21 ras is an essential element of this cascade and when activated directly stimulates the serial phosphorylation and activation of B-Raf, MAP/ERK kinase, and the MAP kinases. A central finding of this study is that extracellular ATP, acting over a P2X purinergic receptor, leads to the phosphorylation and activation of the ERKs. We demonstrate that the mechanism of ERK activation is distinct from that induced by NGF, both by its time course and by its dependence upon extracellular calcium. We were unable to detect any effect of ATP on the activation of other MAP kinase superfamily members, including the stress-activated protein kinases (also termed c-Jun N-terminal kinases or JNKs) or p38 MAP kinase pathways (data not shown).
The linkage of changes in calcium ion levels to the activation of the MAP kinases is illustrative of the complexity of calciumbased signaling events. Presently, several mechanistically distinct signaling processes have been associated with activation of p21 ras and the MAP kinases. The K ϩ -stimulated depolarization of PC12 cells or N-methyl-D-aspartic acid treatment of cortical neurons results in the activation of the MAP kinases as a consequence of calcium influx through L-type channels and the N-methyl-D-aspartic acid receptor, respectively (13,14). The elevation of intracellular calcium levels results in the activation of p21 ras and the subsequent serial activation of protein kinases comprising the MAP kinase cascade. The recent discovery of a calcium-activated protein-tyrosine kinase, Pyk2, has raised the possibility that it too may be a critical intermediate in the activation of p21 ras and the MAP kinases. Lev and colleagues (15) have demonstrated that Pyk2 activation can be stimulated by elevation of intracellular calcium levels. The activated Pyk2 forms complexes with the adapter proteins Shc and Grb2 and the p21 ras nucleotide exchange factor, Sos, leading to MAP kinase activation. The observation of ATP-stimulated, calcium-dependent phosphorylation of Pyk2 strongly suggests the involvement of this enzyme in the P2X2 receptor-driven MAP kinase activation.
In PC12 cells, plasma membrane depolarization has been reported to stimulate the tyrosine phosphorylation of the EGF receptor, which has been postulated to act as a surrogate whose phosphotyrosine residues serve to catalyze the assembly of a signaling complex mediating the activation of p21 ras (21). This is an unusual use of a growth factor receptor in a calciuminitiated signaling pathway. We were unable to confirm this observation and failed to observe any change in EGF receptor tyrosine phosphorylation in response to depolarization, despite a robust stimulation of Pyk2 phosphorylation. Moreover, we found that extracellular ATP did not alter the phosphotyrosine content of the EGF receptor, indicating that the purinoreceptor-mediated calcium influx does not utilize the EGF receptor as a signaling intermediate leading to MAP kinase activation. In some cell types, the activation of heterotrimeric G-proteincoupled receptors also results in the activation of the MAP kinases as a consequence of increased intracellular calcium liberated from internal stores (22). The exact nature of the involvement of tyrosine kinases in this scheme is not presently clear (30), and the specific signaling molecules employed are likely to be cell type-specific.
The mechanisms subserving the biological effects of the ionotrophic P2X receptors have not been extensively investigated, and the present study documents that purinoreceptors in this class employ well characterized signal transduction pathways to mediate their biological effects. A critical unresolved issue is how calcium ion influx is coupled to the upstream elements regulating the activation of the MAP kinases. The recent identification of novel calcium-regulated signaling intermediates has opened new avenues for investigation of these events.