INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

G PROTEIN-COUPLED RECEPTOR (GPCR) agonists can stimulate cell proliferation (7, 10, 38, 45). One of these GPCR agonists is extracellular ATP (5). ATP induces its mitogenic effect by binding to multiple seven-membrane-spanning G protein-coupled P2Y nucleotide receptor subtypes. In addition, ATP modulates vascular tone and neuronal transmission by binding to ligand-gated ion channel P2X receptors (5).

On binding of a cognate ligand to the appropriate GPCR, the G_q subunit of the heterotrimeric G protein can dissociate from the -subunits and activate phospholipase C, which hydrolyses phosphatidylinositol 4,5-bisphosphate to D-myo-inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] and diacylglycerol (7, 10, 38, 45). Ins(1,4,5)P₃ and diacylglycerol induce intracellular Ca²⁺ mobilization and protein kinase C activation, respectively (3). Other cellular responses shown to be induced by GPCR agonists are activation of extracellular signal-regulated kinases (ERK) 1 and 2, tyrosine phosphorylation of signaling proteins, complex formation between growth factor binding protein (Grb2) and insulin receptor substrates (IRSs), and activation of Ras (3, 14, 27, 29). These finding indicate cross-talk between GPCR and receptor tyrosine kinase (RTK) signaling.

Insulin binding to its RTK activates the kinase domain leading to autophosphorylation and subsequent tyrosine phosphorylation of the IRS family and Src homology 2-containing collagen-like (Shc) substrates. The guanine nucleotide exchange protein, son of sevenless (SOS), is constitutively bound to Grb2; SOS becomes associated with the plasma membrane on engagement of Grb2 with the tyrosine-phosphorylated sites of IRSs or Shc. SOS activates Ras, which in turn activates the Raf-mitogen-activated protein or ERK kinase (MEK)-ERK phosphorylation cascade. Insulin also stimulates association of phosphatidylinositol 3-kinase (PI3K) with phosphotyrosyl-IRSs, leading to activation of the lipid kinase with the production and accumulation of phosphatidylinositol phosphate messengers (phosphatidylinositol-3-phosphate; phosphatidylinositol-3,4-bisphosphate; or phosphatidylinositol-3,4,5-trisphosphate) (11, 15, 43). These lipid products have been shown to be required for activation of Akt (also known as protein kinase B) by binding to its pleckstrin homology domain (11). In addition, activation of Akt involves phosphorylation at threonine residue 308 and serine residue 473 by phospholipid-dependent kinases (1, 28). How the ERK and Akt signaling pathways interact and modify each other is currently under investigation.

Stressed or damaged cells and activated platelets can release micromolar quantities of ATP (16) at the site of vascular damage, while insulin is normally or therapeutically present. In the present study, we found that ATP and insulin synergistically stimulate coronary artery smooth muscle cell (CASMC) growth. To understand how ATP and insulin signal the synergistic stimulation of CASMC proliferation, we investigated signaling responses of CASMC stimulated with ATP or a combination of ATP and insulin at the cellular and molecular levels. We further showed that stimulation of CASMC with ATP induces activation of ERK, the upstream kinases MEK and Raf, and the GTP-binding protein Ras and that insulin acts synergistically with ATP in activating the ERK pathway. In addition, ATP inhibits insulin-stimulated Akt phosphorylation, suggesting a possible mechanism for the synergistic proliferative response.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation and culture of CASMC. CASMC were enzymatically isolated from porcine coronary arteries as described previously (47). Briefly, coronary arteries dissected from porcine hearts were denuded of endothelium and placed in physiological buffer [containing (in mM) 10 HEPES (pH 7.4), 138 NaCl, 2 CaCl₂, 1 MgCl₂, 5 KCl, and 10 glucose and 1% each of penicillin and streptomycin]. Cells were isolated by treating arteries with 294 U/ml collagenase in the presence of 0.2% bovine serum albumin, 0.1% soybean trypsin inhibitor, and 0.04% DNase at 37°C in a water bath for 1 h; this procedure was repeated two more times with fresh solutions of collagenase. Dispersed cells were then isolated and placed into culture in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO₂. The isolates were confirmed to be of smooth muscle lineage by actin immunocytochemistry.

DNA synthesis assay. This assay was performed as described previously with minor modifications (47). Briefly, subconfluent cultures of CASMC in 12-well plates were serum starved for 24 h and then stimulated with 100 µM ATP or 100 nM insulin or a combination of both agonists for 42 h; the last 24 h in the presence of 1 µCi [methyl-³H]thymidine. The cells were then washed with ice-cold PBS and solubilized in 0.1% SDS. After addition of trichloroacetic acid to a final concentration of 10%, the precipitates were collected by filtration on glass-fiber discs (Whatman), and radioactivity was determined by scintillation counting.

Phosphorylation of ERK. CASMC grown to confluence were serum starved for 24 h before treatment with 100 µM ATP, 100 nM insulin, or the combination. For experiments that involved the MEK inhibitor PD98059 (BioMol Research Labs), CASMC were treated with 100 µM PD98059 for 60 min before stimulation with agonists. Cells were then lysed in 2× Laemmli sample buffer [100 mM Tris (pH 6.8), 200 mM dithiothreitol (DTT), 4% SDS, 0.2% bromophenol blue, and 20% glycerol], and lysates were boiled and then run on 10% SDS-PAGE gels. Proteins were transferred onto a nitrocellulose membrane, blocked in 5% nonfat milk, Western blotted with anti-phospho ERK1/2 antibody, and detected by enhanced chemiluminescence (New England Biolabs) according to the manufacturer's instructions.

In-gel kinase assay of ERK. The same lysates used for determining ERK phosphorylation were used to determine the kinase activity. Proteins were separated by SDS-PAGE on 10% gels containing 0.5 mg/ml myelin basic protein. SDS was removed by incubating gels at room temperature in a solution that contained 50 mM Tris · HCl (pH 8.0) and 20% isopropanol; three incubations each for 20 min were performed. Proteins were denatured by incubating gels in 6 M guanidine-HCl and 5 mM -mercaptoethanol in 50 mM Tris · HCl (pH 8.0) for 1 h at room temperature and then renatured by incubation in 50 mM Tris · HCl (pH 8.0) containing 50 mM -mercaptoethanol and 0.04% Triton X-100, first at room temperature for 1 h and then at 4°C overnight. The kinase assay was performed by incubating gels in a kinase buffer containing 40 mM HEPES (pH 7.5), 10 mM MgCl₂, 2 mM DTT, 40 µM ATP, and 400 µCi [-³²P]ATP for 1 h at room temperature (19). The gels were then washed five times each for 20 min in 10% trichloroacetic acid containing 1% sodium pyrophosphate, fixed in destain solution (20% methanol and 8% acetic acid), dried, and then were autoradiographed or had radioactivity measured by a phosphorimager (Bio-Rad).

In vitro immunocomplex kinase assays. The kinase activity of Raf and MEK was determined as described previously (4, 24) with minor modification. Cells cultured in 10-cm dishes were stimulated with 100 µM ATP, 100 nM insulin, or the combination and lysed in 25 mM HEPES (pH 7.5), 150 mM NaCl, 50 mM sodium pyrophosphate, 50 mM NaF, 5 mM EDTA, 50 mM -glycerophosphate, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin. After lysates were cleared by centrifugation at 10,000 g, Raf-1 and MEK-1 were immunoprecipitated at 4°C with anti-Raf-1 (C-20, Santa Cruz) and anti-MEK-1 (C-18, Santa Cruz) rabbit polyclonal antibodies (1 µg/ml of lysate) complexed to agarose beads. Samples were then washed three times with lysis buffer, once with lithium chloride buffer [20 mM Tris · HCl (pH 7.5), 500 mM LiCl, 1 mM DTT, and 0.1% Triton X-100], and twice with kinase buffer [40 mM HEPES (pH 7.5), 10 mM MgCl₂, and 1 mM DTT]. The kinase reactions were performed for 15 min at room temperature in a 30-µl volume of kinase buffer that contained 100 µM ATP, 2 µCi [-³²P]ATP, and 1 µg of kinase inactive MEK-1 or ERK2 as appropriate with constant shaking. The reactions were stopped by adding Laemmli sample buffer. Proteins were resolved on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, and were autoradiographed or had radioactivity measured by phosphorimager.

Ras-Raf association assay. The activated GTP-bound form of Ras was determined by its association with the Ras binding domain (RBD) of Raf-1 (residues 1-149) fused to glutathione-S-transferase (GST) (44). Bacteria expressing GST-RBD were lysed by sonication in 20 mM HEPES buffer containing 120 mM NaCl, 10% glycerol, 2 mM EDTA, 0.5% NP-40, and 10 µg/ml each of aprotinin and leupeptin. After lysates were cleared by centrifugation at 10,000 g, GST-RBD was purified on glutathione sepharose beads (10-µl packed volume, ~1 µg GST-RBD/sample) by incubation for 2 h at 4°C. After the beads were washed five times with lysis buffer, CASMC lysate prepared from a confluent 10-cm plate of cells treated with 100 µM ATP, 100 nM insulin, or the combination was added and further incubated for 2 h at 4°C. Agarose beads were then washed three times with lysis buffer, and bound proteins were eluted by boiling for 10 min in Laemmli sample buffer, separated on a 10% SDS-PAGE gel, transferred to nitrocellulose, probed with anti-pan-Ras monoclonal antibody (R02120, Transduction lab), and detected by enhanced chemiluminescence.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We (47) have previously reported that extracellular ATP induces ERK-dependent proliferation of CASMC. In the current study, we investigated the cellular and molecular signaling mechanisms responsible for the synergistic stimulation of CASMC by ATP and insulin in proliferation.

Extracellular ATP and insulin synergistically activate proliferation of CASMC. Stimulation of DNA synthesis as determined by [³H]thymidine incorporation is often used as a criterion for evaluating cell proliferation. In the current study, we investigate the effect of costimulation of CASMC with ATP and insulin on [³H]thymidine incorporation into DNA. ATP and insulin stimulated DNA synthesis four- and twofold over basal, respectively. In costimulated cells, the increase in DNA synthesis was 17-fold over basal (Fig. 1). These results show that ATP and insulin synergize in inducing cell proliferation in CASMC.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   ATP and insulin (Ins) synergistically stimulate DNA synthesis in coronary artery smooth muscle cells (CASMC). Subconfluent cultures of CASMC synchronized by incubation in serum-free medium for 24 h were stimulated with either ATP (100 µM) or insulin (100 nM) or the combination for 18 h at 37°C followed by an additional 24 h with 1 µCi [3H]thymidine. After acid-precipitable material was prepared, stimulation of DNA synthesis was determined by measuring CPM of [3H]thymidine incorporated [in counts per minute (cpm)]. The data presented here are an average of 8 independent experiments (means ± SD).

Synergistic activation of ERK by ATP and insulin. ERK has been shown to play a central role in both peptide growth factor- and GPCR agonist-induced cell proliferation (27). We and others (41, 47) have shown that ERK becomes activated in CASMC stimulated with ATP. To determine whether the synergistic proliferative response in costimulated cells was accompanied by a synergistic ERK response, in-gel kinase assays were performed (see In-gel kinase assay of ERK). Insulin alone had very little effect on ERK activation (twofold over basal), whereas ATP stimulated a sevenfold increase over basal (Fig. 2). Costimulation of CASMC led to a 17-fold increase over basal in ERK activity. These findings suggest a synergism between growth factor- and GPCR agonist-induced ERK activation.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Synergistic activation of extracellular signal-regulated kinase (ERK) by ATP and insulin (IN). Confluent cultures of CASMC were serum starved for 36 h and then stimulated with ATP (100 µM), insulin (100 nM), or the combination for 10 min. ERK activity was determined by in-gel kinase assay (see MATERIALS AND METHODS, In-gel kinase assay of ERK). The results presented are an average of 3 experiments. Con, control.

Synergistic activation of ERK by ATP and insulin is sustained. Previously, we (47) showed that ATP activates ERK in a time-dependent manner with maximal activation at 5 min and returning to the basal level in <20 min. To determine the time course of the observed synergism between ATP and insulin in activating ERK, CASMC were costimulated with ATP and insulin or ATP alone for varying periods of time ranging from 2 min to 12 h (Fig. 3). The general trend was that ERK phosphorylation was sustained for a longer period during costimulation. The level of ERK phosphorylation at 5 min was 16- and 8-fold over basal for the costimulated and ATP-stimulated samples, respectively (Fig. 3, A and B). These results are consistent with the results in Fig. 2: that ATP and insulin synergistically activate ERK. At 10 min, the phosphorylation level was 9-fold in costimulated samples but only 3.5-fold over basal in ATP-stimulated samples. The phosphorylation of ERK returned to near-basal levels in <30 min in the case of ATP stimulation (1.5-fold over basal) but was sustained for up to 2 h, although at a lower level (4-fold over basal), by costimulation (compare Fig. 3A with Fig. 3B, lanes at 30 min and 2 h). These results suggest that costimulation with ATP and insulin induces not only a synergistic response but also a more sustained activation in CASMC.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Time course of ATP and insulin stimulation of ERK. Serum-starved CASMC were stimulated with ATP (100 µM) + insulin (100 nM) (A) or ATP alone (B) for the indicated times. Samples were then analyzed by Western blot using anti-phospho-ERK antibody. Laser scanning densitometry was used to determine the relative intensity of the signal. The Western blots shown are representatives of 3 experiments.

Synergistic activation of MEK by ATP and insulin. MEK has been shown to be the upstream activators of ERK in various cell systems (8, 9, 21, 46). To determine whether MEK was the upstream kinase responsible for ERK activation in CASMC stimulated with ATP or the combination of ATP and insulin, we analyzed ERK phosphorylation after treatment of cells with the MEK selective inhibitor PD98059. Serum-starved CASMC were treated with 100 µM PD98059 for 60 min before stimulation with ATP or a combination of ATP and insulin. PD98059 treatment inhibited ERK phosphorylation induced by ATP (Fig. 4A, lanes 2 and 3) or insulin (lanes 6 and 7). Similarly, PD98059 inhibited the synergistic phosphorylation of ERK induced by ATP and insulin (lanes 4 and 5). These results suggest that MEK is the upstream kinase responsible for ATP- and growth factor-induced ERK activation.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Activation of mitogen-activated protein or ERK kinase (MEK) by ATP and insulin. A: serum-starved CASMC were treated with 100 µM PD98059 for 60 min (lanes 3, 5, and 7) before stimulation with ATP (100 µM) (lanes 2 and 3), insulin (100 nM) (lanes 6 and 7), or a combination of ATP and insulin (lanes 4 and 5). Lane 1: control without treatment with PD98059, ATP, or insulin; pluses and minuses, with and without specific treatment, respectively. Lysates from these cells were analyzed by Western blot with anti-phospho ERK anibody. The Western blot shown is a representative of 5 independent experiments. B: MEK-1 was immunoprecipitated from the indicated lysates, and its enzymatic activity was determined by immunocomplex kinase assay using kinase-inactive ERK2 as a substrate in the presence of [-32P]ATP. The reaction results were separated on a 10% gel, transferred onto a nitrocellulose membrane, and analyzed by 32P quantitation. The data presented are an average of 3 experiments (means ± SD).

ATP and insulin stimulation of Raf activity is additive. Raf-1 has been shown to be the principal activator of MEK in eukaryotic cells (12, 17, 48). Recent reports (31) indicate that Raf-1 is also activated after stimulation of cells with GPCR agonists such as angiotensin II and endothelin-1. To determine whether Raf-1 is activated in CASMC by ATP, insulin, or the combination, we performed a Raf-1 immunocomplex kinase assay. Confluent cultures of CASMC were serum deprived and then stimulated for 5 min with ATP, insulin, or the combination. Lysates prepared from these cells were subjected to immunoprecipitation, and an in vitro Raf-1 kinase assay was performed using a kinase-inactive mutant of human MEK as a substrate (see In vitro immunocomplex kinase assays). ATP stimulated Raf-1 kinase activity by fivefold over basal (Fig. 5), whereas insulin alone had little effect (twofold). In costimulated cells, the kinase activity was sevenfold. These results show that Raf-1 is activated in CASMC by ATP and is the likely mediator of ATP-induced signals upstream of MEK. The additive nature of ATP and insulin activation Raf-1 makes it a common point of the two signaling pathways.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effects of ATP and insulin on Raf-1 activity. Serum-starved confluent cultures of CASMC were stimulated with either ATP (100 µM) or insulin (100 nM) or the combination at 37°C for 5 min. Lysates of these cells were subjected to immunoprecipitation with anti-Raf-1 antibody and then an in vitro immunocomplex kinase assay in the presence of [-32P]ATP and kinase-inactive MEK-1 as substrates. The immunocomplex kinase reactions were then separated on a 10% gel, transferred onto a nitrocellulose membrane, and analyzed by 32P quantitation. The data presented are an average of 3 experiments (means ± SD).

Ras is involved in ATP-induced signaling. Several reports (27, 32) have indicated that Ras is necessary for mediating some signals initiated by GPCR agonists in various cell types. To provide further insight on the activation of the ERK signaling pathway by ATP, we explored the activation of Ras and its interaction with Raf-1 in CASMC stimulated with ATP, insulin, or the combination. The RBD of Raf-1 fused to GST was used for affinity binding studies of activated Ras (see Ras-Raf association assay). ATP stimulated a 5-fold increase over basal in Ras bound to the Raf-RBD, whereas insulin stimulated a 2.5-fold increase (Fig. 6). In CASMC costimulated with ATP and insulin, Ras activation was sevenfold. When aliquots of the same samples were immunoprecipitated with anti-Raf-1 antibody and analyzed by Western blot with anti-Ras monoclonal antibody for coimmunoprecipitation, Ras coimmunoprecipitated with Raf-1 in ATP- and insulin-stimulated and costimulated samples (data not shown). These results suggest that ATP stimulates Ras in CASMC; however, like Raf activation, Ras activation with ATP and insulin is additive with ATP for activation of Ras.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Ras is involved in ATP-induced signaling. Serum-starved confluent cultures of CASMC were stimulated with ATP (100 µM) or insulin (100 nM) or the combination for 5 min at 37°C. Lysates prepared from these cells were incubated with glutathione-S-transferase-Raf-Ras binding domain immobilized on glutathione-agarose for 2 h at 4°C. Bound proteins were eluted by boiling with Laemmli sample-loading buffer, run on a 10% gel, transferred onto a nitrocellulose membrane, and analyzed by Western blot using anti-Ras monoclonal antibody.

ATP induces ERK pathway, whereas it inhibits insulin-stimulated Akt pathway. Akt has been shown to be a downstream effector of PI3K (6, 32). Akt becomes serine-threonine phosphorylated on stimulation of the PI3K pathway (37). In other systems, interactions between the ERK and PI3K signaling pathways have been documented. We took this opportunity to study the effect of ATP, insulin, and combination treatment on the phosphorylation of Akt. Total lysates from CASMC stimulated with ATP, insulin, or the combination were analyzed with anti-phospho-Akt-specific antibody (New England Biolabs). Insulin stimulated the phosphorylation of Akt 10-fold over basal, and ATP stimulated a 2.5-fold increase (Fig. 7A). In cells costimulated with insulin and ATP, the phosphorylation level of Akt was only twofold over basal, suggesting a negative regulatory effect of ATP on insulin-induced Akt activation.

View larger version (83K): [in this window] [in a new window]   Fig. 7.   ATP inhibits insulin-induced Akt phosphorylation. The samples in Fig. 2 were electrophoresed on a 10% gel, transferred onto a nitrocellulose membrane, and analyzed by Western blot with anti-phosphoserine-threonine kinase (Akt) antibody (A). Confluent cultures of CASMC were stimulated with 100 nM insulin and varying concentrations of ATP at 37°C for 5 min. Lysates prepared from these samples were run on a 10% gel and analyzed by Western blot first with anti-phospho-Akt (B) and then with anti-phospho-ERK antibody (C). The Western blots shown are representative of 3 independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Intimal thickening due to abnormal proliferation of vascular smooth muscle cells is the major cause of coronary artery disease (CAD) and cardiovascular complications in diabetics (13, 22, 30, 36, 42). Several growth factors and GPCR agonists, including ATP, have been implicated in inducing proliferation of smooth muscle cells induced by vascular injury (5, 19, 23, 26, 27, 38-40, 45). It is reported that ATP is released in micromolar quantities by activated platelets and damaged or stressed cells at the site of vascular injury. Thus ATP could be a potential modulator of abnormal CASMC proliferation. On this basis, it is logical to hypothesize that extracellular ATP might have pathophysiological significance, especially in disease states such as CAD, atherosclerosis, and vessel restenosis.

We (47) recently showed that ATP induces ERK activation-dependent proliferation of CASMC. In this report, we demonstrate that insulin potentiates the mitogenic effect of ATP in CASMC. These results suggest that RTK- and GPCR-signaling pathways cooperate in inducing mitogenic responses in CASMC. It is possible that, under conditions of vascular damage, ATP, released locally by aggregating platelets and damaged cells, might synergize with insulin and other growth factors in the circulation to induce proliferation of CASMC. Another interesting observation is that insulin, which by itself is a poor mitogen in CASMC, becomes a powerful amplifier of mitogenic signals when present with ATP, a finding that may have pathophysiological relevance in hyperinsulinemic and/or insulin-treated diabetics.

The activation of ERK in CASMC treated with ATP and insulin is not only synergistic in magnitude but also more sustained. This could explain the observed synergism between ATP and insulin in inducing a hyperproliferative response in CASMC. Previous work (2, 23, 33, 40) has shown that ERK, which has sustained activation, translocates to the nucleus and phosphorylates transcription factors such as c-Jun, c-Myc, and Elk. In light of this, it is tempting to speculate that the increased magnitude and sustained activation elicited by the costimulation may prolong the time of ERK action after translocation to the nucleus, thereby enhancing proliferative responses. To our knowledge, this is the first demonstration of synergism between insulin, a RTK agonist, and ATP, a GPCR agonist, inducing CASMC proliferation.

Several reports (18, 21, 29, 38, 48) indicate that Raf-1 becomes activated in cells stimulated with angiotensin II and other GPCR agonists. Our findings are in agreement with these previous reports and further demonstrate that Raf-1 participates in mediating signals (by activating MEK) elicited by ATP in CASMC. We have demonstrated that Ras becomes activated in CASMC stimulated with ATP, insulin, or the combination, suggesting that Ras participates in GPCR agonist-induced signaling. The observation that the synergism between ATP and insulin was less pronounced at the level of Raf-1 than those observed at the level of ERK and MEK may suggest the existence of ATP-induced parallel cascade that converges at Raf or that Raf activity is amplified to cause the larger downstream response.

After evaluating the effect of ATP on the ERK pathway, we thought it was a logical step to assess the activation status of PI3K-Akt pathway, the second major pathway that becomes activated by insulin and other growth factors. ATP did not stimulate, but rather inhibits, insulin-induced Akt phosphorylation in costimulated samples in a concentration-dependent manner. Other reports have shown that activators of some PKC isoforms negatively regulate the PI3K pathway by inducing phosphorylation of serine residue 612 in IRS-1, which blocks tyrosine phosphorylation of the PI3K binding site (25) and consequently the association between IRS1/2 and the p85 subunit of PI3K (11). Thus inhibition of the PI3K-Akt pathway by the P2Y agonist ATP could be by suppressing the interaction of the p85 subunit of PI3K with IRS molecules. Recent reports (20, 35) show that, depending on the differentiation state of the cell, the PI3K-Akt pathway has opposing effects on the Raf-MEK-ERK pathway; it is shown that Akt complexes with Raf and phosphorylates a highly conserved serine residue in the regulatory domain of Raf. Thus these results are consistent with ATP releasing the insulin-stimulated Akt-dependent inhibition of the ERK signaling pathway, leading to synergistic stimulation of CASMC proliferation.

In summary, this study demonstrates that the P2Y agonist ATP and the RTK agonist insulin induce proliferation of CASMC synergistically. Furthermore, this report reveals that the ATP-induced GPCR pathway shares signaling mediators of the ERK pathway to modulate cellular responses, providing further insight into the cross-talk between the RTK and GPCR signaling pathways. On the basis of our results and the works of others (39, 49), we propose that Raf-1 and/or MEK might be direct targets for phosphorylation by protein kinase C, a protein kinase that becomes activated after stimulation of cells with GPCR agonists. The activation of Ras by ATP may be explained by previous reports showing that the -subunits of G_i proteins activate the Ras-Raf-ERK pathway via Src, a cytosolic tyrosine kinase that phosphorylates adapter proteins like Shc. Thus the synergistic mitogenic response in costimulated CASMC could be a result of 1) the cooperation between the Ras and PKC pathways that converge at the level of Ras/Raf and/or 2) ATP-induced release of the inhibitory effect of the PI3K-Akt pathway on the Raf-MEK-ERK pathway. The molecular mechanism(s) as well as the physiological significance of the opposing effect of ATP on the PI3K pathway are unanswered questions that require further investigation.