Conditional Inhibition of the Mitogen-activated Protein Kinase Cascade by Wortmannin

Phosphoinositide (PI) 3-kinase and the mitogen-activated protein (MAP) kinase cascades are activated by many of the same ligands. Several groups have reported involvement of PI 3-kinase in the activation of Erk1 and Erk2, whereas many other groups have shown that activation of Erk1 and Erk2 is not sensitive to inhibitors of PI 3-kinase such as wortmannin. Here we show that wortmannin inhibition of the MAP kinase pathway is cell type- and ligand-specific. Wortmannin blocks platelet-derived growth factor (PDGF)-dependent activation of Raf-1 and the MAP kinase cascade in Chinese hamster ovary cells, which have few PDGF receptors, but has no significant effect on Erk activation in Swiss 3T3 cells, which have high levels of PDGF receptors. However, wortmannin blocks activation of Erk proteins if Swiss 3T3 cells are stimulated with lower, physiological levels of PDGF. These results suggest that PI 3-kinase is in an efficient pathway for activation of MAP kinase, but that MAP kinase can be stimulated by a redundant pathway when a large number of receptors are activated. We present evidence that a protein kinase C family member downstream of phospholipase Cγ is involved in the redundant pathway.


Phosphoinositide (PI) 3-kinase and the mitogen-activated protein (MAP) kinase cascades are activated by many of the same ligands. Several groups have reported involvement of PI 3-kinase in the activation of Erk1 and Erk2, whereas many other groups have shown that activation of Erk1 and Erk2 is not sensitive to inhibitors of PI 3-kinase such as wortmannin. Here we show that wortmannin inhibition of the MAP kinase pathway is cell type-and ligand-specific. Wortmannin blocks platelet-derived growth factor (PDGF)-dependent activation of Raf-1 and the MAP kinase cascade in Chinese hamster ovary cells, which have few PDGF receptors, but has no significant effect on Erk activation in Swiss 3T3 cells, which have high levels of PDGF receptors. However, wortmannin blocks activation of Erk proteins if Swiss
3T3 cells are stimulated with lower, physiological levels of PDGF. These results suggest that PI 3-kinase is in an efficient pathway for activation of MAP kinase, but that MAP kinase can be stimulated by a redundant pathway when a large number of receptors are activated. We present evidence that a protein kinase C family member downstream of phospholipase C␥ is involved in the redundant pathway. PI 1 3-kinase has been implicated as being involved in the signal transduction of virtually all growth factors studied and in the transformation of cells by several oncoproteins (1,2). Activation of PI 3-kinase with growth factors results in the appearance of the lipid products of this enzyme, PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 , within seconds to minutes. There is also a correlation between the elevation in PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 levels and cell transformation. These correlations have suggested an important role for PI 3-kinase in signal transduction pathways leading to cell growth and transformation. Indeed, the catalytic subunit of PI 3-kinase (p110␣) has recently been identified as a retroviral oncogene (3). Overexpression of p110␣ in chick embryo fibroblasts results in constitutive elevation of PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 and cell transformation.
The MAP kinase pathway is another key component in the transduction of signals leading to growth and transformation. This pathway consists of a linear cascade of the protein kinases Raf, MEK, and MAP kinase/Erk; like PI 3-kinase, Erk1 and Erk2 are acutely activated by growth factors and are found constitutively activated in many transformed cell lines. The Erk proteins are phosphorylated and activated by the dual specificity kinase MEK (MAP kinase/Erk kinase), which is phosphorylated and activated by the serine/threonine kinase Raf. Raf is recruited to the membrane of activated cells by direct binding to Ras-GTP. This recruitment by activated Ras is necessary but not sufficient for full activation of Raf (4); therefore, other factors that are necessary for activation of the MAP kinase pathway may feed into the pathway at Raf. A search for other sources that feed into the MAP kinase pathway has yielded several candidates including Src, members of the PKC family, and PI 3-kinase (45).
The relevance of PI 3-kinase for activation of Erk has been controversial. Expression of activated forms of p110␣ PI 3kinase has been reported to stimulate the MAP kinase pathway in one case (5), but not in others (6 -9). A recent study found that overexpression of the p110␥ type of PI 3-kinase resulted in the activation of Erk, but that p110␣ did not (9). Inhibition of PI 3-kinase with wortmannin (10 -16) or dominant-negative PI 3-kinase (9) has been shown to block activation of MAP kinase in some but not all cells (17)(18)(19).
Several direct targets of the lipid products of PI 3-kinase have been identified that could act as intermediates between PI 3-kinase and the MAP kinase pathway. Members of the novel PKC family (calcium-independent), PKC⑀, PKC␦, and PKC (20), have been shown to be activated in vitro by two lipid products of PI 3-kinase, PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 . Inhibition of thrombin-dependent phosphorylation of pleckstrin by wortmannin, combined with the ability of these lipids to stimulate phosphorylation of pleckstrin when added to permeabilized platelets (21,22), suggests that they activate PKC family members in vivo. PDGF activates PKC⑀ (23) and PKC (24) by a mechanism that requires PI 3-kinase. Several PKC family members (25)(26)(27) have already been implicated in the activation of the MAP kinase pathway via phosphorylation of Raf. Another potential intermediate is the serine/threonine protein kinase Akt/PKB, which binds to and is activated by PtdIns-3,4-P 2 in vitro (28,29), and PDGF stimulates this enzyme via activation of PI 3-kinase (30). The serine/threonine kinase p70 S6K has been shown to be downstream of PI 3-kinase (31). Thus, a potential means for PI 3-kinase to feed into the MAP kinase pathway could be via activation of one (or more) of these targets of PI 3-kinase.
We have attempted to better understand the role that PI 3-kinase plays in the activation of the MAP kinase pathway.
We show here that the effect of wortmannin on the activation of Erk varies not only between cell lines, but within a single cell line depending on both the kind and concentration of the growth factor used. We have found that Erk activity is sensitive to wortmannin when relatively few receptors are activated, but resistant if a large number of receptors are activated. We have also found that a down-regulatable PKC contributes to wortmannin-resistant MAP kinase activation.

MATERIALS AND METHODS
Cell Lines-CHO/HIR cells are a CHO clone expressing the human insulin receptor and were maintained in RPMI 1640 medium and 10% fetal calf serum. Balb/c/3T3 clone A31 fibroblasts and NIH/3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium and 10% fetal calf serum. Quiescent cell cultures were established by changing the medium to 0.1% (A31 and NIH/3T3 cells) or 0% (CHO cells) fetal calf serum overnight.
Treatment with Wortmannin-Wortmannin was diluted immediately prior to use in Me 2 SO from a 10 mM stock kept in Me 2 SO at Ϫ70°C. Wortmannin or the Me 2 SO carrier control was added to the cells for 15-20 min before the addition of growth factors.
Immunoprecipitation-Dishes of cells were washed two times with ice-cold phosphate-buffered saline and placed on ice. Cells were immediately lysed in 1 ml of radioimmune precipitation assay buffer (50 mM Tris (pH 7.2), 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 0.5 mM EDTA, and 10% glycerol) collected in an Eppendorf tube, and rocked for 10 min at 4°C. The cell lysate was then spun at 12,000 ϫ g for 5 min at 4°C, and the supernatant was assayed for protein concentration and transferred to another tube. The immunoprecipitating antibody and 30 l of a protein A-Sepharose bead slurry (50%) were added; the suspension was then rocked for 2-3 h. Beads were collected and washed three times with radioimmune precipitation assay buffer and twice with TNE buffer (10 mM Tris (pH 7.2), 100 mM NaCl, 1 mM EDTA) or protein kinase buffer (20 mM Tris (pH 7.0) and 5 mM MgCl 2 ). Radioimmune precipitation assay buffer also contained 1 mM dithiothreitol, 1 mM ZnCl 2 , 1 mM sodium vanadate, 25 mM NaF, 10 mM sodium pyrophosphate, 1 g/ml leupeptin, 1 g/ml aprotinin, and 200 M 4-(2aminoethyl)benzenesulfonyl fluoride added fresh. TNE buffer and protein kinase buffer contained the same concentrations of dithiothreitol, vanadate, NaF, and leupeptin.
Immunoblotting-Immunoblots were developed either by ECL (Renaissance, NEN Life Science Products) followed by exposure to film or by Vistra ECF Western blotting reagent (Amersham Corp.) followed by quantitation on a Molecular Dynamics Storm imaging system.
Immune Complex Protein Kinase Assays-Immunoprecipitated and washed proteins were assayed on the beads. For MAP kinase assays, 40 l of protein kinase buffer with 20 g/ml myelin basic protein were added to the beads, followed by 5 l of a 200 M ATP solution containing 10 Ci of [␥-32 P]ATP. The reaction was incubated at room temperature for 5 min and stopped by adding 12.5 l of 5 ϫ SDS-polyacrylamide gel loading buffer. The mixture was then separated by SDS-polyacrylamide gel electrophoresis, and the wet gel (after fixing) was quantitated on a Bio-Rad Molecular Imager. Results obtained with the Bio-Rad Molecular Imager were linear with results obtained by cutting out radioactive bands and quantitating in a scintillation counter. For Raf kinase assays, 40 l of protein kinase buffer containing 100 mM NaCl, 1 g of glutathione S-transferase-K97A MEK (Upstate Biotechnology, Inc.; eluted from beads with 20 mM glutathione), 15 M ATP, and 10 Ci of [␥-32 P]ATP were added to the washed beads. Reactions were incubated at 30°C for 30 min with intermittent agitation and stopped by the addition of 12.5 l of 5 ϫ SDS-polyacrylamide gel loading buffer. Samples were separated on 8% SDS-polyacrylamide gels, transferred to nitrocellulose, and exposed on a Bio-Rad Molecular Imager.
Lipid Kinase Assays-Lipid kinase assays were performed essentially as described (32). Briefly, 20 l of 0.5 mg/ml sonicated crude bovine brain phosphoinositides (sonicated in 20 mM HEPES (pH 7.0) and 0.1 mM EGTA) were added to immunoprecipitated enzyme (ϳ20-l volume), followed by 10 l of 5 ϫ ATP/MgCl 2 mixture (250 M ATP and 25 mM MgCl 2 in 20 mM HEPES (pH 7.0)) containing 0.5-20 Ci of [␥-32 P]ATP/reaction. Reactions were left at room temperature for 5 min before stopping with 60 l of 2 M HCl. Lipids were extracted by the addition of 160 l of chloroform/methanol (1:1), and the organic phase was collected and analyzed by TLC. The TLC solvent was 65% 1propanol and 35% acetic acid (1 M). The radioactivity on the TLC plate was imaged and quantified using the Bio-Rad Molecular Imager.

Inhibition of MAP Kinase by Wortmannin Varies by Cell Line and Stimulus-
The effect of wortmannin on PDGF-dependent activation of MAP kinase was examined in three cell lines (Fig.  1). In CHO/HIR cells, wortmannin (100 nM) almost completely blocked PDGF-dependent activation of Erk2. In A31 cells, ϳ70% inhibition of MAP kinase was observed. Inhibition of PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 in vivo production correlated with inhibition of MAP kinase activity in A31 cells (Fig. 2). In contrast, wortmannin had little effect on PDGF-dependent activation of Erk proteins in Swiss 3T3 cells (Fig. 1). A trivial explanation for this could be that wortmannin is more effective The cells were then lysed, and Erk2 was immunoprecipitated; an immune complex kinase assay was done using myelin basic protein as substrate. The kinase reaction was stopped after 30 min, and proteins were separated by SDS-polyacrylamide gel electrophoresis; the 32 P incorporated in myelin basic protein was quantitated using a Bio-Rad Molecular Imager. MAPK, MAP kinase. in inhibiting PI 3-kinase in CHO and A31 cells than in Swiss 3T3 cells. However, we find that 100 nM wortmannin is an effective inhibitor of PI 3-kinase activity in all three cell lines as judged by inhibition of PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 production in intact cells (A31 and CHO/HIR cells) or immune complex kinase assays using 4G10 anti-phosphotyrosine antibody (all cell lines) (data not shown). Consistent with this result, insulin-stimulated MAP kinase activation in Swiss 3T3 cells is almost completely inhibited by wortmannin. In contrast, in CHO/HIR cells, where the insulin receptor is overexpressed, wortmannin is less effective in blocking insulin-dependent activation of MAP kinase (Fig. 1). These results suggest that PI 3-kinase is necessary for PDGF-dependent activation of the MAP kinase pathway in CHO and A31 cells, but that in Swiss 3T3 cells, other pathways are utilized for its activation.
These surprising results might be explained by the different levels of expression of receptors for PDGF in the three cell types. CHO cells express low levels of PDGF receptors (Ͻ20,000/cell) (33), and wortmannin is an effective inhibitor of the PDGF response in these cells. Swiss 3T3 cells express high levels of PDGF receptors (400,000/cell) (34), and the PDGF response is resistant to wortmannin in these cells. A31 cells have intermediate levels of PDGF receptors (150,000/cell) (35), and MAP kinase activation is partially inhibited by wortmannin. Likewise, the relative resistance of CHO/HIR cells to wortmannin inhibition of insulin-stimulated MAP kinase activity could be explained by the high level of insulin receptor expression in these cells.
Wortmannin Inhibits MAP Kinase Activation in Swiss 3T3 Cells at Low but Not High PDGF Concentrations-An alternative explanation of the results in Fig. 1 is that the cell lines differ in their composition of PDGF ␣and ␤-receptors, and this could account for differences in wortmannin sensitivity. On the basis of activation with PDGF-AA versus PDGF-BB and immunoprecipitation of Tyr-phosphorylated PDGF receptors with isoform-specific antibodies, CHO cells contain mainly PDGF ␤-receptors, and Swiss 3T3 cells contain mainly PDGF ␣-receptors (data not shown). To test the idea that wortmannin sensitivity of MAP kinase activation depends on the number and not the type of receptors that become stimulated, we reexamined MAP kinase activation in Swiss 3T3 cells at suboptimal concentrations of PDGF. This approach varies the number of receptors stimulated, but not the type. As shown in Fig. 3, when low concentrations of PDGF are used to stimulate the cells, MAP kinase activation is sensitive to wortmannin inhibition. These results suggest that to efficiently activate MAP kinase at low concentrations of growth factor or in cells that have few receptors, PI 3-kinase must be activated. However, PI 3-kinase becomes less important for this pathway when a large number Fig. 1. B and C show the effect of wortmannin on the lipid products of PI 3-kinase. Quiescent A31 cells were labeled with inorganic [ 32 P]phosphate for 3 h and stimulated or not with 10 ng/ml PDGF for 5 min. The cells were lysed in methanol and 1 M HCl; lipids were recovered by extraction with chloroform and deacylated; and the resulting glycerol phosphoinositides were separated by strong anion-exchange high pressure liquid chromatography. The results are presented as a percentage of dpm in PtdIns-4-P plus PtdIns-4,5-P 2 (e.g. PtdIns-3,4,5-P 3 ϫ 100/(dpm PtdIns-4-P ϩ PtdIns-4,5-P 2 )). The average number of counts in PtdIns-4-P and PtdIns-4,5-P 2 was 1.0 ϫ 10 6 and 2.4 ϫ 10 6 cpm/10 6 cells, respectively, and these values did not change significantly with respect to growth factor or wortmannin treatment. The results are from a single experiment representative of three. of receptors become activated, presumably because a second pathway that requires more receptors provides a redundant signal.

FIG. 2. Inhibition of MAP kinase by wortmannin correlates well with its inhibition of the in vivo synthesis of PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 . A shows the effect of wortmannin (Wort) on MAP kinase (MAPK) activation in A31 cells as shown in
PI 3-Kinase Is Activated in Swiss 3T3 Cells at Low PDGF Concentrations-To test this model, we examined whether PI 3-kinase was activated in Swiss 3T3 cells at the low PDGF concentrations at which wortmannin inhibition of MAP kinase is observed. As shown in Fig. 4, recruitment of PI 3-kinase to an anti-Tyr(P)-precipitable complex occurs at very low concentrations of PDGF (half-maximal at 2-3 ng/ml), consistent with a role for PI 3-kinase in the activation of MAP kinase at low PDGF concentrations. In contrast, anti-Tyr(P) precipitation of PLC␥1 saturates at higher concentrations of PDGF (half-maximal at 10 ng/ml). It is possible that PLC␥1 is the redundant signal, parallel to PI 3-kinase, leading to wortmannin-resistant MAP kinase activation at higher PDGF concentrations. This idea is consistent with reports that some PKC family members, which are downstream of PLC␥, can feed into the MAP kinase pathway at Raf (25)(26)(27).

Inhibition of PKC and PI 3-Kinase Together, but Neither Alone, Leads to Inhibition of MAP Kinase in Swiss 3T3 Cells-
The possibility that a PKC family member contributes to PDGFdependent activation of MAP kinase was investigated. Prolonged treatment of cells with the phorbol ester 12-Otetradecanoylphorbol-13-acetate results in degradation of PKC family members. Under the conditions used here, PKC␣ is down-regulated Ͼ95%, and PKC⑀ is down-regulated 80%. PKC down-regulation alone had little effect on PDGF-dependent activation of MAP kinase in Swiss 3T3 cells (Fig. 5). However, the MAP kinase activity in the 12-O-tetradecanoylphorbol-13acetate-treated cells was inhibited by wortmannin. These results are consistent with two redundant pathways for activation of MAP kinase in Swiss 3T3 cells at high PDGF concentrations: a PI 3-kinase-dependent pathway and a PKCdependent pathway that is likely downstream of PLC␥.
Wortmannin Blocks Activation of Raf Kinase-To better define the contribution of PI 3-kinase to the activation of the MAP kinase pathway, the effect of wortmannin on Raf activation was examined in CHO cells. Raf kinase activity peaked within 2 min of PDGF addition (Fig. 6), and wortmannin almost completely inhibited the activation. DISCUSSION The MAP kinase pathway plays a central role in the transduction of signals for growth, differentiation, and other cellular responses. This modular cascade of kinases is a link between receptor signaling events and nuclear events. One of the important links in this pathway to be established was the connection between receptor signaling and the MAP kinase cascade. This link appeared to be found when studies showed that receptor activation of Ras results in Ras-GTP binding to Raf, recruiting it to the membrane (36 -39). While subsequent studies have shown that recruitment of Raf to the membrane is sufficient for its activation (40), other studies have shown that once recruited to the membrane, additional steps are required for full activation of Raf (4). Thus, Ras probably contributes to the activation of Raf by recruiting it to the membrane and inducing a conformational change that facilitates activation by other factors. One of the most pressing challenges in signal transduction is to identify the other factors that contribute to the activation of Raf and the MAP kinase cascade.
Many studies have used wortmannin or dominant-negative forms of PI 3-kinase to address the involvement of this enzyme in the activation of the MAP kinase pathway. While several studies have found that wortmannin (10 -15) or dominantnegative PI 3-kinase (9) effectively inhibits activation of the MAP kinase cascade, many other studies have found that inhibition of PI 3-kinase has no effect on the activation of Erk proteins (17)(18)(19). The results in Fig. 1 address this apparent contradiction. We show that wortmannin sensitivity is clearly dependent on the system under study. The agreement between the results with wortmannin and those with dominant-negative PI 3-kinase have firmly established that PI 3-kinase is required for MAP kinase activation by growth factors in certain FIG. 4. PI 3-kinase is activated at low PDGF concentrations, before PLC␥. As described in the legend to Fig. 3, Swiss 3T3 cells were stimulated with various concentrations of PDGF for 5 min before being lysed. Anti-phosphotyrosine immunoprecipitations were performed; resolved by SDS-polyacrylamide electrophoresis; and blotted with antibodies against p85, the regulatory subunit of PI 3-kinase, and PLC␥1 (PLCg1). The blots were quantitated using a Molecular Dynamics Storm imaging system. cells. However, as we show here, in some cells, inhibition of PI 3-kinase does not affect activation of MAP kinase.
The results that we present are consistent with a model in which PI 3-kinase is required for activation of the MAP kinase cascade under conditions in which relatively few receptors are activated, but is redundant with a parallel pathway when many receptors become activated. This model is supported by comparison of cells with different numbers of PDGF and insulin receptors (Fig. 1) and is directly addressed by varying the number of activated receptors in a single cell type (Fig. 3).
Wortmannin blocks acute (2 min) PDGF-dependent activation of Raf in CHO cells, indicating that, in these cells, PI 3-kinase is required for an early step in the cascade. This result is in agreement with a previous study showing that wortmannin blocks Raf kinase activation by nerve growth factor (10), but not Ras activation by nerve growth factor (41), insulin-like growth factor-1 (10), or insulin (42).
The redundant signal for MAP kinase activation that emerges when a large number of receptors are activated apparently involves a PKC family member. Down-regulation of PKCs did not block PDGF-dependent activation of MAP kinase, unless PI 3-kinase was also inhibited. Thus, PI 3-kinase contributes to Raf kinase and MAP kinase activation by a pathway that does not require a down-regulatable PKC family member. At high levels of stimulated PDGF receptors, a second pathway involving PKC family members circumvents the need for PI 3-kinase. This second pathway is probably mediated by PDGFdependent activation of PLC␥, resulting in elevated Ca 2ϩ and diacylglycerol levels. Phorbol esters are known to activate Raf and MAP kinases in intact cells, and various PKC family members have been shown to activate Raf kinase in vitro (25)(26)(27). Although the result in Fig. 5 indicates that PI 3-kinase-dependent activation of Raf kinase does not involve a down-regulatable PKC, they do not rule out the possibility that a nonconventional PKC or an atypical PKC that is not efficiently downregulated is involved. Both PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 activate nonconventional PKCs in vitro (20), and PI 3-kinase has been shown to be required for PDGF-dependent recruitment of PKC⑀ (23) and PKC (24) to the membrane. PKC⑀ is partially down-regulated under the conditions of Fig. 5 (80%), but could still be contributing to the wortmannin-sensitive pathway. Alternatively, other known or unknown targets of PI 3-kinase could feed into the MAP kinase pathway. Thus, an attractive model is that stimulation of PI 3-kinase provides an efficient pathway for Raf kinase activation at low PDGF levels via stimulation of one of its effectors, whereas activation of PLC␥ at higher PDGF concentration provides a redundant signal via activation of a down-regulatable PKC family member.
The apparent redundancy between PI 3-kinase-and PLC␥dependent pathways for Raf kinase activation is further complicated by evidence that a product of PI 3-kinase is required for maximal activation of PLC␥. Wortmannin has been shown to partially block inositol-3,4,5-P 3 production in several cell types when the pathway stimulated involves PLC␥ (43,44). While these results could be explained by wortmannin inhibition of targets other than PI 3-kinase, Rhee and co-workers 2 has recently found that PtdIns-3,4,5-P 3 activates PLC␥ in vitro by binding to the SH2 domains of this enzyme. Thus, inhibiting PI 3-kinase could prevent direct activation of PKCs by D3 phosphoinositides and also inhibit activation of conventional PKCs by reducing the magnitude of PLC␥ activation. This latter effect is unlikely to explain the wortmannin inhibition of insulin-stimulated MAP kinase since insulin does not activate PLC␥.
Finally, although a requirement for PI 3-kinase in MAP kinase activation is now firmly established in several systems, our results do not imply that activation of PI 3-kinase alone is sufficient to fully activate Raf or MAP kinase. In fact, we do not find significant elevation of MAP kinase activity in chick embryo fibroblasts stably transformed with viral or cellular forms of p110␣ PI 3-kinase, 3 even though these cells have significantly elevated levels of PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 (3) and increased Akt/PKB protein kinase activities (3). This result is in contrast to the recent report that overexpression of p110␥ PI 3-kinase stimulates MAP kinase in COS-7 cells (9). There are several problems with interpreting these types of experiments. It is possible that acute activation of PI 3-kinase is sufficient to activate MAP kinase, but that continual elevation of D3 lipids results in a feedback pathway that inhibits MAP kinase. Conversely, it is possible that the lipid products of PI 3-kinase alone are not sufficient to activate MAP kinase, but that certain cells transformed with PI 3-kinase secrete factors that stimulate MAP kinase in an autocrine manner. Further work will be necessary to address the sufficiency of PI 3-kinase for MAP kinase activation.