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Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
1Correspondence. Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. E-mail: john.exton{at}vanderbilt.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: platelet-derived growth factor • phosphoinositide 3-kinase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. PLD produces phosphatidic acid (PA) and choline upon extracellular stimulation by agents such as lysophosphatidic acid (LPA), platelet-derived growth factor (PDGF), epidermal growth factor, and phorbol ester. Protein kinase C (PKC) and small G-proteins of the ARF and Rho family have been identified as signaling molecules that mediate agonist stimulation of PLD (1)
. Although PLD activation has been studied extensively, the physiological roles of the enzyme are not fully understood. Involvement of PLD in neoplastic transformation has been reported (2)
, but the detailed mechanics of how PLD is involved in cell proliferation signaling remain unclear.
The target of rapamycin (TOR) is known to play a central role in regulating cell growth and proliferation, and TOR activity is linked functionally to various cellular phenomena, including the G0/G1-S transition in the cell cycle, translation, transcription, and actin reorganization. The physiological roles of TOR were initially identified in yeast; the mammalian counterpart (mTOR, also known as FRAP) plays a similar role, with some exceptions. A well-known common function of mTOR and yeast TOR is the regulation of translational initiation. Upon receiving nutritional signals, mTOR increases the phosphorylation of translation regulators p70 S6 kinase 1 (p70S6K) and eIF4E binding protein 1 (4E-BP1, also called PHAS-I), and this results in initiation of protein translation (3
, 4)
. Phosphorylation of these translation regulators is also induced by phosphoinositide 3-kinase (PI3K) -mediated signaling upon the stimulation of cells by mitogens such as growth factors (5)
. Although several reports show that insulin and mitogens in serum can induce phosphorylation of p70S6K and 4E-BP1 by a mTOR-dependent mechanism(s) (6
, 7)
, the pathway(s) upstream of mTOR is now well understood. A recent report has suggested a novel mechanism for regulating mTOR activity by showing that serum can activate mTOR in a PA-dependent manner (8)
. The workers suggested an involvement of PLD in signaling from serum to mTOR by showing the inhibition of p70S6K1 and 4E-BP1 phosphorylation by 1-butanol, which blocks PA production by PLD.
In the present study, we examined mTOR signaling in wild-type Rat-2 fibroblasts and Rat-2 clones in which PLD activity is reduced (9)
in order to correlate PLD function with translational regulation. We have further explored the role of PLD by examining the effects of 1-butanol. We have found differential effects of LPA and PDGF on PLD/mTOR signaling and have obtained evidence that LPA can activate PI3K by a PLD-dependent pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
Rat-2 embryonic fibroblasts were purchased from the American Type Culture Collection (Gaithersburg, MD, USA) and cultured in DMEM containing 10% FBS at 37°C in a humidified atmosphere of air-CO2 (19:1). Rat-2 clones with reduced PLD activity; Rat2V25 and Rat2V29 were maintained in DMEM containing 0.5 mg/mL G418. For serum starvation, cells were incubated in DMEM for 24 h without G418 and incubated for another hour after replacement with fresh DMEM.
Cell growth measurement
Rat-2 and Rat2V25 cells were plated on 6-well plates at a density of 2 x 104 cells/mL. Cells were detached by trypsin-EDTA and counted using a hemocytometer 12 h after the plating and every 24 h thereafter. The average cell number at each time point was obtained from five different countings.
Western blot
Serum-starved or nonstarved Rat-2 cells and their clones were incubated for 1 h in DMEM before stimulation with FBS, LPA, or PDGF. Cells were washed with ice-cold phosphate-buffered saline (PBS) twice, then lysed in SDS sample buffer without 1,4-dithiothreitol. Harvested cell lysates were boiled and the protein concentration was measured using the bicinchoninic acid method. Up to 10 µg of cell lysate was loaded on the SDS-polyacrylamide gels and analyzed by Western blot. A 14% Tris-glycine gel or a 4–20% gradient gel was used for measuring the band shift of 4E-BP1 protein; an 8% gel was used for the band shift of p70S6K1. mTOR phosphorylation was measured by using 6% gels; all other blots were obtained by 4–20% gradient gels.
Measurement of phospholipase D activity
Rat-2 cells or COS-7 cells transiently transfected with pcDNA4C (vector) or Xpress-tagged rPLD1 or rPLD2 cDNA (10)
were starved in DMEM for 24 h in the presence of 1 µCi/mL of [9,10-3H]myristic acid. Cells were washed with DMEM three times and incubated in fresh DMEM for 1 h in the presence or absence of toxin B (11)
. After a pretreatment of 0.3% 1-butanol for 5 min, cells were stimulated with LPA, PDGF, or PMA and the formation of [3H]phosphatidyl butanol was measured as described previously (9)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We examined whether the clones with reduced PLD activity (Rat2V16, Rat2V25, and Rat2V29) had altered growth rates compared with wild-type Rat-2 cells. Figure 1
shows the growth rates of wild-type Rat-2 cells and a representative clone with reduced PLD (Rat2V25). The growth rate of Rat2V25 cells in the presence of 5% FBS was much reduced and closer to the rate of wild-type cells under low-serum (0.5% FBS) conditions. Rat2V25 cells grew even more slowly during culture in the low-serum environment. Similar results were found with the other clones (not shown).2
These results indicate a role for PLD activity in the regulation of cell growth.
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Phosphorylation of p70S6K and 4E-BP1 is reduced in Rat-2 mutant clones
In view of a recent report suggesting that phosphatidic acid (PA) produced by PLD plays an essential role in serum-stimulated translational activation through effects on the mTOR pathway (8)
, we monitored the phosphorylation of two downstream effectors of mTOR (p70S6K and 4E-BP1) in response to stimulation by low (0.5%) and high (5%) concentrations of serum in the PLD-deficient clones. As shown in Fig. 2
A, phosphorylation of p70S6K on Thr389 and of 4E-BP1 on Thr37 was reduced in Rat2V25 and Rat2V29 cells compared with wild-type Rat-2 cells.3
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Since PLD produces phosphatidyl butanol instead of PA in the presence of 1-butanol, this alcohol can be used as an inhibitor of PA production by PLD. Figure 2B
shows the protein band shift of 4E-BP1 due to phosphorylation by FBS. This shift was inhibited by pretreatment with 1-butanol but not by 2-butanol (0.5%), supporting the hypothesis that serum-induced phosphorylation of 4E-BP1 requires PLD activity in Rat-2 fibroblasts. With increasing concentrations of 1-butanol (0.75 and 1.0%), there was a further reduction in phosphorylation, as shown by the appearance of lower bands (Fig. 2B
).
Differential effects of LPA and PDGF on mTOR signaling
Serum stimulation is the mixed effect of various mitogens. To determine whether mitogens that acted through G-protein-coupled receptors and those that interacted with receptors with tyrosine kinase activity induced phosphorylation of p70S6K and 4E-BP1 in a PLD-dependent manner, we compared the effects of LPA and PDGF. As shown in Fig. 3
A, LPA increased the phosphorylation of 4E-BP1, as shown by the band shift, and phosphorylation of this protein on Thr37. PDGF also induced phosphorylation of 4E-BP1 (Fig. 3A
). However, the phosphorylation induced by LPA was inhibited by 1-butanol whereas that caused by PDGF was not. We obtained similar results when we examined phosphorylation of p70S6K by LPA and PDGF (Fig. 3B
). However, the inhibitory effect of 1-butanol on the band shift of p70S6K and on the phosphorylation of Thr 389 and Thr 421/Ser424 of p70S6K induced by LPA was more striking. PDGF-induced phosphorylation of p70S6K was not reproducibly inhibited by 1-butanol. The figure shows that 2-butanol also inhibited the LPA effect, though to a lesser extent than 1-butanol. In other experiments in which tertiary butanol was used as a control, the inhibition was much less (data not shown). Rapamycin blocked the effects of both LPA and PDGF, implying the involvement of mTOR (data not shown).
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We examined the phosphorylation of 4E-BP1 and p70S6K by LPA and PDGF in Rat-2 cells and Rat-2 mutant clones. The phosphorylation of p70S6K at Thr389 was strongly increased by 0.1 and 10 µM LPA in the wild-type cells (Fig. 4
A). However, the phosphorylation increase was much less in the mutant cells. Phosphorylation of 4E-BP1 at Thr37 in response to LPA was decreased in the mutant cells relative to wild-type. In contrast to the situation with LPA, PDGF-induced phosphorylation of p70S6K and 4E-BP1 was not reproducibly altered in the mutant clones (Fig. 4B
). These results supported the results of Fig. 3
by showing that the LPA-induced phosphorylation of p70S6K and 4E-BP1 was PLD dependent whereas the effect of PDGF was not. These results agree with a report showing that LPA-induced phosphorylation of p70S6K in NIH 3T3 cells is susceptible to 1-butanol inhibition (12)
.
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Phosphorylation of mTOR is independent of PLD
p70S6K and 4E-BP1 are phosphorylated via pathways involving either mTOR or P13K signaling (4)
. Since some reports have suggested that mTOR can be phosphorylated by Akt/PKB, which is activated by P13K (13
, 14)
, we examined the phosphorylation of mTOR at Ser2448 to see whether LPA and PDGF regulated mTOR phosphorylation. LPA induced phosphorylation of mTOR, but PDGF had negligible effects on this phosphorylation, although it is a well-known activator of P13K (Fig. 5
A).4
The phosphorylation of mTOR induced by LPA was not inhibited by 1-butanol pretreatment (Fig. 5A
). Furthermore, there was no difference in mTOR phosphorylation when wild-type Rat-2 cells were compared with Rat-2 mutant cells (Fig. 5B)
. On the other hand, phosphorylation of 4E-BP1 was reduced in the same lysates from the mutant cells (Fig. 5B
), in agreement with Fig. 4A
. These findings agree with another report showing that mTOR phosphorylation is not essential for the downstream phosphorylation of p70S6K and 4E-BP1 (14)
. The data suggest that LPA stimulation induces mTOR phosphorylation separately from the phosphorylation of p70S6K and 4E-BP1, which is dependent on PLD.
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Differential effect of PI3K on mTOR, p70S6K, and 4E-BP1 phosphorylation
Since it has been suggested that phosphorylation of mTOR Ser2448 requires signaling through PI3K/Akt (13
, 14)
, we tested whether pretreatment with LY294002, a PI3K inhibitor, could block mTOR phosphorylation. Figure 6
shows that LY294002 had little or no effect on the phosphorylation of mTOR by LPA, in contrast to findings with insulin as a stimulus (13
, 14)
. Another PI3K inhibitor, wortmannin, was also without effect on mTOR phosphorylation induced by both agonists (data not shown).4
In contrast to the findings with mTOR phosphorylation, LY294002 caused large decreases in the phosphorylation of p70S6K and 4E-BP1 induced by LPA and PDGF (Fig. 6)
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LPA-induced phosphorylation of Akt requires PLD activity
Although the foregoing results showed that PLD could be involved in LPA signaling for the initiation of translation, the involvement of PI3K was indicated by the finding that LY294002 suppressed the phosphorylation of p70S6K and 4E-BP1 induced by LPA (Fig. 6)
. We therefore examined whether PLD played a role in the effect of LPA on PI3K activity. As shown in Fig. 7
A, LPA elevated Akt phosphorylation, although the effect was much smaller than that of PDGF. The increase in Akt phosphorylation induced by LPA was inhibited by pretreatment with 1-butanol, but not t-butanol. In contrast, no inhibitory effect of 1-butanol was observed on PDGF-induced phosphorylation. We also compared LPA-induced Akt phosphorylation in wild-type and mutant Rat-2 cells (Fig. 7B
). The phosphorylation of Akt induced by LPA was clearly reduced in the Rat2V25 and Rat2V29 cells. In contrast, the effect of PDGF on the phosphorylation was not affected (Fig. 7C
). The results of Figs. 6
and 7
show that the phosphorylation of Akt by LPA is PLD dependent but the effect of PDGF is not. The data also suggest that LPA signaling to p70S6K and 4E-BP1 can be transmitted by PI3K.
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Differential effects of toxin B on LPA- and PDGF-induced phosphorylation of p70S6K, 4E-BP1, and Akt
Differential involvement of PLD in the effects of LPA and PDGF signaling to mTOR raised the possibility that the effect of PDGF on PLD activity was absent or minimal relative to LPA in Rat 2 cells. However, as shown in Fig. 8
A, PDGF caused a marked stimulation of PLD. Since PLD can be activated by multiple pathways involving protein kinase C and small G-proteins (1)
, we tested the effects of C. difficile toxin B, which inactivates small G-proteins of the Rho family (11)
. The C3 exoenzyme of C. botulinum was tested, but this decreased cell viability to the extent that the results were not reliable. As shown in Fig. 8A
, toxin B significantly impaired the activation of PLD by LPA but had less effect on the activation induced by PDGF. It also inhibited the phosphorylations of p70S6K, 4E-BP1, and Akt induced by LPA, but had no effect on the actions of PDGF. The toxin produced some reduction in the level of RhoA (Fig. 8B
), but its major action was presumed to be the inactivation of Rho family proteins through glycosylation.
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Differential effects of toxin B on PLD1 and PLD2
To see whether the difference between toxin B effects on LPA- and PDGF-induced PLD activation could be due to the involvement of different PLD isozymes, we expressed PLD1 and PLD2 in COS-7 cells and tested the effects of the toxin. Figure 9
shows that expression of both PLD isozymes enhanced PLD activity. However, PLD2 induced higher PLD activity than PLD1 because of its greater expression (data not shown). Toxin B inhibited PLD activity under LPA- and PMA-stimulated conditions in the PLD1-expressing cells, whereas a small enhancement was observed with the PLD2 cells. Smaller effects of the toxin were observed on PLD activity of the untransfected cells. These data suggest that Rho proteins are involved in the activation of PLD1 but not PLD2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, the mechanisms by which PLD is involved in growth and differentiation signaling are not clear. The reduction in growth rate caused by decreased PLD activity (Fig. 1)
supports the requirement of PLD activity for proliferation signal(s), but we could not detect any significant change in the progression from the S to G2/M phase when we monitored cell cycle progress of wild-type and mutant Rat-2 cells after release from arrest at early S phase (data not shown). The idea that PLD may be involved in processes involved in the G1/S transition is suggested by previous (8)
and present evidence that PLD is involved in mTOR signaling, since down-regulation of mTOR and 4E-BP1 signaling causes G1 arrest (15
, 16)
.
LPA and PDGF are well-known activators of PLD in Rat-2 fibroblasts but showed differential effects on mTOR signaling. As shown in Figs. 3
and 4
, LPA-induced phosphorylation of 4E-BP1 is PLD dependent whereas the effect of PDGF is not. A similar difference in the phosphorylation of p70S6K was observed. It is interesting to consider why LPA and PDGF show differential PLD dependency. An obvious possibility is that their signaling pathways to p70S6K and 4E-BP1 are different. This is shown by the effects of toxin B, which impaired the phosphorylation of p70S6K, 4E-BP1, and Akt in response to LPA, but not to PDGF (Fig. 8)
. These data suggest that LPA signaling to mTOR involves Rho family proteins whereas that of PDGF does not. Activation of RhoA by LPA has been demonstrated in several cell lines whereas Rac appears to be the major Rho family member targeted by PDGF (17)
. However, the lack of toxin B effects on PDGF-induced phosphorylation of p70S6K or 4E-BP1 indicates that Rac is not a major factor in PDGF signaling to these proteins; as discussed later, PI3K/Akt seems to be the major pathway involved.
The question then arises as to the nature of the LPA pathway. One possibility is that it involves activation of PLD. This would, of course, be consistent with the effects of inhibition of PLD signaling by 1-butanol and of the reduction of PLD1 activity in Rat2V25 and Rat2V29 cells observed in Figs. 2
3
4
. As illustrated in Fig. 10
, PLD has been reported to activate mTOR through the generation of PA (8)
and a binding site for PA on mTOR has been identified. This is located where the rapamycin-FKBP12 complex binds (8)
, and PA may compete with rapamycin for binding to this site. The results with toxin B indicate the involvement of Rho family proteins in LPA activation of PLD (Fig. 8)
and indicate that PLD1, not PLD2, is the PLD isozyme involved. This view is supported by studies demonstrating that PLD1 can be activated by constitutively active Rho in vivo whereas PLD2 is inhibited (10)
.
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The major pathway from PDGF to p70S6K and 4E-BP1 is probably via PI3K (Fig. 10)
. The activation of PI3K by PDGF and other growth factors through their receptor kinase activity is well established (18
, 19)
; the pathway is supported in the present study by the demonstration that the phosphorylation of Akt, p70S6K, and 4E-BP1 by PDGF is greatly inhibited by LY 294002 (Fig. 6)
. However, the mechanisms by which PI3K activation leads to the phosphorylation of p70S6K and 4E-BP1 apparently do not involve direct effects of Akt, but other protein kinases (4
, 20)
. The question of whether mTOR is directly regulated by P13K/Akt signaling in vivo remains open. The tuberous sclerosis complex has been shown to have an inhibitory effect on mTOR that is reversed by phosphorylation by PI3K/Akt (21
22
23)
. It has also been reported that the effect of the tuberous sclerosis complex on p70S6K is independent of mTOR (24)
.
Figure 8
shows that LY294002 inhibited LPA-mediated phosphorylation of p70S6K and 4E-BP1. Interpretation of these results is complicated by the fact that PI3K inhibitors can act not only on PI3K, but on downstream signaling involving mTOR (7
, 25
, 26)
. Nevertheless, as shown in Fig. 7A
, the effect of LPA on the phosphorylation of Akt is much less than that of PDGF, whereas the effects of the two agonists on the phosphorylation of p70S6K and 4E-BP1 are very similar (Fig. 3)
. This suggests that the contribution of the PI3K/Akt pathway to phosphorylation of these proteins by LPA is minor and that the contrary is true for PDGF. Thus, the PLD-dependent pathway seems to be important for LPA and the PI3K-dependent pathway appears to be important for PDGF (Fig. 10)
.
Another issue is the observation that inhibition of PLD activity decreases the phosphorylation of Akt induced by LPA, but not by PDGF (Fig. 7)
. Although both agonists can induce phosphorylation of Akt, the effect of LPA is much weaker than PDGF, as noted above. The pathways to Akt from LPA and PDGF are very different; our findings (Fig. 7)
and the literature (27
, 28)
indicate they are differentially regulated by PLD. The conventional view is that the PDGF receptor recruits the p110 catalytic subunit of Class 1A PI3Ks to the plasma membrane via the p85 adaptor subunit (18
, 19)
. This generates PIP3 from PIP2, which causes translocation of Akt to the membrane where it is phosphorylated and activated by 3'-phosphoinositide-dependent kinase 1 (18
, 19)
. Agonists such as LPA, which act through G-protein-coupled receptors, use a different mechanism to activate Akt. This involves activation of the Gi/o family of G-proteins to generate ß
subunits that activate PI3K by two mechanisms, namely, interaction with the p101 regulatory subunit to activate the p100 catalytic subunit of Class IB PI3K (29)
or interaction with the p110ß catalytic subunit (30)
.
The effects of toxin B on the stimulation of phosphorylation of Akt, p70S6K, and 4E-BP1 by LPA are attributable to the reduction of PLD1 activation induced by the toxin, but other mechanisms are possible. Rho family proteins Cdc42 and Rac1, but not RhoA, have been shown to activate p70S6K (31)
, but if this were the explanation, toxin B would be expected to affect PDGF, which activates Rac, but not LPA, which activates Rho (17
, 32)
. Protein kinase C can be activated by LPA and PDGF and, in turn, can activate PLD1 (1)
. However, this pathway seems to play a minor role in the phosphorylation of p70S6K and 4E-BP1 since phorbol ester has only a weak effect on this phosphorylation (data not shown) (Fig. 10)
.
Our finding that the signaling pathways from LPA and PDGF to Akt are differentially regulated by PLD is supported by recent reports (27
, 28)
that PLD is involved in the activation of PI3K and Akt by sphingosine 1-phosphate (S1P), but not by insulin-like growth factor I. These agents have similar signaling mechanisms to LPA and PDGF, respectively. These findings and those of the present study suggest that the PLD acts at or upstream of PI3K in LPA signaling. The precise site of PLD action is currently under study, but it would appear to involve Class IB PI3Ks and not Class IA PI3Ks because of the differential involvement of these kinases in LPA and PDGF signaling.
The present findings indicate that LPA can phosphorylate mTOR on Ser 2448. Although PDGF does not induce significant phosphorylation of this residue in the serum-starved cells used in the present study (Fig. 5)
, it does so in nonstarved cells in which the basal phosphorylation is reduced (data not shown). The reasons for this difference are unknown and were not explored. The phosphorylation of mTOR Ser 2448 induced by LPA was not influenced by changes in PLD activity (Fig. 5)
and its role in the activation of p70S6K and 4E-BP1 is unclear (20)
. Although there is evidence that PI3K and Akt are involved in the phosphorylation of mTOR in vitro and in cells stimulated by insulin (7
, 13
, 14)
, the failure of LY294002 to alter the phosphorylation of mTOR induced by LPA or PDGF (Fig. 8)
indicates that Akt does not play a role in this phosphorylation in our fibroblast cell line.
In summary, our results using toxin B, 1-butanol, and cell lines deficient in PLD activity have identified Rho and PLD1 as factors in signaling to mTOR by LPA, but not PDGF (Fig. 10)
. The data are consistent with PI3K and mTOR as sites of PA action and illustrate the diverse cellular actions of PLD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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, and they were not studied further. 
3 Blotting with antibodies to p70S6K (not shown) identified the lower band as this kinase. The nature of the upper phosphorylated band was not investigated, but probably is p85S6K.
4 In other experiments in which nonstarved cells were used, the basal phosphorylation of mTOR was reduced and an effect of PDGF was evident. This was not decreased by the PI3K inhibitor LY294002.
Received for publication August 4, 2003. Accepted for publication October 15, 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunits. Biochem. J. 362,725-731[CrossRef][Medline]
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