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294-301. Phosphorylation of Eukaryotic Elongation Factor 2 Can Be. Regulated by Phosphoinositide 3-Kinase in the Early Stages of. Myoblast Differentiation.
Mol. Cells, Vol. 21, No. 2, pp. 294-301

Molecules and Cells

Communication

©KSMCB 2006

Phosphorylation of Eukaryotic Elongation Factor 2 Can Be Regulated by Phosphoinositide 3-Kinase in the Early Stages of Myoblast Differentiation Joo Hong Woo and Hye Sun Kim* Department of Biological Science, College of Natural Sciences, Ajou University, Suwon 443-749, Korea. (Received October 7, 2005; Accepted January 1, 2006)

We have previously reported that phosphorylation of eukaryotic elongation factor 2 (eEF2) is related to the differentiation of chick embryonic muscle cells in culture. In the present study, we found that eEF2 phosphorylation declined shortly after induction of differentiation of L6 myoblasts, when the cells prepare for terminal differentiation by withdrawing from the cell cycle. This decrease in phosphorylation was prevented by inhibitors of phosphoinositide 3-kinase (PI3-kinase) that strongly inhibit myoblast differentiation. We hypothesized that PI3-kinase plays an important role in myoblast differentiation by regulating eEF2 phosphorylation in the early stages of differentiation. To test this hypothesis, myoblasts were synchronized at in G2/M and cultured in fresh differentiation medium (DM) or growth medium (GM). In DM the released cells accumulated in G0/G1 while in GM they progressed to S phase. In addition, cyclin D1 was more rapidly degraded in DM than in GM, and eEF2 phosphorylation decreased more. Inhibitors of PI3-kinase increased eEF2 phosphorylation, but PI3-kinase became more activated when eEF2 phosphorylation declined. These results suggest that the regulation of L6 myoblast differentiation by PI3-kinase is related to eEF2 phosphorylation. Keywords: Cell Cycle; Eukaryotic Elongation Factor 2 (eEF2); LY 294002; Myoblast Differentiation; Phosphoinositide 3-Kinase (PI3-Kinase).

Introduction We have previously shown that Ca2+/calmodulin-dependent phosphorylation of a 100-kDa protein increased during the * To whom correspondence should be addressed. Tel: 82-31-219-2622; Fax: 82-31-219-1615 E-mail: [email protected]

course of differentiation of chick embryonic muscle cells, and specifically that the level of phosphorylation was closely related to the status of myoblast differentiation (Baek et al., 1994; Kim et al., 1992; 1993). This protein, which was shown to display the most rapid and prominent phosphorylation of any protein during the differentiation of myoblasts, was later identified as eukaryotic elongation factor 2 (eEF2) (Jeon et al., 1994). The activity of eEF2, which is critical for the translocation step in peptide chain elongation, is regulated by phosphorylation (Ryazanov and Davydova, 1989). The only kinase so far identified that can phosphorylate eEF2 is eEF2-kinase, also called Ca2+/calmodulin-dependent protein kinase III or CaM kinase III (Ryazanov, 2002). eEF2 must be dephosphorylated to be active, and when it is phosphorylated translation stops (Ryazanov et al., 1988). It is now apparent that eEF2 is not merely a translation factor: it is also involved in cell cycle regulation, cell differentiation, and many other processes (Gutzkow et al., 2003; Nilsson and Nygard, 1995; Patel et al., 2002). During terminal differentiation, cells leave the cell cycle and usually remain in a G0-like state. Differentiation of skeletal muscle cells is characterized by withdrawal from the cell cycle, activation of genes for musclespecific proteins, and fusion of mononucleated myoblasts to form multinucleated myotubes (Guo et al., 1995; Lassar et al., 1994; Olson and Klein, 1994; Zhang et al., 1999). In the presence of low concentrations of mitogens, muscle-specific gene expression is initiated by myogenic regulatory factors such as Myf5, MyoD, MRF4, and myogenin, members of a family of basic helix-loop-helix transcription factors. Simultaneously, myoblasts irreversibly withdraw from the cell cycle and accumulate in G0/G1. Phosphoinositide 3-kinase (PI3-kinase) is among the factors activated by tyrosine kinase signaling. It catalyzes Abbreviations: DM, differentiation medium; eEF2, eukaryotic elongation factor 2; PI3-kinase, phosphoinositide 3-kinase.

Joo Hong Woo & Hye Sun Kim

phosphorylation of phosphoinositides at the D3 position of the inositol ring, resulting in the production of phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol-3,4,5-triphosphate (PI-3,4,5-P3), which are thought to act as second messengers (Rameh and Cantley, 1999). Activation of PI3-kinase is required for mitogenesis (Valius and Kazlauskas, 1993), anti-apoptosis (Yao and Cooper, 1995), neurite outgrowth in PC12 cells (Kimura et al., 1994), and insulin-stimulated glucose transport of myoblasts and adipocytes (Cortright et al., 1997; Okada et al., 1994). It is believed that PI3-kinase activity is indispensable for terminal differentiation of myoblasts (Kaliman et al., 1996; 1998; Pinset et al., 1997). Inhibition of with PI3kinase by LY294002 or wortmannin, or by a dominantnegative mutant of p85α, the regulatory subunit of PI3kinase, blocks myoblast fusion and expression of musclespecific proteins (Coolican et al., 1997; Jiang et al., 1999; Kaliman et al., 1998). Although accumulating data highlight the pivotal role of PI3-kinase in myoblast differentiation, there is as yet little information about its role in the transition from the proliferation to the differentiation phase. In this study we examined eEF2 phosphorylation in L6 rat skeletal myoblasts, particularly in the early differentiation stages. We observed that eEF2 phosphorylation declined shortly after the induction of differentiation, the point at which cells prepare for terminal differentiation by withdrawing from the cell cycle. We also found that PI3kinase inhibitors, which inhibit both myoblast fusion and the synthesis of muscle-specific proteins, actually increase eEF2 phosphorylation. We hypothesized that the activity of eEF2 in the earlier stages of differentiation is related to withdrawal from the cell cycle, and that PI3kinase is also involved in those processes. To test this hypothesis, cells were synchronized in G2/M phase by incubation with nocodazole (Kiess et al., 1995), released from cell cycle arrest and assayed for eEF2 phosphorylation, cyclin D1 expression, and PI3-kinase activity.

Materials and Methods Cell culture L6 rat skeletal myoblasts were obtained from the American Type Culture Collection (ATCC, USA). They were cultured in growth medium (GM) containing 10% fetal bovine serum (Gibco, USA) for 3 d, and differentiation was induced by transfer to differentiation medium (DM) containing 5% horse serum (Gibco). The time of this transfer was designated 0 h. To measure the extent of myoblast fusion in DM, cells were washed with ice-cold phosphate-buffered saline (PBS) and immediately fixed with a mixture of 95% ethanol, 40% formaldehyde, and acetic acid (20:2:1 by volume). The fixed cells were stained with hematoxylin and observed with an Olympus IMT2 microscope (Olympus, Japan). Cells were considered to be fused only

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if there was clear cytoplasmic continuity and at least three nuclei were present within the myotubes (Shin et al., 2000). Western blotting At the indicated times, cells were washed three times with ice-cold PBS and disrupted by ultrasonication in lysis buffer (50 mM Tris pH 7.5, containing 150 mM NaCl, 5 mM MgCl2, 10 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, 1% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, and 0.1 mM Na3VO4). The lysates were centrifuged at 15,000 × g to remove cell debris. Protein concentrations were determined by the Bradford procedure using the Bio-Rad dye reagent, with bovine serum albumin (BSA) as a standard. Equal amounts of lysate total protein were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels (Laemmli et al., 1970). The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) and incubated with the indicated antibodies. Excess primary antibody was removed by multiple washes, followed by incubation with the appropriate secondary antibody conjugated to horseradish peroxidase (Sigma, USA). The immunoreactive protein bands were visualized by enhanced chemiluminescence (ECL) (Amersham, Sweden). Phosphorylation of eEF2 The assay was performed as previously described (Baek et al., 1994). Briefly, myoblasts were harvested at the indicated times and cell lysates were prepared by ultrasonication in sample buffer (50 mM Tris-HCl buffer, pH 7.5, containing 5 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EDTA, and 10% glycerol). The reaction was started by adding 1.5 µCi of [γ-32P]ATP (Amersham) to cell extract (30 µg protein), incubating the mixture for 5 min at room temperature, and terminating the reaction by adding Laemmli sample buffer. The samples was then electrophoresed on 7−14% polyacrylamide gradient gels in the presence of SDS, stained with Coomassie R250, dried under vacuum, and exposed to X-ray film (Agfa, Belgium). Cell cycle analysis In order to synchronize cells in G2/M, myoblasts were cultured in GM for 2 d, washed in PBS and treated with 40 ng/ml nocodazole for 16 h (Zieve et al., 1980). After washing with PBS to remove the nocodazole, fresh GM or DM was added with or without the PI3-kinase inhibitor, LY294002. Analysis of cell cycle progression was performed according to Kiess et al. (1995), with slight modifications. Briefly, cells were trypsinized and washed three times with PBS, fixed with 70% ethanol overnight at 4°C, and washed with PBS. This was followed by treatment with RNase (500 ng/ml) for 30 min at 37°C, after which the cells were precipitated and resuspended in PBS, stained with 100 µg/ml propidium iodide (Sigma) for 5 min at room temperature and kept on ice for analysis. They were analyzed on a FACScan (BecktonDickinson, USA), and the percentages of cells in G0/G1, S, and G2/M were determined by the SOBR method, as per CellFIT software. The numbers of cells in G0/G1 (DNA content = 2N), S (2N < DNA content < 4N), and G2/M (DNA content = 4N)

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phases are expressed as percentages of total events (10,000 cells), except for sub-G1 cells (DNA content < 2N).

A

PI3-kinase assay PI3-kinase activity was determined by the incorporation of radioactive phosphate from [γ-32P]ATP into exogenous phosphatidylinositol (PI), as described by Woo et al. (2006). Briefly, p85 was immunoprecipitated from extracts with anti-p85 antibody (Upstate Biotechnology Inc., USA). The precipitated enzyme was incubated with 2.5 µg of PI and 5 µCi of [γ-32P]ATP for 10 min at room temperature. The reaction was stopped by the addition of 20 µl 6 N HCl, and lipids were then extracted with a mixture of CHCl3:CH3OH (1:1 by volume). After centrifugation, the lipid extract (50 µl) in the lower phase was applied to an oxalate-pretreated thin layer chromatography plate pre-baked for 1 h at 100°C. The phospholipids were separated by chromatography in CHCl3:CH3OH:H2O:NH4OH (120: 94:23.2:4 by volume) and analyzed using a phosphorimage analyzer (Bio-Rad, USA). The radiolabeled products were identified according to Giorgino et al. (1997).

Results and Discussion PI3-kinase is involved in withdrawal from the cell cycle during myoblast differentiation Growth factors are generally considered to inhibit the terminal differentiation of skeletal muscle cells (De Angelis et al., 1998; Florini et al., 1996; Foulstone et al., 2001). In the present study, L6 myoblasts were cultured in mitogen-rich medium (i.e. GM), which allows continued cell division without cell fusion. About 24−36 h after transfer to mitogen-poor medium (i.e. DM), myoblasts elongate, align with each other and initiate the formation of multinucleated myotubes by spontaneous cell fusion. Maximum fusion is reached after 72−96 h, depending on culture conditions such as medium composition, initial cell density, and cell passage (unpublished data). To examine the role of PI3-kinase in the differentiation of L6 myoblasts in these culture conditions, LY294002 and wortmannin, both inhibitors of PI3-kinase, were added to the culture medium at the time of induction of differentiation. After an additional 72 h of culture, cells were fixed and stained for microscopy. As shown in Fig. 1A, these inhibitors strongly reduced myoblasts fusion; that is, the cells failed to form myotubes. The alignment of the cells did not seem to be impaired, but spontaneous cell fusion did not occur. Inhibition of cell fusion was dose-dependent, with half-maximal inhibition at 0.5 µM of LY294002 and 0.01 µM wortmannin (data not shown). Cell fusion was almost totally blocked at 5 µM of LY294002 and 0.5 µM of wortmannin (Fig. 1A), with fusion indices below 10%, but the cells were still healthy and able to resume fusion upon removal of the agents (data not shown). Differentiation of myoblasts is defined both morphologically, as cell fusion, and biochemically,

B

Fig. 1. Effect of PI3-kinase inhibitors on L6 myoblast differentiation. A. Confluent myoblasts (MB) cultured in GM are induced to differentiate by transfer to DM. At the time of medium change, 5 μM LY294002 (LY) or 0.5 μM wortmannin (W) were added. After 72 h in DM, the cells were fixed, stained with hematoxylin, and observed under a microscope. The degree of fusion is shown in the lower graph. Cells were considered to be fused only if there was clear cytoplasmic continuity and at least three nuclei were present within the myotubes. Bar indicates 50 µm. B. Cells cultured with or without 5 µM LY294002 were harvested after 24 or 96 h in DM. Equal amounts of cell lysate were separated by SDS-PAGE and transferred to PVDF membranes, and the proteins on the membranes were probed with the indicated antibodies. β-Actin was used as a loading control. Results are representative of three independent experiments.

as synthesis and accumulation of muscle-specific proteins. We assessed the effects of LY294002 biochemically by measuring the accumulation of myogenin, an early differentiation marker, and creatine kinase, a late marker. As

Joo Hong Woo & Hye Sun Kim

shown in Fig. 1B, expression of these differentiation markers was completely blocked by LY294002. This demonstrates that the PI3-kinase pathway is a key component of signaling during the differentiation of L6 skeletal muscle cells. PI3-kinase is involved in the control of both cell proliferation and differentiation; addition of LY294002 induced cell cycle arrest in G1 in melanoma, osteosarcoma, and human prostate cancer cells (Casagrande et al., 1998; Gao et al., 2003; Thomas et al., 1997), and PI3-kinase was shown to regulate the cell cycle via Akt, mTOR, and p70 S6 kinase (Gao et al., 2003). In general, cell cycle progression and terminal differentiation are mutually exclusive, and cessation of DNA synthesis and withdrawal from the cell cycle precede the terminal differentiation of myoblasts (Nadal-Ginard, 1978). Cell cycle transitions are controlled by cyclin-dependent kinases (CDKs), which contain both regulatory (cyclin) and catalytic (CDK) subunits. p21 negatively regulates CDK activities and mediates cell cycle arrest by interfering with CDK/cyclin complexes (Waga et al., 1994), and the induction and sustained expression of p21 plays an important role in irreversible withdrawal from the cell cycle and in phenotypic differentiation during myogenesis (Guo et al., 1995; Halevy et al., 1995; Parker et al., 1995). We therefore assayed the expression of p21 as a means of examining the role of PI3kinase in withdrawal from the cell cycle. As shown in Fig. 1B, p21 was expressed in control myoblasts, but LY294002 completely impaired its expression after 24 h of culture. This result suggests that the inhibition of myoblast differentiation by LY294002 is related to the failure to withdraw form the cell cycle. PI3-kinase regulates eEF2 phosphorylation in the early stages of L6 myoblast differentiation Phosphorylation of eEF2 not only acts on protein translation but is involved in various other processes, including cell differentiation and the cell cycle (Gutzkow et al., 2003; Kim et al., 1992; Nilsson and Nygard, 1995). The connection between eEF2 phosphorylation (or the activity of eEF2kinase) and cell cycle progression was first reported in a study of mitogenic stimulation of quiescent human fibroblasts (Palfrey et al., 1987). Treatment of those cells with various mitogens resulted in transient phosphorylation of eEF2. Ryazanov and Spirin later demonstrated that phosphorylation of eEF2 is needed for exit from mitosis to G1. If this does not occur, the cell passes into G0 or the quiescent state (Ryazanov and Spirin, 1993). We have previously shown that eEF2 phosphorylation plays an important role in myoblast differentiation (Baek et al., 1994; Kim et al., 1992; 1993). In that work, we focused on the relationship of eEF2 phosphorylation to myoblast fusion, so that assays were carried out at later stages of differentiation and at 24 h intervals. Here, we examined the role of eEF2 phosphorylation earlier in

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A

B

Fig. 2. Phosphorylation of eEF2 during myoblast differentiation. A. Cells cultured in DM with or without 5 µM LY294002 were harvested at the indicated times, and phosphorylation of eEF2 was assayed by the incorporation of [γ-32P]ATP as described in Materials and Methods. The same aliquots were subjected to Western blot analysis with anti-eEF2 and anti-p85 antibodies. B. The effect of wortmannin on eEF2 phosphorylation was assayed in lysates of the cells cultured for 3 h in DM. Results are representative of three independent experiments.

differentiation. To assess whether eEF2 phosphorylation is related to withdrawal from the cell cycle, cells cultured in DM were harvested and eEF2 phosphorylation was assayed. As shown in Fig. 2A, the level of phosphorylation changed during the course of L6 myoblast differentiation. Although there was some phosphorylation throughout the period of culture, the level fluctuated repeatedly (data not shown). In particular, we consistently observed a decrease in phosphorylation some time prior to 24 h, before the cells started to fuse, although the exact time of this decrease varied slightly according to the culture conditions. We therefore hypothesized that a decrease in eEF2 phosphorylation during the early stages of myoblast differentiation might be related to cell cycle withdrawal. Phosphorylation of eEF2, which inhibits its activity, is primarily mediated by eEF2-kinase (Ryazanov, 2002). Interestingly, the activity of eEF2-kinase is in turn also blocked by phosphorylation (Nairn et al., 1985; Tuazon et al., 1989). Several upstream kinases, including stressactivated protein kinase 4 (SAPK 4), ribosomal protein S6 kinase (p70 S6 kinase), p90RSK1, and AMP-activated protein kinase (AMPK), inhibit eEF2-kinase by phosphorylating different Ser residues (Browne et al., 2004; Knebel et al., 2001; Wang et al., 2001). It has been reported that PI3kinase regulates the cell cycle through Akt, mTOR, and p70 S6 kinase (Gao et al., 2003). Because there is overlap in the pathways leading to eEF2 phosphorylation and activating

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PI3-kinase, we tested whether PI3-kinase was involved in eEF2 phosphorylation. At the time when differentiation was induced, the cells were cultured in DM with or without 5 µM LY294002 and assayed for eEF2 phosphorylation. Interestingly, the early decline in phosphorylation before 24 h did not occur in the presence of LY294002 (Fig. 2A); 3 h after differentiation induction, phosphorylation was actually more than 2-fold higher than in the untreated control cells, although it ultimately declined (data not shown). Since wortmannin, the other PI3-kinase inhibitor, is unstable in growth media; we could not assay its effect on eEF2 phosphorylation throughout the course of differentiation. Instead, its effect was examined after 3 h in DM. As shown in Fig. 2B, it also increased the phosphorylation of eEF2. The concentration of eEF2 protein itself did not change significantly during the course of differentiation, either in control or inhibitor-treated cells. These results show that PI3-kinase is involved in eEF2 phosphorylation in the course of L6 myoblast differentiation. PI3-kinase is involved in regulation of the cell cycle by eEF2 phosphorylation Cells are not normally in a uniform cell cycle phase when differentiation is induced. To examine whether the decrease of eEF2 phosphorylation occurs only in differentiating myoblasts, cells were first synchronized in G2/M phase by treatment with nocodazole, which inhibits cell cycle progression by depolymerizing microtubules (Zieve et al., 1980). Before the addition of nocodazole (P), myoblasts occupied a spectrum of cell cycle phases: 23.9% in G2/M, 30.6% in S, and 45.5% in G0/G1 (Fig. 3A). After 16 h of incubation with nocodazole, however, 78.8% of the cells were in G2/M, with 14.2% in S and only 7.0% in G0/G1. When the nocodazole-arrested cells were transferred to fresh GM or DM, they progressed to the other phases of the cell cycle. After 6 h in DM and GM, the fraction of cells in G2/M decreased to 48.3 and 41.7%, respectively. At the same time, G0/G1 cells increased to 39.1 and 43.9%, respectively. The difference became more prominent after 24 h. In GM, the proportion of cells in the S phase increased to 39.4%, and the percentages in G2/M and G0/G1 phases were 28.2 and 32.4%, respectively. In DM, however, the percentage of cells in S phase remained at 12.6%, and 46.5% were in G0/G1. Interestingly, only 7.1% of the cells were in S phase after 24 h in fresh DM containing LY294002, with 53.1% in G0/G1, and no myoblast differentiation took place (see Fig. 1). These results suggest that LY294002 keeps the cells arrested in G1 phase and does not permit them to withdraw from the cell cycle. To confirm the observed cell cycle kinetics, we measured the expression of cyclin D1 by Western blot analysis. The D-type cyclins are closely related proteins whose expression is induced by mitogens and growth factors (Altucci et al., 1996; Lanahan et al., 1992; Lavoie et al., 1996; Matsushime et al., 1991; Musgrove et al., 1993)

A

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C

D

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Fig. 3. Phosphorylation of eEF2, and activity of PI3-kinase in synchronized cells. A. Myoblasts were synchronized in G2/M by treatment with nocodazole, and then released from arrest by transfer to fresh GM or DM. At the indicated times, the cells were subjected to cell cycle analysis as described in Materials and Methods. P indicates just prior to nocodazole treatment and 0 h indicates just after the incubation in nocodazole. The time of release from nocodazole is indicated. B. Cells were harvested from DM or GM at the indicated times after release from nocodazole arrest. Expression of cyclin D1 was measured by Western blotting with anti-cyclin D1 antibody. C. Cells were harvested after the indicated times in fresh DM or GM and assayed for expression and phosphorylation of eEF2 as previously described. β-actin was used as a loading control. The same aliquots were also assayed for expression of cyclin D1. D. eEF2 phosphorylation and protein concentration were assayed as described above using cells cultured in DM for 12 h without (C) or with 10 µM LY294002 (LY), 0.5 μM wortmannin (W), 1 μM API-2 (A) or 0.1 μM rapamycin (R). E. Cells were harvested after the indicated times in DM. PI3kinase activity phosphorylating exogenous PI to PI3P was assayed as described in Materials and Methods. The radiolabeled PI3P was identified by preincubating p85 immunocomplex with 5 µM LY294002 for 30 min (LY). The expression of p85 or myogenin was assessed by Western blotting with anti-p85 or the antimyogenin antibodies, respectively. All results are representative of at least three independent experiments.

and down-regulated by growth factor deprivation or by anti-mitogens (Muise-Helmericks et al., 1998; Miyatake

Joo Hong Woo & Hye Sun Kim

et al., 1995; Watts et al., 1994). Accumulation of cyclin D1 is required for progression through the G1 phase, and commits the cell to enter S phase (Quelle et al., 1993). As shown in Fig. 3B, cyclin D1 rapidly declined after nocodazole release in DM and had almost disappeared after about 24 h. In contrast, there was a high concentration of cyclin D1 after more than 24 h in GM. These results show that myoblasts in DM withdraw from the cell cycle and initiate differentiation, whereas in GM they enter S phase of the next cell cycle. We also examined eEF2 phosphorylation in the synchronized cells. The extent of eEF2 phosphorylation changed in both fresh DM and GM without significant changes in the concentration of the protein (Fig. 3C). However, the pattern of phosphorylation was somewhat different in DM and GM. At 12 h, there was a pronounced decrease in eEF2 phosphorylation in DM but not in GM. Although the exact time and degree of the decrease varied slightly in several experiments, the pattern of decrease was consistent. Recently, mTOR has also been recognized as a key molecule in skeletal muscle differentiation. Thus rapamycin, an inhibitor of mTOR, blocks the differentiation of various myoblast cell lines (Canicio et al., 1998; Conejo et al., 2001; Cuenda and Cohen, 1999; Sarker and Lee, 2004; Shu et al., 2002). The two best-known downstream effectors of mTOR are ribosomal S6 kinase 1 and the initiation factor eIF4E-binding protein 1 (Hay and Sonenberg, 2004). Activation of S6-kinase 1 results in phosphorylation and inhibition of eEF2 kinase (Wang et al., 2001). The PI3-kinase/Akt pathway is linked to mTOR in various types of cell (Fingar and Blenis, 2004; Gao et al., 2003). We therefore examined the effects of the PI3kinase/mTOR signaling pathway on eEF2 phosphorylation. At the time of release from arrest, cells were given medium with or without LY294002, wortmannin, API-2 (an Akt inhibitor) or rapamycin. As shown in Fig. 3D, each of these inhibitors increased the phosphorylation of eEF2, with little effect on the concentration of eEF2 protein. These results suggest that the PI3-kinase/mTOR signaling pathway has a connection with eEF2 phosphorylation. Finally, we examined PI3-kinase activity under the same conditions. Activity phosphorylating exogenous PI to PI3P increased with time in DM (Fig. 3E), and myogenin was expressed from 12 h after release, indicating that normal myoblast differentiation occurs under these conditions. The observations that LY294002 promotes the accumulation of G0/G1 phase-arrested cells (Fig. 3A) and that it blocks the expression of p21 (Fig. 1B) suggest that this inhibitor can block the progression through the cell cycle that is needed for the terminal differentiation of myoblasts. This agent did not actually induce myoblast differentiation. Of interest was the finding that it increased eEF2 phosphorylation early in differentiation and that it stimulated PI3-kinase activity. There have been few reports on the regulation of eEF2 phosphorylation by PI3-kinase,

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and neither eEF2 kinase nor eEF2 are direct substrates for PI3-kinase. However, because PI3-kinase is a well-known regulator of the cell cycle, it is plausible that eEF2 is linked to downstream events in the PI3-kinase pathway. In conclusion, we suggest that the downregulation of eEF2 phosphorylation is required for the early stages of myoblast differentiation, and that PI3-kinase can regulate the phosphorylation of eEF2. Further studies should be performed both to investigate other components of the pathway linking PI3-kinase and eEF2 phosphorylation and to elucidate the precise function of eEF2 phosphorylation in withdrawal from the cell cycle during myoblast differentiation.

Acknowledgment This work was supported by a Korea Research Foundation Grant (R05-2003-000-11880-0) funded by the Korean Government.

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