Chapter 5 TGF-β1-Mediated Regulation of Cell Cycle-Sensitive Translational Components: Regulation of P70 S6 Kinase and Eukaryotic Translational Initiation Factor (EIF)-2α

Abstract

Protein synthesis was monitored by tritiated-leucine incorporation under transforming growth factor (TGF)-β1 treatment in C3H-10T½ murine embryonic fibroblasts. TGF-β1 induced protein synthesis as early as 4 hours. Rapamycin significantly reduced, but did not completely abolish TGF-β1-mediated protein synthesis. Both rapamycin-sensitive and rapamycin-independent pathways may be operational where rapamycin-independent protein synthesis may partially compensate for rapamycin-inhibited protein synthesis.

Rapamycin inhibition of protein synthesis correlated with decreased p70 ribosomal S6 kinase levels in C3H-10T½ fibroblasts. While wortmannin did not overcome TGF-β1-mediated protein synthesis, wortmannin also decreased p70 S6 kinase levels in these fibroblasts. At the level of protein synthesis, rapamycin-sensitive pathways diverge from wortmannin-sensitive pathways. Based on observation of p70 S6 kinase levels, rapamycin signaling converged with wortmannin signaling.

TGF-β1 induced phosphorylation of p70 S6 kinase at the pseudosubstrate domain (threonine-421/serine-424.) Phosphorylation at the linker domain (threonine-389) remained constitutive. Rapamycin, but not wortmannin, reduced TGF-β1-mediated phosphorylation at the pseudosubstrate domain. These results implicated a role for p70 S6K activation in mediating rapamycin inhibitory effects against TGF-β1, particularly through phosphorylation at threonine-421/serine-424.

The stress-inducible eukaryotic translation initiation factor, eIF-2α was examined under TGF-β1 treatment and was observed to accumulate in a delayed kinetics manner. Both rapamycin and wortmannin accelerated the accumulation of eIF-2α by TGF-β1. Thus, increasing eIF-2α protein levels by these G1 inhibitory drugs may exert a stress-like response to encourage eIF-2α-dependent (cap-independent) protein synthesis.

Introduction

The relationship between increased protein synthesis and G1 progression was first proposed as an increased functional demand hypothesis (Novi, 1976) whereby extracellular signals activated pre-existing translational components to induce de novo synthesis of additional components required in achieving critical cell mass for G1/S phase progression. Indeed, the importance of inducible protein synthesis in cell proliferation is highlighted in viral infection models where early events of host invasion involved taking over the regulation of host protein synthesis machinery for redirection towards viral-specific translation.

In studies of cell cycle-sensitive translational regulation, eukaryotic translation factors have received much attention. With the emergence of the immunosuppressant drug rapamycin as a tool in studying G1-related signaling mechanisms, the eukaryotic ribosomal kinase p70 S6K became a focal point in studying mitogen-stimulated protein synthesis necessary for cell cycle progression. The p70 S6 kinase phosphorylates the prominent S6 riboprotein in eukaryotic ribosomes. This ubiquitously expressed kinase is sensitive to external stimuli and was identified to be a critical regulator of G1/S transition (Reinhard et al, 1992). Threonine-421 and serine-424 at the C-terminal auto-inhibitory domain correlated with mitogen-induced phosphorylation (Ferrari et al, 1992). Phosphorylation at threonine-389 in the linker region was observed to be sensitive to both rapamycin and wortmannin (Han et al, 1995). Regulation of p70 S6 kinase therefore may be at the level of protein abundance or phosphorylation.

Measurements of cellular metabolic rates under TGF-β1-stimulated cell cycle progression showed that induced glycolysis depended upon protein synthesis, as addition of cycloheximide abolished this TGF-β1 effect (Boerner et al, 1985). Furthermore, radiolabeling assays using methyl-aminoisubutyrate (MeAIB), a nonmetabolized substrate of the system A neutral amino acid transporter, demonstrated a cell-cycle sensitive nature of this amino acid transporter (Racker et al, 1985). Effective stimulation of C3H fibroblasts by TGF-β1 makes this an ideal in vitro platform for dissecting biochemical mechanisms of TGF-β1-stimulated cell cycle progression. Early studies in these fibroblasts showed Na+/K+ ATPase-dependent ion movements (Leister et al, 1985; Wenner et al, 1981) and amino acid transport (Schenerman et al, 1988) correlated witcell cycle entry. Increasing amino acid availability and transport by TGF-β1 suggests that TGF-β1 may shift the bioenergetics balance towards G1/S transition. Substrate availability therefore is an important contribution in cell-cycle regulated protein synthesis.

Eukaryotic protein synthesis is regulated both at the binding of tRNA_i^met to the 40S ribosomal subunit and at the binding of mRNA to the 43S preinitiation complex (Pain, 1996). EIF-2 proteins mediate formation of the preinitiation complex in a ternary structure involving GTP hydrolysis where the nucleotide exchange factor eIF-2B exchanges GDP for GTP. EIF-4 proteins mediate mRNA binding to ribosomes where eIF-4E binds the 5'-m7GTP cap structure present in mammalian mRNAs. Studies on the effects of altering eIF-4E activity in cells suggest a correlation between eIF-4E activation and the translation of selected proteins including cyclin D1.

Amino acid-sensitive protein synthesis was proposed to be contributed by changes in eIF-2 components rather than eIF-4. Changes in global protein synthesis were suggested to be modulated through eIF-2B activity (Kimball et al, 1998). EIF-2B activity was well characterized to be regulated by eIF-2α phosphorylation: phosphorylation of eIF-2α converts this translation factor from a substrate to a competitive inhibitor (Pain, 1996). However, this phosphorylation event could not reconciled with total inhibition of eIF-2B due to the low molar concentration of phosphorylated eIF-2α relative to the concentration of total protein. Other unknown mechanisms may be operative, including changes in eIF-2α species receptive to phosphorylation by eIF-2α kinases. Still, changes in eIF-2α levels may therefore indirectly affect eIF-2B activity by increasing the phosphorylated species of eIF-2α. In the present study, the effects of TGF-β1 on p70 S6K regulation and on eIF-2α levels were examined in C3H fibroblasts.

Materials and Methods

Cell Culture and Growth Factor Treatment

Cells were cultured and treated with growth factor and inhibitors as described in Materials and Methods, Chapter 2.

Radiolabeling Assay

Radiolabeling solutions and radiolabeling assay were prepared and implemented according to Materials and Methods, Chapter 2.

Immunoblot Analysis

Whole cell extracts were isolated and immunoblot analyses were carried out according to Materials and Methods, Chapter 2.

Results

Rapamycin inhibited TGF-β1-stimulated protein synthesis

Since G1/S cell cycle progression correlated with increased protein synthesis to achieve critical mass, changes in protein synthesis often underlie biochemical mechanisms of mitogenic and anti-mitogenic effects. Although TGF-β1 exhibited delayed cell cycle entry kinetics (S-phase entry begins at 16 hours and reaches maximal DNA synthesis at 32 hours), amino acid uptake and protein synthesis occurred shortly after growth factor addition.

To determine whether rapamycin could inhibit TGF-β1-mediated protein synthesis, incorporation of tritiated leucine into total cellular protein were monitored in radiolabeling assays. TGF-β1 significantly induced protein synthesis when compared with control cells receiving no growth factor and this effect was reduced by nanomolar amount (10 nM) of rapamycin, Figure 5-1. While rapamycin-reduction of this TGF-β1 effect was significant, the inhibition was incomplete. This suggests that TGF-β1-stimulated protein synthesis occurred via a rapamycin-sensitive pathway as well as other pathways that partially compensated rapamycin-inhibited protein synthesis. Another cell cycle inhibitor, wortmannin, did not significantly inhibit TGF-β1-stimulated protein synthesis. Therefore, the phosphatidylinositol (PI) 3-kinase regulated pathway diverged from the TGF-β1 signaling pathway.

To determine whether the observed inhibitory effects of rapamycin was dependent on pre-incubation, cells were treated first with TGF-β1 for designated times before the addition of rapamycin. According to one-hour pulse labeling experiments, rapamycin inhibition occurred even after growth factor addition, Figure 5-2. Points of interception of TGF-β1-sensitive protein synthesis and leucine uptake by rapamycin was therefore independent of inhibitor addition-time relative to growth factor treatment. Temporal independence of rapamycin addition in reducing the TGF-β1 effect supports the presence of a rapamycin-mediated pathway in sustained TGF-β1 signaling.

TGF-β1-mediated up-regulation of p70 S6 kinase was inhibited by rapamycin and wortmannin

Ribosomal p70 S6 kinase is a cell cycle-regulated translational ribosomal component sensitive to mitogens and hormones. To determine the effect of TGF-β1 on p70 S6K protein level, total cellular protein were obtained from quiescent C3H fibroblasts stimulated with TGF-β1 for immunoblot analysis. Prior to initiation of S-phase entry, at 10 hours TGF-β1 up-regulated p70 S6K, Figure 5-3. Rapamycin reduced p70 S6K protein to control levels. Even as wortmannin did not significantly inhibit TGF-β1-stimulated protein synthesis from leucine incorporation data, wortmannin also down-regulated p70 S6K, Figure 5-3. At the point of S-phase entry at 16 hours, rapamycin- and wortmannin-treated p70 S6K levels were markedly less than that observed in control cells, suggesting that these drugs also affected basal expression of p70 S6K. Whereas leucine incorporation data suggested pathways divergence between rapamycin signaling and wortmannin signaling, these pathways may converge at the level of p70 S6K abundance. However, the inhibition of TGF-β1-stimulated p70 S6K by wortmannin could not account for continued protein synthesis. Inhibition of phosphorylation may prevent activation of this ribosomal kinase.

TGF-β1-stimulated p70 S6 kinase phosphorylation at T421/S424 was reduced by rapamycin but not wortmannin

Phosphorylation of p70 S6K occurs at 3 domains: the catalytic domain (T229), linker domain (T389), and the serine-proline rich pseudosubstrate domain (T421/S424). Phosphorylation at the linker domain closely correlated with in vivo activation of p70 S6K and phosphorylation of the pseudosubstrate domain relieves auto-inhibition in response to mitogens. To determine whether TGF-β1-stimulated cell cycle entry corresponded positively with p70 S6K phosphorylation in C3H fibroblasts, antibodies were obtained that were specific for the phosphorylated forms of p70 S6K. These antibodies would provide information on the phosphorylation state of p70 S6K at two locales: the linker domain (Threonine 389) and the pseudosubstrate domain (Threonine 421/Serine 424). The phosphorylation of the linker region (T389) was observed to be constitutive whereas phosphorylation of the pseudosubstrate domain (T421/S424) was stimulated by TGF-β1, Figure 5-4. Rapamycin reduced p70 S6K phosphorylation at this domain while wortmannin was without effect. This paralleled the radiolabeling assay results for inhibition of protein synthesis by rapamycin and the lack of effect by wortmannin at S-phase entry. TGF-β1 induced phosphorylation of the pseudosubstrate domain of p70 S6K correlated with the stimulation of protein synthesis and implicated a role for p70 S6K activation in mediating TGF-β1-stimulated cell cycle entry.

Rapamycin and wortmannin up-regulated eukaryotic translation initiation factor eIF-2α levels against TGF-β1 stimulation

While eukaryotic translation initiation factor-4E (eIF-4E) was recognized to couple mitogenic cell cycling signals to activating translation, eIF-2α is another component in the rate limiting step of protein synthesis that remains important for studying protein synthesis particularly under nutrient deprivation. In yeast where global cellular protein synthesis was inhibited, a small percentage of stress-inducible proteins including eIF-2α were translated for an adaptive cellular profile characteristic of nutrient starvation. Previous observations showed that TGF-β1 did not affect eIF-4E levels whereas EGF up-regulated eIF-4E protein (unpublished results). Eukaryotic initiation factor-2α was therefore examined to determine whether TGF-β1 and rapamycin altered this protein.

Rapamycin and wortmannin up-regulated eIF-2α while TGF-β1 alone did not induce eIF-2α until 10 hours, Figure 5-5. EIF-2α level was low at thirty minutes and by five hours both rapamycin and wortmannin significantly up-regulated eIF-2α protein levels in TGF-β1 stimulated cells whereas cells with TGF-β1 alone remained low for eIF-2α, Figure 5-5. EIF-2α levels in control cells remained low, comparable with those treated with TGF-β1. These observations suggest that wortmannin and rapamycin may accelerate a 'stress like' response in these cells.

The presence of eIF-2α protein levels may also contribute to the delayed cell cycle entry kinetics characteristic for TGF-β1 compared with early acting growth factors. Rapamycin accumulated eIF-2α against TGF-α, an EGF-like growth factor, Figure 5-6. Concomitant addition of TGF-β1 and TGF-α showed that TGF-β1 induced eIF-2α in the presence of TGF-α while TGF-α alone did not induce eIF-2α. This further strengthens a role for eIF-2α in mediating the delayed-kinetic response in TGF-β1 modulation of early acting growth factors such as TGF-α. The presence of rapamycin may increase eIF-2α levels in cells stimulated through serine/threonine kinase receptor-mediated signaling events including TGF-β1-signaling as well as cells stimulated through tyrosine kinasreceptor-mediated signaling events involving TGF-α/EGF-like mitogens. Eukaryotic initiation factor-2α may thus represent a point of convergence in TGF-β1-signaling pathways and EGF-like signaling pathways.

Discussion

These studies demonstrate that in C3H fibroblasts, TGF-β1 stimulated protein synthesis that was inhibited by rapamycin but not wortmannin. In cells where TGF-β1 and rapamycin inhibited INF-g production, TGF-β1 and rapamycin were proposed to share related signaling pathways although TGF-β1 may signal in a distinct mechanism from that by rapamycin (Dumont and Kastner, 1994). The observation that rapamycin only partially eliminated TGF-β1-stimulated DNA synthesis correlated with partial inhibition of leucine incorporation by TGF-β1 and illustrated the intimate relationship between protein synthesis and S-phase entry. Compensating pathways in the TGF-β1 network may be present in the cross talk between TGF-β1 and other growth factors such as TGF-α, EGF and PDGF.

Examination of p70 S6K levels also showed that TGF-β1 up-regulated p70 S6K prior to and at cell-cycle entry. Both rapamycin and wortmannin down-regulated p70 S6K, suggesting that at the level of protein availability, p70 S6K regulation was both rapamycin- and wortmannin-sensitive. However, total protein synthesis monitored with tritiated leucine incorporation showed that wortmannin was ineffective against TGF-β1-mediated protein synthesis. Since wortmannin also down-regulated TGF-β1 mediated p70 S6K induction, the effects of these drugs on specific phosphorylation of p70 S6may determine whether rapamycin or wortmannin was effective TGF-β1. Limiting p70 S6K protein levels may render post-translational regulation more sensitive to extracellular signals.

Reduced phosphorylation further impacted on protein synthesis at the G1/S transition due to the already limited p70 S6K level.

An interesting observation in the effect of TGF-β1 on p70 S6K was differential phosphorylation: threonine-389 was constitutively phosphorylated but threonine-421/serine-424 appeared to be modifiable in the TGF-β1/rapamycin "push-pull" dynamic. Divergence in wortmannin and rapamycin pathways also occurred at p70 S6K phosphorylation, although cell cycle components native to this divergence remain to be identified. Nevertheless, distinguishing rapamycin and wortmannin effects on TGF-β1 was in part due to phosphorylation of the pseudosubstrate domain of p70 S6K and suggested signal-specific assignment of domain activation.

The eukaryotic translation initiation factor-2 (eIF-2) is a component in the rate-limiting step of regulating protein synthesis. During suppression of global protein synthesis under cellular stress, a small percentage of proteins were observed to be specifically induced and translated in a cap-independent mechanism. In Yeast, post-transcriptional and -translational regulation of cell cycle-sensitive factors correlated with vulnerability of eIF-2 translation factors to various stresses including heat shock and nutrient starvation. The mammalian counterparts of these yeast translational factors may therefore be similarly regulated. An intriguing causal possibility in delayed cell cycle kinetics observed under TGF-β1 treatment was the temporal regulation of eIF-2α by TGF-β1.

While TGF-β1 treatment appeared to induce eIF-2α levels by 10 hours post-growth factor addition, pre-incubation with rapamycin or wortmannin apparently accelerated the onset of eIF-2α. Low eIF-2α levels observed under all conditions at thirty minutes confirmed the accumulation of eIF-2α as an inductive process. If drug treatment correlated with the onset of eIF-2α expression native to a stress-response, the induction of eIF-2α by TGF-β1 at ten hours may suggest simulation of a stress-response by TGF-β1, to support a biochemical mechanism underlying the delayed cell-cycle entry kinetics characteristic of TGF-β1.

Concomitant addition of TGF-β1 with early acting growth factors including EGF and PDGF resulted in a shift in S-phase entry kinetics of these early acting growth factors where TGF-β1 delayed EGF- and PDGF-mediated S-phase entry (Kim et al, 1993). C3H fibroblasts also showed a correlation between TGF-β1-mediated cell cycle progression with alterations in phosphorylation of the retinoblastoma protein, pRB (Kim et al, 1994), and the down-regulation of the cyclin-cdk inhibitory protein p27kip1. The eIF-2α induction pattern under TGF-β1 and TGF-α parallels that for p27kip1 induction undeTGF-β1 and EGF treatment. Supporting the role for eIF-2α in mediating TGF-β1-delayed cell cycle entry is the induction of eIF-2α under concomitant treatment of TGF-β1 and TGF-α.

Since the presence of eIF-2α may reflect stress experienced by the cell, the point of rapamycin sensitivity against TGF-β1 must lie upstream p27kip1 degradation and downstream eIF-2α induction. p27kip1 may possibly be regulated at the level of an eIF-2α kinase. This is an attractive model since p27kip1 protein accumulated during stress and heat shock, and under such conditions where global protein synthesis halts, there must be specific accumulation of proteins characteristic to the perturbed cellular profile. Furthermore, within the upstream regions of p27kip1, three potential CTG start sites unique to eIF-2α initiation mechanisms are found (unpublished observation). If p27kip1 was indeed regulated at the level of eIF-2α, such a relationship would place p27kip1 regulation downstream various metabolic pathways regulating eIF-2-specific translation initiation. Future experiments will help evaluate this possibility by determining whether the upstream elements in the p27kip1 gene may serve as regulatory points for eIF-2α-governed mechanisms.

Integration of Eukaryotic Initiation Factors with Metabolism and Cell Cycle

Glucose metabolism illustrates the regulatory hierarchy in the cell upon glucose presentation. Glucose is converted by the cell for energy through glycolysis and oxidative phosphorylation. The expenditure of 2 ATP during glycolysis results in the generation of 4 ATP by converting 1,3-bisphosphoglycerate to 3-phosphoglycerate and phosphoenolpyruvate to pyruvate. Since each intermediate is a 3-carbon compound, two reactions per glucose result with a net production of 2 ATP. While glycolysis is not the major event in energy generation, its importance in generating metabolic and anabolic intermediates should not be overlooked. In addition, glucose and glycolytic intermediates are potential regulatory signals in specific cellular events controlling proliferation and differentiation.

One key regulatory enzyme in glycolysis is phosphofructokinase (PFK), a rate-limiting enzyme that commits the cell to glycolysis. Phosphofructokinase catalyzes fructose-6-phosphate to fructose-1,6-bisphosphate through ATP hydrolysis. ATP inhibits PFK through AMP amplification (there is approximately 50 times more cellular AMP than ATP and small increases in ATP concentration lead to a large amplification of AMP production from adenylate cyclase).

Protein synthesis is a cell cycle-regulated process, and amino acid deficiency leads to the accumulation of uncharged tRNA in the cell. Phosphofructokinase has been demonstrated to be inhibited by uncharged tRNA (Rabinovitz, 1995) to halt glycolysis and glucose uptake. In addition, the product of PFK, fructose-1,6-bisphosphate has been shown to stimulate protein synthesis at the level of GDP-binding to release eIF-2B from the inactive GDP-eIF-2B complex. Transformed cells are known to exhibit higher levels of metabolic activity when compared with normal cells (Warburg hypothesis) and are often not subjected to the same inhibitory mechanisms operative in the normal phenotype. The PFK-tRNA connection is consistent with this observation.

Following the pathways in regulating PFK and the initiation of protein synthesis, complexity of cell cycle progression processes becomes apparent. The interactive dynamics between positive and negative components of the cell cycle determine growth arrest or cell cycle progression. The relationship between PFK and these cell cycle components may be examined in light of eukaryotic translation initiation factors that govern each component. Presently, cyclin D1 has been shown to be regulated by a MAP kinase-dependent pathway and induced by activating the cap-dependent translation initiation factor, eIF-4E (Rousseau et al, 1996). Various inhibitory and mitogenic signals were observed to affect eIF-4E activity, although the pathways and intermediates leading to activation are not definitive. P27kip1 regulation was shown to occur at the protein level, through accumulation, turnover, and degradation - most likely through a ubiquitin-dependent mechanism (Pagano et al, 1995). An issue that has not been addressed was whether p27kip1 may also be regulated at the translation-initiation level, and therefore subject to regulatory mechanisms exerted on eIFs.

Based on the GCN2 model in yeast cells (Hinnebusch, 1994), p27kip1 may be regulated at the level of eIF-2α or eIF-2α kinase during starvation or stress-induced responses. If p27kip1 is indeed regulated at the level of eIF-2α, a link between PFK activity and p27kip1 regulation may be established. The significance of such a relationship points to the possibility of p27kip1 as a nutrient sensor in a cycling cell. Further investigations are necessary to evaluate this possibility especially in cells where p27kip1 was an endpoint in cell cycle progression.

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