Department of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, Pennsylvania 17033
Submitted 21 January 2004 ; accepted in final form 24 June 2004
ABSTRACT
Anti-retroviral therapy promotes clinical, immunologic, and virologic improvement in human immunodeficiency virus-infected patients. Whereas this therapy adversely affects carbohydrate and lipid metabolism, the effects of anti-retroviral drugs on muscle protein synthesis and degradation have not been reported. To examine these processes, we treated C2C12 myocytes with increasing concentrations of the protease inhibitor indinavir for 1 or 2 days. Treatment of myocytes with a therapeutic concentration of indinavir (20 µM) for 24 h decreased basal protein synthesis by 18%, whereas a 42% decline was observed after 48 h. A similar decrement, albeit quantitatively smaller, was detected with other protease inhibitors. Indinavir did not alter the rate of proteolysis. Likewise, indinavir did not impair the anabolic effect of insulin-like growth factor-I on protein synthesis. Mechanistically, indinavir decreased the phosphorylation of the S6 ribosomal protein (rpS6), and this reduction was associated with a decreased phosphorylation of p70S6 kinase and p90rsk as well as the upstream regulators ERK1/2 and MEK1/2. Indinavir also decreased the phosphorylation of Mnk1 and its upstream effectors, p38 MAPK and ERK1/2. Indinavir did not affect the phosphorylation of mTOR or 4E-BP1, but it did decrease the amount of the active eukaryotic initiation factor eIF4G-eIF4E complex. In conclusion, indinavir decreased protein synthesis in myocytes. This decrease was associated with the disruption of the ERK1/2 and p38 MAPK pathways and a reduction in both the level of functional eIF4F complex and rpS6 phosphorylation.
anti-retroviral drugs; mitogen-activated protein kinases; translation initiation
Despite HAART being the cornerstone of current AIDS therapy, it is becoming increasingly evident that HAART is associated with metabolic toxicity (16). The toxic effects of these treatments have commanded little attention in the past, because of the dramatic increase in patient survival. However, concern regarding the adverse effects of these drugs is mounting, because this ultimately impacts the quality of life and long-term survival in patients. The use of HAART regimens is correlated with a wide range of alterations in glucose homeostasis, and these often produce a diabetes-like condition. Of particular interest are those in vivo and in vitro studies demonstrating that the PI indinavir markedly alters the ability of insulin to stimulate glucose uptake in adipose tissue and muscle via alterations in GLUT-4 activity (33, 34). Likewise, there is now extensive literature pertaining to disturbances in lipid metabolism caused by PIs. These derangements include elevated plasma concentrations of triglycerides and disrupted cellular cholesterol homeostasis, as well as fat redistribution (3, 19, 48). In contrast, the ability of PIs to regulate protein metabolism has not been as thoroughly investigated, although there is evidence that HIV-related wasting still occurs in patients treated with these drugs (32).
Protein synthesis is a complex process that includes transcription, translation, and signal transduction events. Translation of mRNA on the ribosome involves three stages: initiation, elongation, and termination. Regulation of the initiation step is the primary determinant in controlling the rate of protein synthesis (13, 27). As such, one of the key steps in translation involves the phosphorylation of the S6 ribosomal protein (rpS6), which leads to upregulation of ribosome biogenesis and an increased translational capacity of the cell.
p70S6K and p90rsk are serine/threonine-signaling intermediate kinases that phosphorylate rpS6 (9, 37). Although p70S6K is relatively specific for the phosphorylation of rpS6, the p90rsk kinase has a wide range of substrates (11). Phosphorylation at various sites of p70S6K and p90rsk is important for their kinase activation and function. In the case of p70S6K, this activation is believed to be regulated by several upstream kinases. For example, one pathway is regulated by the mammalian target of rapamycin (mTOR), as evidenced by the use of the immunosuppressant rapamycin. This drug inhibits mTOR and blocks the activation of p70S6K, particularly the phosphorylation at the T389 residue (15, 41, 46). Another possible pathway involves the activation of a series of multisite phosphorylations by proline-directed kinases such as mitogen-activated protein kinases (MAPKs). Recently, a number of studies demonstrated that the MAPK signaling pathway is important for the activation of both p70S6K1 and p70S6K2 in cardiac myocytes and smooth muscle cells (8, 18, 46). Likewise, p90rsk is a known substrate of MAPKs both in vivo and in vitro (10, 11, 26).
Another key step in the translational control of protein synthesis occurs at the level of the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is a trimeric complex consisting of eIF4E, eIF4A, and eIF4G. Subsequently, eIF4F binds to the 5' cap structure, and this interaction is required for recruiting the 40S subunit onto the mRNA molecule. eIF4G functions as a scaffold protein that bridges the mRNAs to ribosomes via interaction with eIF4E and eIF3 (29). In addition, eIF4G interacts with other polypeptides involved in translation, such as eIF4A, the poly(A) binding protein, and Mnk1. eIF4E function is regulated in part by its association with the repressor binding protein, referred to as 4E-BP1. Phosphorylation of this protein hinders the formation of the functional complex eIF4F by blocking the binding of eIF4E to eIF4G (14). This regulatory step is altered under catabolic conditions, as evidenced by a decreased eIF4G-eIF4E interaction and increased formation of the inactive 4E-BP1-eIF4E complex (23). Furthermore, this pathway appears to be regulated in part via a MAPK-dependent pathway. Mnk1, a protein kinase, interacts with eIF4G and is responsible for the phosphorylation of eIF4E. Mnk1, in turn, is activated by both ERK1/2 and p38 MAPK (12, 38).
The aim of present study was to determine the effects of various PIs in general and indinavir in particular on protein synthesis in mouse C2C12 myocytes. We also investigated the protein synthesis-related signaling events mediated by indinavir. Various PIs decreased protein synthesis after 1- or 2-day treatments. Indinavir-induced impairment of protein synthesis was associated with alterations in both MEK/ERK and p38 MAPK pathways. The level of ERK1/2 phosphorylation was decreased after indinavir treatment. As expected, this response was associated with a decreased phosphorylation of p70S6K1, p90rsk, and their downstream target, rpS6. The p38 MAPK pathway was also affected by indinavir, and this influenced the phosphorylation of the downstream target Mnk1. Likewise, the interaction of the eIF4G-eIF4E complex was impaired by indinavir.
MATERIALS AND METHODS
Reagents. Indinavir was a generous gift from Merck (Rahway, NJ). Nelfinavir, saquinavir, amprenavir, and ritonavir were obtained through the National Institutes of Health AIDS Research and Reference Reagent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, Bethesda, MD). The majority of the antibodies used in this study were purchased from Cell Signaling Technology (Beverly, MA). These included monoclonal antibodies that recognize the phosphorylated form of p38 MAPK (T180/Y182) and ERK1/2 (T202/Y204) as well as polyclonal antibodies specific for phosphorylated (p)-S6 ribosomal protein (S235/S236), p-eIF4E (S209), p-eIF4G (S1108), p-p70S6K (T389, T421/S424), p-p90rsk1 (T359/S363), p-Mnk1 (T197/T202), p-mTOR (S2448), and p-MEK1/2 (S217/S221). Antibodies to total mTOR, p90rsk (RSK13), p38 MAPK, and ERK1/2 were also obtained from Cell Signaling Technology, whereas p70S6K1, p70S6K2, and Mnk1 were from Santa Cruz Biotechnology (Santa Cruz, CA). The p38 MAPK inhibitor SB-202190 was purchased from EMD Biosciences (San Diego, CA). Propidium iodide was obtained from Sigma (St. Louis, MO), and RNase was obtained from Roche (Indianapolis, IN). Human recombinant insulin-like growth factor (IGF)-I was provided by Genentech (San Francisco, CA). [35S]methionine/cysteine (>1,000 Ci/mol) was obtained from MP Biomedicals (Aurora, OH). Cell culture medium and fetal bovine serum (FBS) were from GIBCO Invitrogen (Carlsbad, CA).
Cell culture. C2C12 mouse myocytes were purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM containing 10% FBS, 100 U/ml penicillin, 100 µg/ml penicillin, and 25 µg/ml amphotericin. The effect of various PIs on protein synthesis was determined with minor modification as previously described (17). Briefly, cells were subcultured in 24-well plates to 90% confluence. Most studies were conducted using cells at the myoblast stage, but selected experiments were repeated using differentiated myotubes. Cells were then incubated in 1% FBS medium alone (control) or in media containing one of the anti-retroviral agents described in Reagents. Drugs were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO in the culture was <0.1%. Preliminary studies indicated that this concentration did not have a significant effect on protein synthesis. For 1-day experiments, cells were incubated in the presence of the drug and radioisotope for 24 h before being harvested. For 2-day experiments, cells were preincubated for 1 day with one of the above agents and then radiolabeled with fresh medium on the second day in the continued presence of this agent. Cells were labeled with 10 µCi [35S]methionine/cysteine in DMEM per well for 24 h unless otherwise indicated. The media contained "cold" methionine and cysteine at concentrations of 30 and 65 mg/l, respectively. Preliminary studies showed that the rate of radiolabel incorporation into protein was linear between 1 and 24 h (data not shown), indicating that there were no significant changes in the specific activity of the precursor pool. Hence, all subsequent studies were conducted using the 24-h labeling protocol. At the end of the experiment, cells were collected and precipitated in 10% TCA. The incorporation of [35S]methionine/cysteine into TCA-precipitable protein was determined using liquid scintillation counting. The results were then compared with those of the appropriate time-matched control group, and data were expressed as percentages of the control value. DNA synthesis was determined as described by Rauch et al. (40), with minor modification.
Protein degradation. C2C12 myocytes were subcultured in 24-well plates as described previously (17). Cells were pulse labeled with [35S]methionine/cysteine in the absence or presence of indinavir for 1 or 24 h to determine the rate of degradation of short- and long-half-life proteins, respectively. Some cells were collected at this time (pulse), whereas for other cells, the radiolabeled medium was removed and replaced with fresh medium lacking radioactive methionine/cysteine (chase). Myocytes were chased for various times in the absence (control) or presence of this drug. Cells were collected and precipitated in 10% TCA, and the TCA-precipitable counts were determined as described in Cell culture.
Western blot analysis. To study the signaling pathways known to regulate protein synthesis, we subcultured C2C12 myocytes in six-well plates in the presence of indinavir for 24 h. Thereafter, cells were changed to serum-free medium in the continued presence of indinavir and collected after 2025 min in 2x Laemmli sample buffer. Cell lysates were electrophoresed on denaturing polyacrylamide gels and transferred to nitrocellulose. The resulting blots were blocked with 5% nonfat dry milk and incubated with the antibodies of interest described in Reagents. Unbound primary antibody was removed by washing with Tris-buffered saline (TBS) containing 0.05% Tween 20 (ICI Americas, Wilmington, DE), and blots were incubated with anti-rabbit or anti-mouse immunoglobulin conjugated with horseradish peroxidase. Blots were briefly incubated with an enhanced chemiluminescent detection system (Amersham, Amersham, UK) and exposed to Kodak X-ray film (Rochester, NY). The film was scanned (ScanMaker 4; Microtek, Los Angeles, CA) and analyzed with NIH Image 1.6 software.
Immunoprecipitation of the eIF4E and eIF4G complex.
For quantification of the eIF4G-eIF4E complex, myocytes were grown in 100-mm plates as described and then collected in 20 mM HEPES buffer containing 100 mM KCl, 2 mM EGTA, 0.2 mM EDTA, 50 mM NaF, 50 mM -glycerolphosphate, 1 mM DTT, and protease inhibitor cocktail (Sigma). The supernatants were immunoprecipitated with an anti-eIF4E monoclonal antibody overnight. The antibody-antigen complex was captured by incubation for 1 h with protein G-Sepharose (Amersham Biosciences, Piscataway, NJ). The beads were washed in TBS, and proteins were eluted using 2x Laemmli sample buffer. Precipitated material was examined by Western blot analysis as described.
Cell viability and cell cycle progression. C2C12 myocytes were subcultured in six-well plates to 90% confluence. Some cells were treated with indinavir, whereas other cells were maintained in media lacking this agent. Time-matched samples were collected, and the cell number was determined using a hemocytometer.
To investigate the effect of indinavir on the cell cycle, we cultured C2C12 cells in six-well plates to 75% confluence in the presence or absence of indinavir for 24 h. Cells were harvested by centrifugation, washed, fixed in 70% cold ethanol, and incubated with 40 µg/ml propidium iodide and 5 µg/ml RNase. Samples were kept in the dark at room temperature for 30 min and analyzed for the cell-cycle profile with the use of a FACScan instrument (Becton Dickinson, San Jose, CA). DNA content was determined using Cell Quest 3.3 data-aquisition flow cytometry software (Becton Dickinson) and analyzed with ModFilt DNA analysis software (Verity Software House, Topsham, ME). Results for the different phases of cells in indinavir-treated cultures or the control group are expressed as percentages of total cycling cells.
Statistical analysis. For experimental protocols with more than two groups, statistical significance was determined using one-way ANOVA followed by Dunnett's test to compare all data with the appropriate time-matched control group. For experiments with only two groups, an unpaired Student's t-test was performed. Data are presented as means ± SE. Mean values were considered significantly different at P < 0.05.
RESULTS
Effect of protease inhibitors on basal protein synthesis. To determine whether various protease inhibitors altered the basal rate of protein synthesis, we incubated C2C12 myoblasts with therapeutic concentrations of indinavir, nelfinavir, amprenavir, saquinavir, or ritonavir for 2 days. The drug concentrations utilized for this and subsequent experiments were based on plasma levels reported for patients receiving these treatments (22, 43). Treatment of cells with various protease inhibitors significantly decreased protein synthesis 1542%, compared with rates in control cells (Fig. 1A). Of the drugs tested, indinavir appeared to have the most pronounced adverse effect on protein synthesis. Therefore, we focused all subsequent studies on the metabolic effects of indinavir. Figure 1B shows that a 24-h incubation with 10 µM indinavir significantly decreased protein synthesis (18%) relative to control values. This decline was more pronounced when cells were exposed to indinavir for 48 h, with protein synthesis being impaired by 42%. Incubation of myocytes with 20 and 40 µM indinavir produced changes in protein synthesis comparable to those seen in cells treated with 10 µM indinavir at both time points.
|
Effects of indinavir on cell viability, proliferation, and cell cycle progression. To ensure that the observed effects on protein synthesis were not caused by a decline in cell number, we performed cell counts on plates of myocytes incubated in the presence or absence of 20 µM indinavir for 1 or 2 days. Treatment of cells with indinavir in 1% FBS medium for 2 days did not affect cell viability (76.0 ± 2.3 x 104 cells/well) relative to time-matched untreated control cells (74.8 ± 3.4 x 104 cells/well), indicating that the reduction in protein synthesis was not due to a decrease in cell number. Results from indinavir-treated cells at day 1 showed a similar lack of effect on cell number (data not shown). The viability of myocytes was further confirmed by the observation that treatment of cells with indinavir in medium containing 10% FBS did not inhibit cell proliferation (Fig. 2A). Likewise, DNA synthesis in indinavir-treated myocytes was not affected (91.5% ± 3) relative to control values (100% ± 6), as assessed by [3H]thymidine uptake.
|
Finally, we determined whether indinavir exerted any effect on cell cycle progression. For these experiments, C2C12 cells were treated in the presence or absence of 20 µM indinavir for 24 h, and cells were examined using fluorescence-activated cell sorting (FACS) analysis. Data in Table 1 show that treatment of cells with indinavir did not affect the cell cycle profile of C2C12 myocytes compared with the control group.
|
|
|
|
|
|
|
|
|
|
|
In this study we investigated the effect of the anti-retroviral drug indinavir on protein metabolism and signal transduction in cultured myocytes. Our results demonstrate that indinavir decreased the basal rate of protein synthesis in cultured myocytes after a 24- or 48-h exposure. However, this treatment did not alter proteolysis. Myocytes treated with other PIs such as nelfinavir, saquinavir, amprenavir, and ritonavir also inhibited basal protein synthesis. This comparable response indicates that as a class, these drugs can adversely influence muscle protein metabolism under in vitro conditions.
The effects of indinavir have been tested in a number of cell lines. In the present study we used C2C12 myoblasts, which are a well-characterized model system. These cells embody a muscle precursor phenotype, much like satellite cells that are resident in mature muscle. In addition, we found that indinavir also decreased protein synthesis in myotubes, although the response was less dramatic. Likewise, treatment of adipocytes with indinavir has been shown to have a negative effect on protein synthesis (19). On the other hand, past studies reported that treatment of nonmuscle cells for 2 h with the PI saquinavir did not adversely affect protein synthesis or protein content (39). This apparent discrepancy may be due to the difference in cell type or the much shorter duration of the experimental protocol. It is noteworthy that the indinavir-induced decrease in protein synthesis did not result from a change in cell viability. These data are in agreement with previous reports that treatment of nondifferentiated adipocytes with indinavir for several days did not alter cell number (2). Furthermore, our data showed that indinavir did not affect the cell cycle progression. This finding is consistent with the result reported by Chavan et al. (4) that treatment of Jurkat or PM1 T-cell lines with 550 µM indinavir did not influence their cell cycle profiles.
The mechanisms by which indinavir alters muscle protein synthesis have not been investigated previously. In general, upregulation of the biosynthetic apparatus is needed to support cell growth and proliferation. One essential component of the protein synthetic machinery is rpS6, a downstream substrate of p70S6K. The phosphorylation of rpS6 by p70S6K has a positive effect on the translation of 5'-TOP mRNAs and hence increases the overall translational capacity of cells (20). Our data clearly demonstrate that indinavir produces a consistent, albeit modest, decrease in the phosphorylation of rpS6. These data suggest that the indinavir-induced impairment in protein synthesis might be due in part to decreases in the level of the ribosomal biosynthetic machinery.
A number of in vivo and in vitro studies indicate that the activity of p70S6K1 is regulated by phosphorylation, and this is important for the maintenance of normal rates of protein synthesis. The p70S6K1 activation process relies on the sequential phosphorylation of multiple sites located in different domains of the kinase. One set of phosphorylation sites is within the linker region and catalytic domain (15, 36). The phosphorylation sites of the second set reside within the autoinhibitory domain, and these have been suggested to be important for the subsequent phosphorylation of T389 in the linker region (25). Our results show that indinavir decreased the phosphorylation of the T424/S421 sites located in the autoinhibitory domain as well as the T389 site in the linker region. A portion of this decline was attributed to a decrease in the total p70S6K1 protein level. On the other hand, this inhibitory effect could be due to a decreased upstream kinase activity and/or an increased upstream phosphatase activity. In this regard, we found that the indinavir-induced decrease in phosphorylation of sites within the autoinhibitory domain was more dramatic than that observed in the linker region, indicating that these sites may be regulated by different upstream kinases.
Previous studies (9, 37) have shown that both p70S6K and p90rsk phosphorylate the same serine residue of rpS6, indicating that p90rsk is also an upstream kinase of rpS6. Similar to p70S6K, phosphorylation of various sites in p90rsk is important for its functional activity. In the current study, we demonstrated that indinavir decreases phosphorylation of p90rsk at T359 and S363 located in the linker region. This implies that the function of this kinase is diminished and that the inhibition may contribute to the decreased phosphorylation of rpS6. In contrast, one additional phosphorylation site (S381) in the same region of p90rsk was unaffected (data not shown). Previous studies showed that the phosphorylation of S381 is important for activation of the NH2-terminal domain (6). However, the specific role of various phosphorylation sites in the activation of p90rsk is not well established.
Although p70S6K and p90rsk share some amino acid sequence similarity, they appear to be regulated by different kinase cascades. Several lines of evidence indicate that phosphorylation of these intermediate kinases is regulated by various upstream effectors (7). Whereas p90rsk is mainly activated by the MAPK pathway (9, 10), the regulation of p70S6K is complex and not fully understood. For example, at least two major signaling pathways have been described for the phosphorylation and activation of p70S6K1. The first pathway involves phosphatidylinositol 3-kinase and its downstream target, PDK1, and the second essential pathway is mediated by Akt/mTOR (1, 36). Previous studies (5, 24) reported that the MAPK pathway is not required for the phosphorylation of p70S6K1. However, it was demonstrated recently that MAPK family members, such as MEK/ERK, are important for p70S6K1 and p70S6K2 activation (8, 45). In the present study, indinavir decreased the phosphorylation of p70S6K1 (T421 and S424) in the autoinhibitory domain, which contains the consensus serine/threonine-proline motif. Therefore, these sites can be phosphorylated by proline-directed protein kinases such as MAPK kinases (31). Our data demonstrate that the impairment observed in p70S6K1 phosphorylation is associated with a decreased phosphorylation of both MEK1/2 and ERK1/2, suggesting that MAPKs are involved in the regulation of p70S6K1. This finding is in agreement with earlier studies in which indinavir inhibited insulin signal transduction at the level of MAPK activation in adipocytes (2). On the other hand, past studies have shown that inhibition of mTOR with rapamycin blocks the T389, but not the T421 and S424, phosphorylation of p70S6K1. These data suggest that mTOR is responsible for the activation of T389 (18). In the present study, mTOR activity was not directly assessed and although indinavir did not alter mTOR phosphorylation, we cannot exclude the possibility that this PI decreased p70S6K phosphorylation via a change in mTOR activity.
The binding of mRNA to the 43S preinitiation complex is mediated by the eIF4F complex. Among the three subunits of eIF4F, eIF4E is the least abundant and is considered to be rate limiting in the binding of mRNA to ribosomes. However, the phosphorylation of eIF4E is not necessarily required for its function because unphosphorylated eIF4E can also stimulate translation. In addition, there is no direct evidence that phosphorylation at S209 has any effect on translation (13, 28). In the present study, indinavir increased the phosphorylation of eIF4E at S209 at the same time that the rate of protein synthesis was decreased. These results are consistent with previous findings that anisomycin or arsenite increased eIF4E phosphorylation, despite decreased rates of translation (30, 47). Collectively, these data indicate that phosphorylated eIF4E is not necessarily correlated with increased rate of translation.
In our study, the indinavir-induced decrease in protein synthesis was also associated with an impaired formation of the active eIF4F complex. Indeed, we found that the protease inhibitor decreased the amount of p-eIF4G associated with eIF4E. As stated above, eIF4E can be regulated by binding to the repressor protein 4E-BP1, which enhances the formation of an inactive 4E-BP1-eIF4E complex by decreasing the (hyperphosphorylated)-isoform of 4E-BP1. However, in the present study, there was no detectable alteration in 4E-BP1 phosphorylation. Therefore, the indinavir-induced decrease in protein synthesis does not appear to be mediated through 4E-BP1. It is possible that the action of indinavir targets other proteins, as yet unidentified, that regulate the formation of the eIF4G-eIF4E complex. Furthermore, it is possible that this complex may be regulated by upstream kinases, including Mnk1 and MAPKs. In this regard, it has been demonstrated (12) that Mnk1 is phosphorylated and activated by upstream effectors ERK1/2 and p38 MAP kinases, and this association is supported by our data (see Fig. 8A and 10C). It has been reported (13, 38) that eIF4G binds to Mnk1. Therefore, Mnk1 might regulate the formation of the active eIF4G-eIF4E complex in addition to its role in phosphorylating eIF4E.
In summary, incubation of C2C12 myocytes with indinavir for 24 or 48 h inhibited basal protein synthesis without a concomitant change in protein degradation. Indinavir-impaired protein synthesis was associated with defective MEK1/2, ERK1/2, and p38 MAPK signaling that may account for the reduction in rpS6 phosphorylation. In addition, indinavir decreased the formation of the eIF4F complex, and this change was independent of a change in 4E-BP1 phosphorylation. Hence, in addition to altering carbohydrate and lipid metabolism, indinavir has a pronounced effect on protein synthesis that appears to be mediated by multiple defects in translation initiation. It remains to be determined whether such changes occur under in vivo conditions.
GRANTS
This study was supported by National Institute of Allergy and Infectious Diseases Grant AA-11290.
ACKNOWLEDGMENTS
We thank D. Huber for technical assistance. We thank Drs. Jefferson and Kimball (Penn State University College of Medicine) for the generous gift of eIF4E antibody.
FOOTNOTES
Address for reprint requests and other correspondence: L. Q. Hong-Brown, Dept. of Cellular and Molecular Physiology (H166), 500 Univ. Drive, Hershey, PA 17033 (E-mail: lqh10{at}psu.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
1. Alessi D, Cuenda A, Cohen P, Dudley DT, and Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogen activated protein kinase kinases in vitro and in vivo. J Biol Chem 270: 2748927494, 1995.
2. Caron M, Auclair M, Vigouroux C, Glorian M, Forest C, and Capeau J. The HIV protease inhibitor indinavir impairs sterol regulatory element-binding protein-q intranuclear localization, inhibits preadipocyte differentiation, and induces insulin resistance. Diabetes 50: 13781388, 2001.
3. Carr A, Samaras k Burton S, Law M, Freund J, Chisholm DJ, and Cooper DA. A syndrome of peripheral lipodystrophy, hyperlipidemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS 12: F51F58, 1998.[CrossRef][ISI][Medline]
4. Chavan S, Kodoth S, Pahwa R, and Pahwa S. The HIV protease inhibitor indinavir inhibits cell-cycle progression in vitro in lymphocytes of HIV-infected and uninfected individuals. Blood 98: 383389, 2001.
5. Chung J, Grammer TC, Lemon KP, Kazlauskas A, and Blenis J. PDGF- and insulin-dependent p70S6K activation mediated by phosphatidylinositol-3-OH kinase. Nature 370: 7175, 1994.[CrossRef][ISI][Medline]
6. Dalby KN, Morrice N, Caudwell FB, Avruch J, and Cohen P. Identification of regulatory phosphorylation sites in mitogen activated protein kinase (MAPK)-activated protein kinase-1a/p90rsk that are inducible by MAPK. J Biol Chem 273: 14961505, 1998.
7. Dufner A and Thomas G. Ribosomal S6 kinase signaling and the control of translation. Exp Cell Res 253: 100109, 1999.[CrossRef][ISI][Medline]
8. Eguchi S, Iwasaki H, Ueno H, Frank GD, Motley ED, Eguchi K, Marumo F, Hirata Y, and Inagami T. Intracellular signaling of angiotensin II-induced p70S6 kinase phosphorylation at Ser411 in vascular smooth muscle cells. J Biol Chem 274: 3684336851, 1999.
9. Erikson E and Maller JL. A protein kinase from Xenopus eggs specific for ribosomal protein S6. Proc Natl Acad Sci USA 82: 742746, 1985.[Abstract]
10. Fan HY, Tong C, Lian L, Li SW, Gao WX, Cheng Y, Chen DY, Schatten H, and Sun QY. Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization. Biol Reprod 68: 968977, 2003.
11. Frodin M and Gammeltoft S. Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction. Mol Cell Endocrinol 151: 6577, 1999.[CrossRef][ISI][Medline]
12. Fukunaga R and Hunter T. MNK1, a new MAP kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates. EMBO J 16: 19211933, 1997.
13. Gingras AC, Raught B, and Sonenberg N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68: 913963, 1999.[CrossRef][ISI][Medline]
14. Haghighat A, Mader S, Pause A, and Sonenberg N. Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E. EMBO J 14: 57015709, 1995.[Abstract]
15. Han JW, Pearson RB, Dennis PB, and Thomas G. Rapamycin, wortmannin, and the methylxanthine SQ 20006 inactivate p70S6K by inducing dephosphorylation of the same subset of sites. J Biol Chem 270: 2139621403, 1995.
16. Herman JS and Esterbrook PJ. The metabolic toxicities of antiretroviral therapy. Int J STD AIDS 12: 555564, 2001.[CrossRef][ISI][Medline]
17. Hong-Brown LQ, Frost RA, and Lang CH. Alcohol impairs protein synthesis and degradation in cultured skeletal muscle cells. Alcohol Clin Exp Res 25: 13731382, 2001.[CrossRef][ISI][Medline]
18. Iijima Y, Laser M, Shiraishi H, Willey CD, Sundaravadive B, Xu L, McDermott PJ, and Kuppuswamy D. c-raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells. J Biol Chem 277: 2306523075, 2002.
19. Janneh O, Hoggard PG, Tjia JF, Jones SP, Khoo SH, Maher B, Back DJ, and Pirmohamed M. Intracellular disposition and metabolic effects of zidovudine, stavudine and four protease inhibitors in cultured adipocytes. Antivir Ther 8: 417426, 2003.[Medline]
20. Jefferies HBJ, Fumagalli S, Dennis PB, Reinhard C, Pearson RB, and Thomas G. Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70S6K. EMBO J 16: 36933704, 1997.
21. Jordan R, Gold L, Cummins C, and Hyde C. Systematic review and meta-analysis of evidence for increasing numbers of drugs in antiretroviral combination therapy. Br Med J 324: 747748, 2002.
22. Justesen US, Pedersen C, and Kliltgaard NA. Simultaneous quantitative determination of the HIV protease inhibitors indinavir, amprenavir, ritonavir, lopinavir, saquinavir, nelfinavir and the nelfinavir active metabolite M8 in plasma by liquid chromatography. J Chromatograph 783: 491500, 2003.[CrossRef][ISI]
23. Kumar V, Frost RA, and Lang CL. Alcohol impairs insulin and IFG-I stimulation of S6K1 but not 4E-BP1 in skeletal muscle. Am J Physiol Endocrinol Metab 283: E917E928, 2002.
24. Lenormand P, McMahon M, and Pouyssegur J. Oncogenic Raf-1 activates p70S6 kinase via a mitogen-activated protein kinase-independent pathway. J Biol Chem 271: 1576215768, 1996.
25. Mahalingam M and Templeton DJ. Constitutive activation of S6 kinase by deletion of amino-terminal autoinhibitory and rapamycin sensitivity domains. Mol Cell Biol 16: 405413, 1996.[Abstract]
26. Marshall CJ. MAP kinase kinase kinase, MAP kinase kinase, MAP kinase. Curr Topics Genet Dev 4: 8289, 1994.[CrossRef]
27. Mathews MB, Sonenberg N, and Hershey JWB. Origins and principles of translational control. In: Translational Control of Gene Expression, edited by Sonenberg N, Hershey JWB, and Mathews MB. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 2000, p. 131.
28. McKendrick L, Morley SJ, Pain VM, Kagis R, and Joshi B. Phosphorylation of eukaryotic initiation factor-4E (eIF4E) at Ser 209 is not required for protein synthesis in vitro and in vivo. Eur J Biochem 268: 53755385, 2001.
29. Morley SJ, Curtis PS, and Pain VM. eIF4G: translation's mystery factor begins to yield its secrets. RNA 3: 10851104, 1997.[ISI][Medline]
30. Morley SJ and McKendrick L. Involvement of stress-activated protein kinase and p38/ERK mitogen-activated protein kinase signaling pathways in the enhanced phosphorylation of initiation factor 4E in NIH 3T3 cells. J Biol Chem 272: 1788717893, 1997.
31. Muhkopadhayay NK, Price DJ, Kyriakis JM, Pelech Sanghera J SL, and Avruch J. An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70S6 kinases. J Biol Chem 267: 33253335, 1992.
32. Mulligan K, Tai VW, and Schambelan M. Cross-sectional and longitudinal evaluation of body composition in men with HIV infection. J AIDS Hum Retro 15: 4348, 1997.
33. Murata H, Hruz PW, and Mueckler M. The mechanism of insulin resistance caused by HIV protease inhibitor therapy. J Biol Chem 275: 2025120254, 2000.
34. Noor MA, Seneviratne T, Aweeka FT, Lo JC, Schwartz JM, Mulligan K, Schambelan M, and Grunfeld C. Indinavir acutely inhibits insulin-stimulated glucose disposal in humans: a randomized, placebo-controlled study. AIDS 16: F1F8, 2002.[CrossRef][ISI][Medline]
35. Palella FJ, Delaney KM, Moorman AC, Loveless MO, Fuhrer J, Satten GA, Aschman DJ, and Holmberg SD. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N Engl J Med 338: 853860, 1998.
36. Pullen N, Dennis PB, Andjekovic M, Dufner A, Kozma SC, Hemmings BA, and Thomas G. Phosphorylation and activation of p70S6K by PDK1. Science 279: 707710, 1998.
37. Pullen N and Thomas G. The modular phosphorylation and activation of p70S6K. FEBS Lett 410: 7882, 1997.[CrossRef][ISI][Medline]
38. Pyronnet S, Imatake H, Gingras AC, Fukunaga R, Hunter T, and Sonenberg N. Human eukaryotic translation initiation factor 4G (eIF4G) recruits Mnk1 to phosphorylate eIF4E. EMBO J 18: 270279, 1999.
39. Ranganathan S and Kern PA. The HIV protease inhibitor saquinavir impairs lipid metabolism and glucose transport in cultured adipocytes. J Endocrinol 172: 155162, 2002.
40. Rauch BH, Millette E, Kenagy RD, Daum G, and Clowes AW. Thrombin - and factor Xa-induced DNA synthesis is mediated by transactivation of fibroblast growth factor receptor-1 human vascular smooth muscle cells. Circ Res 94: 340345, 2004.
41. Saitoh S, Brennan N, Cantrell D, Dennis PB, and Thomas G. Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site. J Biol Chem 277: 2010420112, 2002.
42. Schwarcz DK, Hsu LC, Vittinghoff E, and Katz MH. Impact of protease inhibitors and other antiretroviral treatments on acquired immunodeficiency syndrome survival in San Francisco, California, 19871996. Am J Epidemiol 152: 178185, 2000.
43. Turner ML, Reed-Walker K, King JR, and Acosta EP. Simultaneous determination of nine antiretroviral compounds in human plasma using liquid chromatography. J Chromatogr 784: 331341, 2003.[CrossRef]
44. Von Manteuffel SR, Dennis PB, Pullen N, Gingras AC, Sonenberg N, and Thomas G. The insulin-induced signaling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcate at a rapamycin-sensitive point immediately upstream of p70S6K. Mol Cell Biol 17: 54265436, 1997.[Abstract]
45. Wang L, Gout I, and Proud CG. Cross-talk between the ERK and p70S6 kinase (S6K) signaling pathways. J Biol Chem 276: 3267032677, 2001.
46. Wang L and Proud CG. Ras/Erk signaling is essential for activation of protein synthesis by Gq protein-coupled receptor agonists in adult cardiomyocytes. Circ Res 91: 821829, 2002.
47. Wang X, Flynn A, Waskiewicz AJ, Webb BLJ, Vries RG, Baines IA, Cooper JA, and Proud CG. The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathway. J Biol Chem 273: 93739377, 1998.
48. Williams K, Rao YP, Natarajan R, Pandak WM, and Hylemon PB. Indinavir alters sterol and fatty acid homeostatic mechanisms in primary rat hepatocytes by increasing levels of activated sterol regulatory element-binding proteins and decreasing cholesterol 7-hydroxylase mRNA levels. Biochem Pharmacol 67: 255267, 2004.[CrossRef][ISI][Medline]