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PRAS40 Regulates Protein Synthesis and Cell Cycle in …
PRAS40 is an mTOR binding protein that has complex effects on cell metabolism. Our study tests the hypothesis that PRAS40 knockdown (KD) in C2C12 myocytes will increase protein synthesis via upregulation of the mTOR-S6K1 pathway. PRAS40 KD was achieved using lentiviruses to deliver short hairpin (sh)-RNA targeting PRAS40 or a scrambled control. C2C12 cells were used as either myoblasts or differentiated to myotubes. Knockdown reduced PRAS40 mRNA and protein content by >80% of time-matched control values but did not alter the phosphorylation of mTOR substrates, 4E-BP1 or S6K1, in neither myoblasts nor myotubes. No change in protein synthesis in myotubes was detected, as measured by the incorporation of 35S-methionine. In contrast, protein synthesis was reduced 25% in myoblasts. PRAS40 KD in myoblasts also decreased proliferation rate with an increased percent of cells retained in the G1 phase. PRAS40 KD myoblasts were larger in diameter and had a decreased rate of myotube formation as assessed by myosin heavy chain content. Immunoblotting revealed a 25–30% decrease in total p21 and S807/811 phosphorylated Rb protein considered critical for G1 to S phase progression. Reduction in protein synthesis was not due to increased apoptosis, since cleaved caspase-3 and DNA laddering did not differ between groups. In contrast, the protein content of LC3B-II was decreased by 30% in the PRAS40 KD myoblasts, suggesting a decreased rate of autophagy. Our results suggest that a reduction in PRAS40 specifically impairs myoblast protein synthesis, cell cycle, proliferation and differentiation to myotubes.
Exposure of muscle to growth factors and nutrients increases protein translation initiation via the mTOR pathway, thereby stimulating protein synthesis (). In response to growth factor signaling, the phosphoinositide 3-kinase (PI3K) pathway enhances Akt via phosphoinositide-dependent kinase. Activated Akt then phosphorylates PRAS40 on T246, releasing PRAS40 from the mTOR/raptor complex and enhances its binding to the cellular anchor protein 14-3-3 (,). Conversely, in the absence of growth factors, PRAS40 is hypo-phosphorylated and remains bound to mTOR-raptor and thereby inhibits binding of other mTOR substrates, such as the ribosomal protein S6 kinase (S6K1) and the translational repressor eukaryotic initiation factor 4E binding protein (4E-BP1), thereby suppressing cap-dependent protein translation initiation ().
Protein Synthesis -Translation and Regulation
As a cell approaches the end of the G1 phase it is controlled at a vital checkpoint, called G1/S, where the cell determines whether or not to replicate its DNA. At this checkpoint the cell is checked for DNA damage to ensure that it has all the necessary cellular machinery to allow for successful cell division. As a result of this check, which involves the interactions of various proteins, a "molecular switch" is toggled on or off. Cells with intact DNA continue to S phase; cells with damaged DNA that cannot be repaired are arrested and "commit suicide" through apoptosis, or programmed cell death. A second such checkpoint occurs at the G2 phase following the synthesis of DNA in S phase but before cell division in M phase. Cells use a complex set of enzymes called kinases to control various steps in the cell cycle. Cyclin Dependent Kinases, or CDKs, are a specific enzyme family that use signals to switch on cell cycle mechanisms. CDKs themselves are activated by forming complexes with cyclins, another group of regulatory proteins only present for short periods in the cell cycle. When functioning properly, cell cycle regulatory proteins act as the body's own tumor suppressors by controlling cell growth and inducing the death of damaged cells. Genetic mutations causing the malfunction or absence of one or more of the regulatory proteins at cell cycle checkpoints can result in the "molecular switch" being turned permanently on, permitting uncontrolled multiplication of the cell, leading to carcinogenesis, or tumor development.
Using ModuleFinder a larger picture of the effects of these chemical stresses on the expression of mitochondrial components becomes evident. In the defined subset of co-expressed genes the induction of the alternative transport chain components is coupled to the induction of transcripts encoding for eight different substrate dehydrogenases, providing new avenues for NADH generation, or in the case of the electron transfer flavoprotein (At1g50940), provision of electrons to ubiquinone. Significantly, the new carbon substrates for these NADH generating pathways, while including the organic acids of the TCA cycle, are likely to be generated by catabolism of amino acids. Enzymes involved in valine, isoleucine, cysteine, tyrosine, alanine and glutamate catabolism are induced. Concomitant with this change in substrate for energy generation is the upregulation of transcripts for 4 mitochondrial carrier proteins, most of unknown function. Down-regulation is observed for components of the classical electron transport chain complexes I and III, a separate set of five mitochondrial substrate carriers (most of unknown function) and lipid biosynthesis pathways for phosphotidylglyerol and phosphotidylethanolamine. Interestingly, both genes for NAD-malic enzyme (At4g00570, At2g13560) are down-regulated. This protein normally bridges the TCA cycle to allow the anaplerotic removal of organic acids for functions elsewhere in the cell. Together the insights from this analysis suggests that these simple chemical inhibitors appear to initiate the signals for a complicated re-organisation of mitochondrial function within the plant cell that can now been investigated independently.
A Science Odyssey: You Try It: DNA Workshop - PBS
In summary, PRAS40 knockdown in differentiated myotubes did not alter protein synthesis. In contrast, PRAS40 knockdown in C2C12 myoblasts decreased protein synthesis independent of a change in the phosphorylation of S6K1 and 4E-BP1, suggesting that PRAS40 is not a negative regulator of mTOR-mediated translation initiation in this cell type. Moreover, both myoblasts and myotubes remained responsive to anabolic and catabolic stimuli when PRAS40 was reduced. Knockdown of PRAS40 inhibited G1 to S phase transition of cell cycle and lowered proliferation rate in myoblasts, supporting the contention that PRAS40 is required for this aspect of mTOR signaling. Our data suggest that PRAS40 knockdown in C2C12 myoblasts impairs the ability of mTOR to regulate cell size and proliferation and that PRAS40 is required for these mTOR-associated functions. We confirm that PRAS40 plays an important role in regulation of cell size and show that it also affects cell proliferation and differentiation. Understanding the role of PRAS40 in proliferation and differentiation of myocytes as outlined here may prove important in designing new strategies to manage the muscle wasting associated with catabolic insults such as sepsis, alcohol abuse and aging.
These results are consistent with those of Williamson et al. () showing that prolonged G1/G0 and reduced p21 expression in C2C12 myocytes produced by AICAR decreased cell cycling and delayed myotube formation. To determine whether PRAS40 knockdown would delay myoblast fusion and thereby myotube formation, we monitored the progression and ability of these cells to form myotubes in culture. Time lapse imaging and Western blotting analysis for myosin heavy chain (a marker for matured myotubes) indicated that knockdown of PRAS40 in C2C12 myoblasts delayed myotube formation. Autophagy is another mTOR regulated cellular event that plays an important role in differentiation of myoblasts to mature myocytes (–). Our results indicate that PRAS40 KD decreases autophagy in myoblasts, as inferred from the reduction in the LC3BII/LC3B-I ratio. These changes suggest PRAS40 regulates muscle proliferation and differentiation via regulation of cell cycle and autophagy regulatory proteins.
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An embryonic cell divides again and again
C2C12 myoblasts (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco’s minimum essential medium (DMEM; Invitrogen; Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL) and streptomycin (100 μg/mL) (all from Mediatech, Herndon, VA, USA) under 5% CO2 at 37°C. Myoblasts were subcultured and when 100% confluent, the cells were switched to differentiation medium consisting of DMEM with the above antibiotics-antimycotics and 2% horse serum (Hyclone, Logan, UT, USA) to promote myoblast fusion and differentiation to myotubes. Cells were differentiated for 6 d before experimental manipulation. Myotubes were provided with fresh differentiation medium on day 6 and experiments were performed on day 7. To simulate basal mTOR activity, experiments measuring protein synthesis and the phosphorylation of mTOR substrates were performed with serum-free DMEM without antibiotics-antimycotics. 5-Aminoimidazol-4-carboximide ribonucleoside (AICAR; Toronto Research Chemicals, Ontario, Canada), when present, was added at a final concentration of 2 mmol/L for 8 h. Insulinlike growth factor (IGF)-I, when present, was used at final concentration of 100 ng/mL for the last 20 min of the experiment. These doses maximally suppress and activate protein synthesis in C2C12 cells, respectively (,).
Where there was one cell there are two, then four, then eight,..
The necessity of mTOR activation and subsequent phosphorylation of S6K1 and 4E-BP1 has been demonstrated previously; however, the role of PRAS40 in mTORC1 is poorly defined. Present data place PRAS40 either at the level of mTOR (as an Akt substrate) or as a direct downstream substrate of mTOR, where it is phosphorylated on S183 (,). Several reports have implicated PRAS40 as a negative regulator of mTOR via its inhibition of mTOR substrates, while in contrast, others have shown PRAS40 is required for mTOR signaling (). These opposing data have given rise to controversies regarding the role of PRAS40 in regulating protein translation initiation via mTORC1. Despite several reports implicating PRAS40 as a regulator of protein translation initiation in a variety of cells, there is a paucity of information related to its role in skeletal muscle. Given the pivotal role mTOR plays in response to environmental cues in regulating protein translation initiation, cell cycle and proliferation, it is reasonable to suspect that one or more of these mTOR functions are altered by PRAS40 in myocytes. Therefore, the purpose of our current investigation was to examine changes in C2C12 myocyte protein synthesis, cell proliferation and cell cycle in response to PRAS40 knockdown using short hairpin (sh)-RNA–based in vitro experimental approaches.
Unfolded protein response - Wikipedia
Knockdown of PRAS40 in differentiated myotubes did not alter global protein synthesis compared with scramble controls, as measured by 35S-methionine incorporation into protein (). To determine whether the responsiveness of the PRAS40 knockdown cells to external stimuli was altered, cells were incubated with either an anabolic (IGF-I) or catabolic (AICAR) agent. Addition of IGF-I to the myotubes increased protein synthesis, whereas AICAR inhibited protein synthesis (). Contrary to expectations, the magnitude of the changes produced by these agents in myotubes was the same in both control and PRAS40 knockdown cells. To confirm protein synthesis data, we performed Western blotting for mTOR and its substrates and binding partners. PRAS40 knockdown cells remained responsive to both types of stimuli and their response was similar and comparable to the scramble controls (). For example, IGF-I increased phosphorylation of S6K1 (T389) and PRAS40 (T246), while AICAR increased raptor phosphorylation (S792).
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