PRAS40 Regulates Protein Synthesis and Cell Cycle in C2C12 Myoblasts
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.
We thank Drs. Ly Hong-Brown and Robert Frost for discussions and critical readings of the manuscript. We thank Danuta Huber and Anne Pruznak for technical support, Dr. David Spector for technical help with viral purification and transfection, Dr. Arun Das with MTT assay and reagents, and Dr. Samina Alam for help with the DNA laddering assay and reagents. We also thank David Stanford of the Penn State Flow Cytometry Core facility for help with cell cycle analysis imaging. This work was supported in part by grants from the National Institutes of Health (GM38032 and AA11290) to CH Lang and Pennsylvania Department of Health using Tobacco Settlement Funds (AA Kazi). The Department specifically disclaims responsibility for any analyses, interpretations or conclusions.
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