PRAS40 regulates protein synthesis, cell cycle, proliferation and myogenic differentiation in C2C12 myocytes

Abstract

PRAS40 is an mTOR binding protein which has complex effects on cell metabolism. Our study tested the hypothesis that PRAS40 knockdown (KD) in C2C12 myocytes will increase protein synthesis via up‐regulation of mTOR‐S6K1 pathway. PRAS40 KD was achieved using lentiviruses to deliver shRNA targeting PRAS40 or a scrambled control. C2C12 cells were used as either myoblasts or differentiated to myotubes. KD 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 either myoblasts or myotubes. No change in protein synthesis in myotubes was detected. In contrast, protein synthesis was reduced 25% in myoblasts as measured by the incorporation of 35S‐methionine. PRAS40 KD in myoblasts also decreased proliferation rate with increased percent of cells retained in 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 25–30% decrease in total p21 and phosphorylated Rb protein considered critical for G1 – S phase progression. Reduction in protein synthesis was not due to increased apoptosis as cleaved caspase‐3 and DNA laddering were not different between groups. Our results suggest that a reduction in PRAS40 specifically impairs myoblast protein synthesis, cell cycle, proliferation, and differentiation to myotubes.