Untitled

Abstract

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 35 S-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.

PRAS40 signaling in tumor

“…Therefore mTORC1 phosphorylates PRAS40 to enhance protein synthesis and simultaneously to facilitate the assembly of the β subunits for forming immunoproteasomes [ 70 ]. Consistent with this report, PRAS40 deletion decreases protein synthesis in C2C12 myoblasts [ 53 ].…”

“…Malla R et al reported that mTOR activation was increased in the liver tissues of PRAS40 knockout mice [ 44 ] (Figure 1A ). However, PRAS40 knockdown does not make any alteration on mTOR pathway in myoblasts [ 53 ].…”

“…PRAS40 (T246A) transgenic mice have less response to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal hyperproliferation and skin tumor promotion [ 51 ]. The data in myoblasts show that PRAS40 knockdown results in a greater proportion of cells in G1/G0 phase of the cell cycle and fewer cells in the active S phase compared with control values [ 53 ]. Abnormal expression or activation of PRAS40 in tumor may promote the cellular proliferation by deregulating cell cycle.…”

Amplification and Demultiplexing in Insulin-regulated Akt Protein Kinase Pathway in Adipocytes

“…5, A and B ). Phosphorylation of PRAS40, a regulator of mTORC1 and protein synthesis (36, 37), was less sensitive to insulin compared with protein synthesis per se (Fig. 5 A ).…”

Characterization of early autophagy signaling by quantitative phosphoproteomics

“…Several well-described interactions are reconstructed. Furthermore, the inferred network also suggests new interactions between e.g., MTORC1 and proteins involved in translation via AKT1S1 36 indicating that this strategy might serve as an approach to extract new hypotheses about putative interactions.…”