PRAS40 regulates protein synthesis and cell cycle in C2C12 myoblasts

Abid A. Kazi, Charles H. Lang

Research output: Contribution to journalArticle

24 Citations (Scopus)

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 yoblasts 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.

Original languageEnglish (US)
Pages (from-to)359-371
Number of pages13
JournalMolecular Medicine
Volume16
Issue number9-10
DOIs
StatePublished - Sep 1 2010

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Cell Cycle Proteins
Myoblasts
Skeletal Muscle Fibers
Proteins
TOR Serine-Threonine Kinases
Retinoblastoma Protein
Lentivirus
Myosin Heavy Chains
Autophagy
G1 Phase
S Phase
Immunoblotting
Caspase 3
Methionine
Muscle Cells
Small Interfering RNA
Cell Differentiation
Cell Cycle
Carrier Proteins
Up-Regulation

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Genetics(clinical)

Cite this

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title = "PRAS40 regulates protein synthesis and cell cycle in C2C12 myoblasts",
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 yoblasts 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.",
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PRAS40 regulates protein synthesis and cell cycle in C2C12 myoblasts. / Kazi, Abid A.; Lang, Charles H.

In: Molecular Medicine, Vol. 16, No. 9-10, 01.09.2010, p. 359-371.

Research output: Contribution to journalArticle

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