Molecular Medicine

, Volume 16, Issue 9–10, pp 359–371 | Cite as

PRAS40 Regulates Protein Synthesis and Cell Cycle in C2C12 Myoblasts

  • Abid A. Kazi
  • Charles H. Lang
Research Article


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.


  1. 1.
    Kimball SR, Jefferson LS. (2006) Signaling pathways and molecular mechanisms through which branched-chain amino acids mediate translational control of protein synthesis. J. Nutr. 136 (Suppl. 1): 227S–31S.CrossRefGoogle Scholar
  2. 2.
    Anthony JC, et al. (2002) Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine. Am. J. Physiol. Endocrinol. Metab. 282:E1092–101.CrossRefGoogle Scholar
  3. 3.
    Krawiec BJ, Frost RA, Vary TC, Jefferson LS, Lang CH. (2005) Hindlimb casting decreases muscle mass in part by proteasome-dependent proteolysis but independent of protein synthesis. Am. J. Physiol. Endocrinol. Metab. 289:E969–80.CrossRefGoogle Scholar
  4. 4.
    Dunlop EA, Tee AT. (2009) Mammalian target of rapamycin complex 1: signalling inputs, substrates and feedback mechanisms. Cell Signal 21:827–35.CrossRefGoogle Scholar
  5. 5.
    Gingras AC, Raught B, Sonenberg N. (2004) mTOR signaling to translation. Curr. Top. Microbiol. Immunol. 279:169–97.PubMedGoogle Scholar
  6. 6.
    Hay N, Sonenberg N. (2004) Upstream and downstream of mTOR. Genes Dev. 18:1926–45.CrossRefGoogle Scholar
  7. 7.
    Hall MN. (2008) mTOR: what does it do? Transplant. Proc. 40 (Suppl. 10):S5–8.CrossRefGoogle Scholar
  8. 8.
    Schmelzle T, Hall MN. (2000) TOR, a central controller of cell growth. Cell 103:253–62.CrossRefGoogle Scholar
  9. 9.
    Balasubramanian S, et al. (2009) mTOR in growth and protection of hypertrophying myocardium. Cardiovasc. Hematol. Agents Med. Chem. 7:52–63.CrossRefGoogle Scholar
  10. 10.
    Holz MK, Ballif BA, Gygi SP, Blenis J. (2005) mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events. Cell 123:569–80.CrossRefGoogle Scholar
  11. 11.
    Kim DH, et al. (2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110:163–75.CrossRefGoogle Scholar
  12. 12.
    Peterson TR, et al. (2009) DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell 137:873–86.CrossRefGoogle Scholar
  13. 13.
    Lang CH, et al. (2003) Alcohol impairs leucinemediated phosphorylation of 4E-BP1, S6K1, eIF4G, and mTOR in skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 285:E1205–15.CrossRefGoogle Scholar
  14. 14.
    Kovacina KS, et al. (2003) Identification of a proline-rich Akt substrate as a 14-3-3 binding partner. J. Biol. Chem. 278:10189–94.CrossRefGoogle Scholar
  15. 15.
    Sancak Y, et al. (2007) PRAS40 is an insulinregulated inhibitor of the mTORC1 protein kinase. Mol. Cell. 25:903–15.CrossRefGoogle Scholar
  16. 16.
    Wang L, Harris TE, Lawrence JC Jr. (2008) Regulation of proline-rich Akt substrate of 40 kDa (PRAS40) function by mammalian target of rapamycin complex 1 (mTORC1)-mediated phosphorylation. J. Biol. Chem. 283:15619–27.CrossRefGoogle Scholar
  17. 17.
    Oshiro N, et al. (2007) The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1. J. Biol. Chem. 282:20329–39.CrossRefGoogle Scholar
  18. 18.
    Fonseca BD, Smith EM, Lee VH, MacKintosh C, Proud CG. (2007) PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex. J. Biol. Chem. 282:24514–24.CrossRefGoogle Scholar
  19. 19.
    Williamson DL, Bolster DR, Kimball SR, Jefferson LS. (2006) Time course changes in signaling pathways and protein synthesis in C2C12 myotubes following AMPK activation by AICAR. Am. J. Physiol. Endocrinol. Metab. 291:E80–9.CrossRefGoogle Scholar
  20. 20.
    Frost RA, Lang CH, Gelato MC. (1997) Transient exposure of human myoblasts to tumor necrosis factor-alpha inhibits serum and insulin-like growth factor-I stimulated protein synthesis. Endocrinology 138:4153–9.CrossRefGoogle Scholar
  21. 21.
    Vander Haar E, Lee SI, Bandhakavi S, Griffin TJ, Kim DH. (2007) Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell. Biol. 9:316–23.CrossRefGoogle Scholar
  22. 22.
    Hong-Brown LQ, Brown CR, Huber DS, Lang CH. (2007) Alcohol regulates eukaryotic elongation factor 2 phosphorylation via an AMP-activated protein kinase-dependent mechanism in C2C12 skeletal myocytes. J. Biol. Chem. 282:3702–12.CrossRefGoogle Scholar
  23. 23.
    Giegerich R, Meyer F, Schleiermacher C. (1996) GeneFisher—software support for the detection of postulated genes. Proc. Int. Conf. Intell. Syst. Mol. Biol. 4:68–77.PubMedGoogle Scholar
  24. 24.
    Krawiec BJ, Nystrom GJ, Frost RA, Jefferson LS, Lang CH. (2007) AMP-activated protein kinase agonists increase mRNA content of the muscle-specific ubiquitin ligases MAFbx and MuRF1 in C2C12 cells. Am. J. Physiol. Endocrinol. Metab. 292:E1555–67.CrossRefGoogle Scholar
  25. 25.
    Lang CH, Frost RA, Vary TC. (2008) Acute alcohol intoxication increases REDD1 in skeletal muscle. Alcohol Clin. Exp. Res. 32:796–805.CrossRefGoogle Scholar
  26. 26.
    Das A, Desai D, Pittman B, Amin S, El-Bayoumy K. (2003) Comparison of the chemopreventive efficacies of 1,4-phenylenebis(methylene)selenocyanate and selenium-enriched yeast on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced lung tumorigenesis in A/J mouse. Nutr. Cancer 46:179–85.CrossRefGoogle Scholar
  27. 27.
    Zhivotovsky B, Nicotera P, Bellomo G, Hanson K, Orrenius S. (1993) Ca2+ and endonuclease activation in radiation-induced lymphoid cell death. Exp. Cell. Res. 207:163–70.CrossRefGoogle Scholar
  28. 28.
    Srinivas V, Bohensky J, Shapiro IM. (2009) Autophagy: a new phase in the maturation of growth plate chondrocytes is regulated by HIF, mTOR and AMP kinase. Cells Tissues Organs 189:88–92.CrossRefGoogle Scholar
  29. 29.
    Zeng M, Zhou JN. (2008) Roles of autophagy and mTOR signaling in neuronal differentiation of mouse neuroblastoma cells. Cell Signal 20:659–65.CrossRefGoogle Scholar
  30. 30.
    Shaw RJ, Cantley LC. (2006) Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 441:424–30.CrossRefGoogle Scholar
  31. 31.
    Fingar DC, et al. (2004) mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E. Mol. Cell. Biol. 24:200–16.CrossRefGoogle Scholar
  32. 32.
    Fingar DC, Salama S, Tsou C, Harlow E, Blenis J. (2002) Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes. Dev. 16:1472–87.CrossRefGoogle Scholar
  33. 33.
    Madhunapantula SV, Sharma A, Robertson GP. (2007) PRAS40 deregulates apoptosis in malignant melanoma. Cancer Res. 67:3626–36.CrossRefGoogle Scholar
  34. 34.
    Thedieck K, et al. (2007) PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis. PLoS One 2:e1217.CrossRefGoogle Scholar
  35. 35.
    Sartori R, et al. (2009) Smad2 and 3 transcription factors control muscle mass in adulthood. Am. J. Physiol. Cell. Physiol. 296:C1248–57.CrossRefGoogle Scholar
  36. 36.
    Welle S, Burgess K, Thornton CA, Tawil R. (2009) Relation between extent of myostatin depletion and muscle growth in mature mice. Am. J. Physiol. Endocrinol. Metab. 297:935–40.CrossRefGoogle Scholar
  37. 37.
    Hentges KE, etal. (2001) FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse. Proc. Natl. Acad. Sci. U. S. A. 98:13796–801.CrossRefGoogle Scholar
  38. 38.
    Zhang LH, Lin FR. (2009) Effect of rapamycin on leukemia cell lines [in Chinese]. Zhongguo Shi Yan Xue Ye Xue Za Zhi 17:870–3.PubMedGoogle Scholar
  39. 39.
    Shafer A, Zhou C, Gehrig PA, Boggess JF, Bae-Jump VL. (2010) Rapamycin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and induction of apoptosis. Int. J. Cancer 126:1144–54.PubMedPubMedCentralGoogle Scholar
  40. 40.
    Rosner M, Fuchs C, Siegel N, Valli A, Hengstschlager M. (2009) Functional interaction of mammalian target of rapamycin complexes in regulating mammalian cell size and cell cycle. Hum. Mol. Genet. 18:3298–310.CrossRefGoogle Scholar
  41. 41.
    Imamura K, Ogura T, Kishimoto A, Kaminishi M, Esumi H. (2001) Cell cycle regulation via p53 phosphorylation by a 5′-AMP activated protein kinase activator, 5-aminoimidazole- 4-carboxamide-1-beta-D-ribofuranoside, in a human hepatocellular carcinoma cell line. Biochem. Biophys. Res. Commun. 287:562–7.CrossRefGoogle Scholar
  42. 42.
    Williamson DL, Butler DC, Alway SE. (2009) AMPK inhibits myoblast differentiation through a PGC-1alpha-dependent mechanism. Am. J. Physiol. Endocrinol. Metab. 297:E304–14.CrossRefGoogle Scholar
  43. 43.
    Deng C, Zhang P, Harper JW, Elledge SJ, Leder P. (1995) Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 82:675–84.CrossRefGoogle Scholar
  44. 44.
    Zhang P, et al. (1999) p21(CIP1) and p57(KIP2) control muscle differentiation at the myogenin step. Genes. Dev. 13:213–24.CrossRefGoogle Scholar
  45. 45.
    Hawke TJ, et al. (2003) p21 is essential for normal myogenic progenitor cell function in regenerating skeletal muscle. Am. J. Physiol. Cell. Physiol. 285:C1019–27.CrossRefGoogle Scholar
  46. 46.
    Lee YJ, et al. (2009) Involvement of a p53-independent and post-transcriptional upregulation for p21WAF/CIP1 following destabilization of the actin cytoskeleton. Int. J. Oncol. 34:581–9.PubMedGoogle Scholar
  47. 47.
    Parker SB, et al. (1995) p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells. Science 267:1024–7.CrossRefGoogle Scholar
  48. 48.
    Mammucari C, Schiaffino S, Sandri M. (2008) Downstream of Akt: FoxO3 and mTOR in the regulation of autophagy in skeletal muscle. Autophagy 4:524–6.CrossRefGoogle Scholar
  49. 49.
    Annovazzi L, et al. (2009) mTOR, S6 and AKT expression in relation to proliferation and apoptosis/autophagy in glioma. Anticancer Res. 29:3087–94.PubMedGoogle Scholar
  50. 50.
    Jung CH, Ro SH, Cao J, Otto NM, Kim DH. (2010) mTOR regulation of autophagy. FEBS Lett. 584:1287–95.CrossRefGoogle Scholar
  51. 51.
    Ravikumar B, et al. (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 36:585–95.CrossRefGoogle Scholar

Copyright information

© The Feinstein Institute for Medical Research 2010

Authors and Affiliations

  1. 1.Department of Cellular and Molecular PhysiologyPennsylvania State University College of MedicineHersheyUSA

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