Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

PRR5L degradation promotes mTORC2-mediated PKC-δ phosphorylation and cell migration downstream of Gα12

Nature Cell Biology volume 14, pages 686–696 (2012)Cite this article

Subjects

Abstract

Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates AGC protein kinases including protein kinase C (PKC) and regulates cellular functions such as cell migration. However, its regulation remains poorly understood. Here we show that lysophosphatidic acid (LPA) induces two phases of PKC-δ hydrophobic motif phosphorylation. The late phase is mediated by Gα12, which specifically activates ARAF, leading to upregulation of the RFFL E3 ubiquitin ligase and subsequent ubiquitylation and degradation of the PRR5L subunit of mTORC2. Destabilization of PRR5L, a suppressor of mTORC2-mediated hydrophobic motif phosphorylation of PKC-δ, but not AKT, results in PKC-δ hydrophobic motif phosphorylation and activation. This Gα12-mediated signalling pathway for mTORC2 regulation is critically important for fibroblast migration and pulmonary fibrosis development.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Gα12 is required for LPA-induced late phase PKC hydrophobic motif phosphorylation and activation.
Figure 2: LPA and Gα12 destabilize PRR5L by inducing PRR5L ubiquitylation.
Figure 3: PRR5L inhibits mTORC2-mediated PKC-δ hydrophobic motif phosphorylation.
Figure 4: PRR5 does not regulate PKC-δ hydrophobic motif phosphorylation.
Figure 5: RFFL is the E3 ligase for PRR5L.
Figure 6: RAF upregulates RFFL expression through MEK and ERK.
Figure 7: The RFFL–PRR5L pathway has an important role in regulation of cell migration.
Figure 8: Gα12 has an important role in bleomycin-induced pulmonary fibrosis.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Polak, P. & Hall, M. N. mTOR and the control of whole body metabolism. Curr. Opin. Cell Biol. 21, 209–218 (2009).

    Article  CAS  Google Scholar 

  2. Wullschleger, S., Loewith, R. & Hall, M. N. TOR signaling in growth and metabolism. Cell 124, 471–484 (2006).

    Article  CAS  Google Scholar 

  3. Zoncu, R., Efeyan, A. & Sabatini, D. M. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 12, 21–35 (2011).

    Article  CAS  Google Scholar 

  4. Foster, K. G. & Fingar, D. C. Mammalian target of rapamycin (mTOR): conducting the cellular signaling symphony. J. Biol. Chem. 285, 14071–14077 (2010).

    Article  CAS  Google Scholar 

  5. Huang, J. & Manning, B. D. A complex interplay between Akt, TSC2 and the two mTOR complexes. Biochem. Soc. Trans. 37, 217–222 (2009).

    Article  CAS  Google Scholar 

  6. Fonseca, B. D., Smith, E. M., Lee, V. H., MacKintosh, C. & Proud, C. G. PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex. J. Biol. Chem. 282, 24514–24524 (2007).

    Article  CAS  Google Scholar 

  7. Hara, K. et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110, 177–189 (2002).

    Article  CAS  Google Scholar 

  8. Kim, D. H. et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110, 163–175 (2002).

    Article  CAS  Google Scholar 

  9. Kim, D. H. et al. GβL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol. Cell 11, 895–904 (2003).

    Article  CAS  Google Scholar 

  10. Kovacina, K. S. et al. Identification of a proline-rich Akt substrate as a 14-3-3 binding partner. J. Biol. Chem. 278, 10189–10194 (2003).

    Article  CAS  Google Scholar 

  11. Jacinto, E. et al. SIN1/MIP1 maintains rictor–mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell 127, 125–137 (2006).

    Article  CAS  Google Scholar 

  12. Pearce, L. R. et al. Identification of Protor as a novel Rictor-binding component of mTOR complex-2. Biochem J 405, 513–522 (2007).

    Article  CAS  Google Scholar 

  13. Sarbassov, D. D. et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr. Biol. 14, 1296–1302 (2004).

    Article  CAS  Google Scholar 

  14. Woo, S. Y. et al. PRR5, a novel component of mTOR complex 2, regulates platelet-derived growth factor receptor β expression and signaling. J. Biol. Chem. 282, 25604–25612 (2007).

    Article  CAS  Google Scholar 

  15. Yang, Q., Inoki, K., Ikenoue, T. & Guan, K. L. Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity. Genes Dev. 20, 2820–2832 (2006).

    Article  CAS  Google Scholar 

  16. Jacinto, E. & Lorberg, A. TOR regulation of AGC kinases in yeast and mammals. Biochem. J. 410, 19–37 (2008).

    Article  CAS  Google Scholar 

  17. Mamane, Y., Petroulakis, E., LeBacquer, O. & Sonenberg, N. mTOR, translation initiation and cancer. Oncogene 25, 6416–6422 (2006).

    Article  CAS  Google Scholar 

  18. Tremblay, F. & Marette, A. Amino acid and insulin signaling via the mTOR/p70 S6 kinase pathway. A negative feedback mechanism leading to insulin resistance in skeletal muscle cells. J. Biol. Chem. 276, 38052–38060 (2001).

    CAS  PubMed  Google Scholar 

  19. Um, S. H. et al. Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 431, 200–205 (2004).

    Article  CAS  Google Scholar 

  20. Facchinetti, V. et al. The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C. EMBO J. 27, 1932–1943 (2008).

    Article  CAS  Google Scholar 

  21. Garcia-Martinez, J. M. & Alessi, D. R. mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). Biochem. J. 416, 375–385 (2008).

    Article  CAS  Google Scholar 

  22. Ikenoue, T., Inoki, K., Yang, Q., Zhou, X. & Guan, K. L. Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling. EMBO J. 27, 1919–1931 (2008).

    Article  CAS  Google Scholar 

  23. Loewith, R. et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 10, 457–468 (2002).

    Article  CAS  Google Scholar 

  24. Sarbassov, D. D., Guertin, D. A., Ali, S. M. & Sabatini, D. M. Phosphorylation and regulation of Akt/PKB by the rictor–mTOR complex. Science 307, 1098–1101 (2005).

    Article  CAS  Google Scholar 

  25. Jacinto, E. et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat. Cell Biol. 6, 1122–1128 (2004).

    Article  CAS  Google Scholar 

  26. Schmidt, A., Kunz, J. & Hall, M. N. TOR2 is required for organization of the actin cytoskeleton in yeast. Proc. Natl Acad. Sci. USA 93, 13780–13785 (1996).

    Article  CAS  Google Scholar 

  27. Liu, L., Das, S., Losert, W. & Parent, C. A. mTORC2 regulates neutrophil chemotaxis in a cAMP- and RhoA-dependent fashion. Dev. cell 19, 845–857 (2010).

    Article  CAS  Google Scholar 

  28. Copp, J., Manning, G. & Hunter, T. TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2. Cancer Res. 69, 1821–1827 (2009).

    Article  CAS  Google Scholar 

  29. Soliman, G. A. et al. mTOR Ser-2481 autophosphorylation monitors mTORC-specific catalytic activity and clarifies rapamycin mechanism of action. J. Biol. Chem. 285, 7866–7879 (2010).

    Article  CAS  Google Scholar 

  30. Gan, X., Wang, J., Su, B. & Wu, D. Evidence for direct activation of mTORC2 kinase activity by phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 286, 10998–11002 (2011).

    Article  CAS  Google Scholar 

  31. Zinzalla, V., Stracka, D., Oppliger, W. & Hall, M. N. Activation of mTORC2 by association with the ribosome. Cell 144, 757–768 (2011).

    Article  CAS  Google Scholar 

  32. Oh, W. J. et al. mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide. EMBO J. 29, 3939–3951 (2010).

    Article  CAS  Google Scholar 

  33. Worzfeld, T., Wettschureck, N. & Offermanns, S. G(12)/G(13)-mediated signalling in mammalian physiology and disease. Trends Pharmacol. Sci. 29, 582–589 (2008).

    Article  CAS  Google Scholar 

  34. Riobo, N. A. & Manning, D. R. Receptors coupled to heterotrimeric G proteins of the G12 family. Trends Pharmacol. Sci. 26, 146–154 (2005).

    Article  CAS  Google Scholar 

  35. Choi, J. W. et al. LPA receptors: subtypes and biological actions. Annu. Rev. Pharmacol. Toxicol. 50, 157–186 (2010).

    Article  CAS  Google Scholar 

  36. Karagiosis, S. A., Chrisler, W. B., Bollinger, N. & Karin, N. J. Lysophosphatidic acid-induced ERK activation and chemotaxis in MC3T3-E1 preosteoblastsare independent of EGF receptor transactivation. J. Cell Physiol. 219, 716–723 (2009).

    Article  CAS  Google Scholar 

  37. Herroeder, S. et al. Guanine nucleotide-binding proteins of the G12 family shape immune functions by controlling CD4+ T cell adhesiveness and motility. Immunity 30, 708–720 (2009).

    Article  CAS  Google Scholar 

  38. Gong, H. et al. G protein subunit Gα13 binds to integrin αIIbβ3 and mediates integrin ‘outside-in’ signaling. Science 327, 340–343 (2010).

    Article  CAS  Google Scholar 

  39. Moers, A. et al. G13 is an essential mediator of platelet activation in hemostasis and thrombosis. Nat. Med. 9, 1418–1422 (2003).

    Article  CAS  Google Scholar 

  40. Shan, D. et al. The G protein Gα13 is required for growth factor-induced cell migration. Dev. cell 10, 707–718 (2006).

    Article  CAS  Google Scholar 

  41. Radhika, V., Hee Ha, J., Jayaraman, M., Tsim, S. T. & Dhanasekaran, N. Mitogenic signaling by lysophosphatidic acid (LPA) involves Gα12 . Oncogene 24, 4597–4603 (2005).

    Article  CAS  Google Scholar 

  42. Ki, S. H., Choi, M. J., Lee, C. H. & Kim, S. G. Gα12 specifically regulates COX-2 induction by sphingosine 1-phosphate. Role for JNK-dependent PKC-αion and degradation of IκBα. J. Biol. Chem. 282, 1938–1947 (2007).

    Article  CAS  Google Scholar 

  43. Won, H. Y., Min, H. J., Lee, W. H., Kim, S. G. & Hwang, E. S. Gα12 is critical for TCR-induced IL-2 production and differentiation of T helper 2 and T helper 17 cells. Biochem. Biophys. Res. Com. 394, 811–816 (2010).

    Article  CAS  Google Scholar 

  44. Tager, A. M. et al. The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak. Nat. Med. 14, 45–54 (2008).

    Article  CAS  Google Scholar 

  45. Neubert, K. et al. The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis. Nat. Med. 14, 748–755 (2008).

    Article  CAS  Google Scholar 

  46. Janes, M. R. et al. Effective and selective targeting of leukemia cells using a TORC1/2 kinase inhibitor. Nat. Med. 16, 205–213 (2010).

    Article  CAS  Google Scholar 

  47. Coumailleau, F. et al. Over-expression of Rififylin, a new RING finger and FYVE-like domain-containing protein, inhibits recycling from the endocytic recycling compartment. Mol. Biol. Cell 15, 4444–4456 (2004).

    Article  CAS  Google Scholar 

  48. McDonald, E. R. 3rd & El-Deiry, W. S. Suppression of caspase-8- and -10-associated RING proteins results in sensitization to death ligands and inhibition of tumor cell growth. Proc. Natl Acad. Sci. USA 101, 6170–6175 (2004).

    Article  CAS  Google Scholar 

  49. Yang, W. et al. CARPs are ubiquitin ligases that promote MDM2-independent p53 and phospho-p53ser20 degradation. J. Biol. Chem. 282, 3273–3281 (2007).

    Article  CAS  Google Scholar 

  50. Liao, W. et al. CARP-2 is an endosome-associated ubiquitin ligase for RIP and regulates TNF-induced NF-κB activation. Curr. Biol. CB 18, 641–649 (2008).

    Article  CAS  Google Scholar 

  51. Woo, S. Y. et al. PRR5, a novel component of mTOR complex 2, regulates platelet-derived growth factor receptor β expression and signaling. J. Biol. Chem. 282, 25604–25612 (2007).

    Article  CAS  Google Scholar 

  52. Thedieck, K. et al. PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis. PLoS One 2, e1217 (2007).

    Article  Google Scholar 

  53. Pearce, L. R. et al. Identification of Protor as a novel Rictor-binding component of mTOR complex-2. Biochem. J. 405, 513–522 (2007).

    Article  CAS  Google Scholar 

  54. Pearce, L. R., Sommer, E. M., Sakamoto, K., Wullschleger, S. & Alessi, D. R. Protor-1 is required for efficient mTORC2-mediated activation of SGK1 in the kidney. Biochem. J. 436, 169–179 (2011).

    Article  CAS  Google Scholar 

  55. Bai, X. et al. Protein kinase C{δ} deficiency accelerates neointimal lesions of mouse injured artery involving delayed reendothelialization and vasohibin-1 accumulation. Arterioscler. Thromb. Vasc. Biol. 30, 2467–2474 (2010).

    Article  CAS  Google Scholar 

  56. Zhao, C. T. et al. PKCδ regulates cortical radial migration by stabilizing the Cdk5 activator p35. Proc. Natl Acad. Sci. USA 106, 21353–21358 (2009).

    Article  CAS  Google Scholar 

  57. Liu, B. et al. Protein kinase C-δ regulates migration and proliferation of vascular smooth muscle cells through the extracellular signal-regulated kinase 1/2. J. Vasc. Surg. 45, 160–168 (2007).

    Article  Google Scholar 

  58. Chou, W. H. et al. Neutrophil protein kinase C δ as a mediator of stroke-reperfusion injury. J. Clin. Invest. 114, 49–56 (2004).

    Article  CAS  Google Scholar 

  59. Zhang, Y. et al. Different roles of G protein subunits β1 and β2 in neutrophil function revealed by gene expression silencing in primary mouse neutrophils. J. Biol. Chem. 285, 24805–24814 (2010).

    Article  CAS  Google Scholar 

  60. Hart, M. J. et al. Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Gα13 . Science 280, 2112–2114 (1998).

    Article  CAS  Google Scholar 

  61. Kozasa, T. et al. p115 RhoGEF, a GTPase activating protein for Gα12 and Gα13 . Science 280, 2109–2111 (1998).

    Article  CAS  Google Scholar 

  62. Gu, J. L., Muller, S., Mancino, V., Offermanns, S. & Simon, M. I. Interaction of Gα(12) with Gα(13) and Gα(q) signaling pathways. Proc. Natl Acad. Sci. USA 99, 9352–9357 (2002).

    Article  CAS  Google Scholar 

  63. Tager, A. M. et al. Inhibition of pulmonary fibrosis by the chemokine IP-10/CXCL10. Am. J. Respir. Cell Mol. Biol. 31, 395–404 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Su for discussion and the anti-MAPKAP1 antibody, and M. Orsulak for technical help. This work is supported by National Institutes of Health grants (HL070694, HL080706 and CA139395 to D.W. and GM61454 and GM074001 to T.K.) and an Ellison Medical Foundation grant (AG-SS-2190-08 to M.I.S.).

Author information

Authors and Affiliations

  1. Department of Pharmacology and Program in Vascular Biology and Therapeutics, Yale School of Medicine, New Haven, Connecticut 06520, USA

    Xiaoqing Gan, Jiyong Wang, Chen Wang & Dianqing Wu

  2. MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK

    Eeva Sommer & Dario Alessi

  3. Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois 60612, USA

    Tohru Kozasa

  4. Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153, Japan

    Tohru Kozasa

  5. National Cancer institute, National Institutes of Health, Bethesda, Maryland 20892, USA

    Srinivasa Srinivasula

  6. Department of Pharmacology, Max-Planck-Institute for Heart and Lung Research, Ludwigstr. 43, 61231 Bad Nauheim, Germany

    Stefan Offermanns

  7. Department of Pharmacology, University of California at San Diego, La Jolla, California 92093, USA

    Melvin I. Simon

Authors
  1. Xiaoqing Gan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiyong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chen Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eeva Sommer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tohru Kozasa
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Srinivasa Srinivasula
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dario Alessi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stefan Offermanns
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Melvin I. Simon
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Dianqing Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.G. conceived the initial idea, planned and carried out some of the experiments, analysed the data and participated in manuscript preparation. J.W. and C.W. carried out some of the experiments. E.S., T.K., S.S. and D.A. provided experimental materials. S.O. and M.I.S. provided research materials and comments and helped in manuscript preparation. D.W. conceived the initial idea, planed the experiments, analysed the data and prepared the manuscript.

Corresponding author

Correspondence to Dianqing Wu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2714 kb)

Supplementary Table 1

Supplementary Information (XLS 36 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gan, X., Wang, J., Wang, C. et al. PRR5L degradation promotes mTORC2-mediated PKC-δ phosphorylation and cell migration downstream of Gα12. Nat Cell Biol 14, 686–696 (2012). https://doi.org/10.1038/ncb2507

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2507

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing