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.

  • Original Article
  • Published:

Calpain small-1 modulates Akt/FoxO3A signaling and apoptosis through PP2A

Oncogene volume 28, pages 721–733 (2009)Cite this article

Abstract

Here, we show that FoxO3A transcription factor is upregulated upon calpain small-1 (CAPNS1) depletion both in mouse embryonic fibroblasts (MEFs) and in the human mammary carcinoma cell line MCF-7. On starvation, CAPNS1 depletion is associated with a higher rate of FoxO3A dephosphorylation and translocation to the nucleus and to a sharper increase in the levels of p27Kip1 and Bim, the products of two FoxO target genes. Notably, FoxO3A depletion in CAPNS1−/− MEFs reduces both the induction of Bim and apoptosis. Both okadaic acid treatment and silencing of the protein phosphatase 2A (PP2A) catalytic subunit can partially reduce starvation-induced FoxO3A activation and apoptosis in CAPNS1−/− fibroblasts. PP2A associates more tightly with Akt in CAPNS1 knockout cells, indicating that PP2A is involved in calpain-mediated FoxO regulation. Finally, we show that PP2A regulatory subunits B56 alpha and gamma are in vitro substrates of calpain, and calpain regulates B56 alpha stability in vivo, suggesting a direct role of calpain in the regulation of PP2A function. In conclusion, for the first time we report that CAPNS1 interferes with PP2A-Akt interaction consequently affecting FoxO3A-dependent cell death. Calpain inhibition might therefore be exploited as a tool to induce apoptosis in tumors sensitive to FoxO activation.

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
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

References

  • Altomare DA, Testa JR . (2005). Perturbations of the AKT signaling pathway in human cancer. Oncogene 24: 7455–7464.

    Article  CAS  PubMed  Google Scholar 

  • Andrabi S, Gjoerup OV, Kean JA, Roberts TM, Schaffhausen B . (2007). Protein phosphatase 2A regulates life and death decisions via Akt in a context-dependent manner. Proc Natl Acad Sci USA 104: 19011–19016.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Arthur JS, Elce JS, Hegadorn C, Williams K, Greer PA . (2000). Disruption of the murine calpain small subunit gene, Capn4: calpain is essential for embryonic development but not for cell growth and division. Mol Cell Biol 20: 4474–4481.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Barreyro FJ, Kobayashi S, Bronk SF, Werneburg NW, Malhi H, Gores GJ . (2007). Transcriptional regulation of Bim by FoxO3A mediates hepatocyte lipoapoptosis. J Biol Chem 282: 27141–27154.

    Article  CAS  PubMed  Google Scholar 

  • Biggs III WH, Meisenhelder J, Hunter T, Cavenee WK, Arden KC . (1999). Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci USA 96: 7421–7426.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brummelkamp TR, Bernards R, Agami R . (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550–553.

    Article  CAS  PubMed  Google Scholar 

  • Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS et al. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96: 857–868.

    Article  CAS  PubMed  Google Scholar 

  • Carragher NO, Fonseca BD, Frame MC . (2004). Calpain activity is generally elevated during transformation but has oncogene-specific biological functions. Neoplasia 6: 53–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Darnell GA, Schroder WA, Antalis TM, Lambley E, Major L, Gardner J et al. (2007). Human papillomavirus E7 requires the protease calpain to degrade the retinoblastoma protein. J Biol Chem 282: 37492–37500.

    Article  CAS  PubMed  Google Scholar 

  • Demarchi F, Bertoli C, Copetti T, Eskelinen EL, Schneider C . (2007). Calpain as a novel regulator of autophagosome formation. Autophagy 3: 235–237.

    Article  CAS  PubMed  Google Scholar 

  • Demarchi F, Bertoli C, Copetti T, Tanida I, Brancolini C, Eskelinen EL et al. (2006). Calpain is required for macroautophagy in mammalian cells. J Cell Biol 175: 595–605.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Demarchi F, Bertoli C, Greer PA, Schneider C . (2005). Ceramide triggers an NF-kappaB-dependent survival pathway through calpain. Cell Death Differ 12: 512–522.

    Article  CAS  PubMed  Google Scholar 

  • Dijkers PF, Birkenkamp KU, Lam EW, Thomas NS, Lammers JW, Koenderman L et al. (2002). FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity. J Cell Biol 156: 531–542.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dourdin N, Bhatt AK, Dutt P, Greer PA, Arthur JS, Elce JS et al. (2001). Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts. J Biol Chem 276: 48382–48388.

    Article  CAS  PubMed  Google Scholar 

  • Essafi A, Fernandez de Mattos S, Hassen YA, Soeiro I, Mufti GJ, Thomas NS et al. (2005). Direct transcriptional regulation of Bim by FoxO3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells. Oncogene 24: 2317–2329.

    Article  CAS  PubMed  Google Scholar 

  • Gao T, Furnari F, Newton AC . (2005). PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol Cell 18: 13–24.

    Article  CAS  PubMed  Google Scholar 

  • Goll DE, Thompson VF, Li H, Wei W, Cong J . (2003). The calpain system. Physiol Rev 83: 731–801.

    Article  CAS  PubMed  Google Scholar 

  • Greer EL, Brunet A . (2005). FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24: 7410–7425.

    Article  CAS  PubMed  Google Scholar 

  • Guan N, Korukonda R, Hurh E, Schmittgen TD, Donkor IO, Dalton JT . (2006). Apoptosis induced by novel aldehyde calpain inhibitors in human tumor cell lines. Int J Oncol 29: 655–663.

    CAS  PubMed  Google Scholar 

  • Harriman JF, Liu XL, Aleo MD, Machaca K, Schnellmann RG . (2002). Endoplasmic reticulum Ca(2+) signaling and calpains mediate renal cell death. Cell Death Differ 9: 734–741.

    Article  CAS  PubMed  Google Scholar 

  • Janssens V, Goris J . (2001). Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 353: 417–439.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kops GJ, Burgering BM . (1999). Forkhead transcription factors: new insights into protein kinase B (c-akt) signaling. J Mol Med 77: 656–665.

    Article  CAS  PubMed  Google Scholar 

  • Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI et al. (1997). PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275: 1943–1947.

    Article  CAS  PubMed  Google Scholar 

  • Libertini SJ, Robinson BS, Dhillon NK, Glick D, George M, Dandekar S et al. (2005). Cyclin E both regulates and is regulated by calpain 2, a protease associated with metastatic breast cancer phenotype. Cancer Res 65: 10700–10708.

    Article  CAS  PubMed  Google Scholar 

  • Manning BD, Cantley LC . (2007). AKT/PKB signaling: navigating downstream. Cell 129: 1261–1274.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Millward TA, Zolnierowicz S, Hemmings BA . (1999). Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci 24: 186–191.

    Article  CAS  PubMed  Google Scholar 

  • Mumby M . (2007). PP2A: unveiling a reluctant tumor suppressor. Cell 130: 21–24.

    Article  CAS  PubMed  Google Scholar 

  • Nakamura N, Ramaswamy S, Vazquez F, Signoretti S, Loda M, Sellers WR . (2000). Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN. Mol Cell Biol 20: 8969–8982.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Paik JH, Kollipara R, Chu G, Ji H, Xiao Y, Ding Z et al. (2007). FoxOs are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell 128: 309–323.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reagan-Shaw S, Ahmad N . (2006). RNA interference-mediated depletion of phosphoinositide 3-kinase activates forkhead box class O transcription factors and induces cell cycle arrest and apoptosis in breast carcinoma cells. Cancer Res 66: 1062–1069.

    Article  CAS  PubMed  Google Scholar 

  • Resjo S, Goransson O, Harndahl L, Zolnierowicz S, Manganiello V, Degerman E . (2002). Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes. Cell Signal 14: 231–238.

    Article  CAS  PubMed  Google Scholar 

  • Rocher G, Letourneux C, Lenormand P, Porteu F . (2007). Inhibition of B56-containing protein phosphatase 2As by the early response gene IEX-1 leads to control of Akt activity. J Biol Chem 282: 5468–5477.

    Article  CAS  PubMed  Google Scholar 

  • Sablina AA, Chen W, Arroyo JD, Corral L, Hector M, Bulmer SE et al. (2007). The tumor suppressor PP2A Abeta regulates the RalA GTPase. Cell 129: 969–982.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A et al. (2004). Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117: 399–412.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sato S, Fujita N, Tsuruo T . (2000). Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci USA 97: 10832–10837.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Seeling JM, Miller JR, Gil R, Moon RT, White R, Virshup DM . (1999). Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A. Science 283: 2089–2091.

    Article  CAS  PubMed  Google Scholar 

  • Sunters A, Fernandez de Mattos S, Stahl M, Brosens JJ, Zoumpoulidou G, Saunders CA et al. (2003). FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. J Biol Chem 278: 49795–49805.

    Article  CAS  PubMed  Google Scholar 

  • Tan Y, Wu C, De Veyra T, Greer PA . (2006). Ubiquitous calpains promote both apoptosis and survival signals in response to different cell death stimuli. J Biol Chem 281: 17689–17698.

    Article  CAS  PubMed  Google Scholar 

  • Tang ED, Nunez G, Barr FG, Guan KL . (1999). Negative regulation of the forkhead transcription factor FKHR by Akt. J Biol Chem 274: 16741–16746.

    Article  CAS  PubMed  Google Scholar 

  • Trotman LC, Alimonti A, Scaglioni PP, Koutcher JA, Cordon-Cardo C, Pandolfi PP . (2006). Identification of a tumour suppressor network opposing nuclear Akt function. Nature 441: 523–527.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Trotman LC, Niki M, Dotan ZA, Koutcher JA, Di Cristofano A, Xiao A et al. (2003). Pten dose dictates cancer progression in the prostate. PLoS Biol 1: E59.

    Article  PubMed  PubMed Central  Google Scholar 

  • van der Horst A, Burgering BM . (2007). Stressing the role of FoxO proteins in lifespan and disease. Nat Rev Mol Cell Biol 8: 440–450.

    Article  CAS  PubMed  Google Scholar 

  • Van Kanegan MJ, Adams DG, Wadzinski BE, Strack S . (2005). Distinct protein phosphatase 2A heterotrimers modulate growth factor signaling to extracellular signal-regulated kinases and Akt. J Biol Chem 280: 36029–36036.

    Article  CAS  PubMed  Google Scholar 

  • Xu L, Deng X . (2006). Suppression of cancer cell migration and invasion by protein phosphatase 2A through dephosphorylation of mu- and m-calpains. J Biol Chem 281: 35567–35575.

    Article  CAS  PubMed  Google Scholar 

  • Yan L, Lavin VA, Moser LR, Cui Q, Kanies C, Yang E . (2008). PP2A regulates the pro-apoptotic activity of FOXO1. J Biol Chem 283: 7411–7420.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yin KJ, Hsu CY, Hu XY, Chen H, Chen SW, Xu J et al. (2006). Protein phosphatase 2A regulates bim expression via the Akt/FKHRL1 signaling pathway in amyloid-beta peptide-induced cerebrovascular endothelial cell death. J Neurosci 26: 2290–2299.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • You H, Pellegrini M, Tsuchihara K, Yamamoto K, Hacker G, Erlacher M et al. (2006). FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal. J Exp Med 203: 1657–1663.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zatz M, Starling A . (2005). Calpains and disease. N Engl J Med 352: 2413–2423.

    Article  CAS  PubMed  Google Scholar 

  • Zhang X, Gan L, Pan H, Guo S, He X, Olson ST et al. (2002). Phosphorylation of serine 256 suppresses transactivation by FKHR (FOXO1) by multiple mechanisms. Direct and indirect effects on nuclear/cytoplasmic shuttling and DNA binding. J Biol Chem 277: 45276–45284.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We acknowledge Silvano Piazza for help in statistics. CB was a recipient of an FIRC (Fondazione italiana per la ricerca sul cancro) fellowship. This study was supported by PRIN (Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale) 2006-prot. 2006053543_003, and TRANSFOG (Translational and Functinal Onco-Genomics) EU Contract 503438, AIRC (Associazione italiana per la ricerca sul cancro) Grant proposal 2007 Cod. 4752, and Progetto FAR CT cod. MIUR prog. 10036.

Author information

Authors and Affiliations

  1. Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), Trieste, Italy

    C Bertoli, T Copetti, F Demarchi & C Schneider

  2. Department of Oncology, Cancer Research-United Kingdom Laboratories, Imperial College London, Hammersmith Hospital, London, UK

    E W-F Lam

  3. Dipartimento di Scienze e Tecnologie Biomediche, Universita’ degli Studi di Udine, Udine, Italy

    C Schneider

Authors
  1. C Bertoli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T Copetti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E W-F Lam
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F Demarchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C Schneider
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to F Demarchi or C Schneider.

Additional information

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bertoli, C., Copetti, T., Lam, EF. et al. Calpain small-1 modulates Akt/FoxO3A signaling and apoptosis through PP2A. Oncogene 28, 721–733 (2009). https://doi.org/10.1038/onc.2008.425

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2008.425

Keywords

This article is cited by

Search

Advanced search

Quick links