Content-Length: 373084 | pFad | https://doi.org/10.1038%2F35070019

ma=86400 Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing | Nature Cell Biology
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:

Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing

Nature Cell Biology volume 3, pages 346–352 (2001)Cite this article

Abstract

Increased phosphorylation of myosin light chain (MLC) is necessary for the dynamic membrane blebbing that is observed at the onset of apoptosis. Here we identify ROCK I, an effector of the small GTPase Rho, as a new substrate for caspases. ROCK I is cleaved by caspase-3 at a conserved DETD1113/G sequence and its carboxy-terminal inhibitory domain is removed, resulting in deregulated and constitutive kinase activity. ROCK proteins are known to regulate MLC-phosphorylation, and apoptotic cells exhibit a gradual increase in levels of phosphorylated MLC concomitant with ROCK I cleavage. This phosphorylation, as well as membrane blebbing, is abrogated by inhibition of caspases or ROCK proteins, but both processes are independent of Rho activity. We also show that expression of active truncated ROCK I induces cell blebbing. Thus, activation of ROCK I by caspase-3 seems to be responsible for bleb formation in apoptotic cells.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: ROCK is cleaved by caspases during apoptosis.
Figure 2: Caspase cleavage removes a C-terminal domain from ROCK I.
Figure 3: Caspase-3 is required for cleavage of ROCK.
Figure 4: ROCK I is cleaved at Asp 1113.
Figure 5: Apoptotic cell blebbing is abrogated by inactivation of ROCK or inhibition of capases.
Figure 6: The Mr 130K kinase fragment of ROCK I binds to activated Rho but its activity is Rho-independent.
Figure 7: MLC is phosphorylated concomitantly with ROCK cleavage in a ROCK-dependent, caspase-dependent, Rho-independent manner.
Figure 8: Expression of active ROCK I induces membrane blebbing.

Similar content being viewed by others

References

  1. Rao, L., Perez, D. & White, E. Lamin proteolysis facilitates nuclear events during apoptosis. J. Cell. Biol. 135, 1441–1455 (1996).

    Article  CAS  Google Scholar 

  2. Enari, M. et al. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43–50 (1998) (published erratum Nature 393, 396).

    Article  CAS  Google Scholar 

  3. Liu, X., Zou, H., Slaughter, C. & Wang, X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89, 175–184 (1997).

    Article  CAS  Google Scholar 

  4. Rudel, T. & Bokoch, G. M. Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 276, 1571–1574 (1997).

    Article  CAS  Google Scholar 

  5. Lee, N. et al. Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis. Proc. Natl Acad. Sci. USA 94, 13642–13647 (1997).

    Article  CAS  Google Scholar 

  6. Rudolf, E., Peychl, J., Novak, J. & Cervinka, M. Apoptosis — when the cells begin to dance. Front. Biosci. 5, F1–F2 (2000).

    Article  CAS  Google Scholar 

  7. Kerr, J. F., Winterford, C. M. & Harmon, B. V. Apoptosis. Its significance in cancer and cancer therapy. Cancer 73, 2013–2026 (1994) (published erratum Cancer 73, 3108).

    Article  CAS  Google Scholar 

  8. Majno, G. & Joris, I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am. J. Pathol. 146, 3–15 (1995).

    CAS  Google Scholar 

  9. Zheng, T. S. et al. Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo. Proc. Natl Acad. Sci. USA 95, 13618–13623 (1998).

    Article  CAS  Google Scholar 

  10. Janicke, R. U., Ng, P., Sprengart, M. L. & Porter, A. G. Caspase-3 is required for alpha-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis. J. Biol. Chem. 273, 15540–15545 (1998).

    Article  CAS  Google Scholar 

  11. Laster, S. M. & Mackenzie, J. M. Jr Bleb formation and F-actin distribution during mitosis and tumor necrosis factor-induced apoptosis. Microsc. Res. Tech. 34, 272–280 (1996).

    Article  CAS  Google Scholar 

  12. Mills, J. C., Stone, N. L., Erhardt, J. & Pittman, R. N. Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J. Cell. Biol. 140, 627–636 (1998).

    Article  CAS  Google Scholar 

  13. Huot, J. et al. SAPK2/p38-dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis. J. Cell. Biol. 143, 1361–1373 (1998).

    Article  CAS  Google Scholar 

  14. Mills, J. C., Stone, N. L. & Pittman, R. N. Extranuclear apoptosis. The role of the cytoplasm in the execution phase. J. Cell. Biol. 146, 703–708 (1999).

    Article  CAS  Google Scholar 

  15. Kohama, K., Ye, L. H., Hayakawa, K. & Okagaki, T. Myosin light chain kinase: an actin-binding protein that regulates an ATP-dependent interaction with myosin. Trends Pharmacol. Sci. 17, 284–287 (1996).

    Article  CAS  Google Scholar 

  16. Alessi, D., MacDougall, L. K., Sola, M. M., Ikebe, M. & Cohen, P. The control of protein phosphatase-1 by targetting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. Eur. J. Biochem. 210, 1023–1035 (1992).

    Article  CAS  Google Scholar 

  17. Amano, M. et al. Phosphorylation and activation of myosin by Rho-associated kinase (Rho–kinase). J. Biol. Chem. 271, 20246–20249 (1996).

    Article  CAS  Google Scholar 

  18. Kimura, K. et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho–kinase). Science 273, 245–248 (1996).

    Article  CAS  Google Scholar 

  19. Kawano, Y. et al. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho–kinase in vivo. J. Cell. Biol. 147, 1023–1038 (1999).

    Article  CAS  Google Scholar 

  20. Ishizaki, T. et al. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J. 15, 1885–1893 (1996).

    Article  CAS  Google Scholar 

  21. Leung, T., Manser, E., Tan, L. & Lim, L. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J. Biol. Chem. 270, 29051–29054 (1995).

    Article  CAS  Google Scholar 

  22. Leung, T., Chen, X. Q., Manser, E. & Lim, L. The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol. Cell. Biol. 16, 5313–5327 (1996).

    Article  CAS  Google Scholar 

  23. Janicke, R. U., Sprengart, M. L., Wati, M. R. & Porter, A. G. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J. Biol. Chem. 273, 9357–9360 (1998).

    Article  CAS  Google Scholar 

  24. Scaffidi, C., Krammer, P. H. & Peter, M. E. Isolation and analysis of components of CD95 (APO-1/Fas) death-inducing signaling complex. Methods 17, 287–291 (1999).

    Article  CAS  Google Scholar 

  25. Ishizaki, T. et al. p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions. FEBS Lett. 404, 118–124 (1997).

    Article  CAS  Google Scholar 

  26. Uehata, M. et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389, 990–994 (1997).

    Article  CAS  Google Scholar 

  27. Itoh, K. et al. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nature Med. 5, 221–225 (1999).

    Article  CAS  Google Scholar 

  28. Klages, B., Brandt, U., Simon, M. I., Schultz, G. & Offermanns, S. Activation of G12/G13 results in shape change and Rho/Rho–kinase-mediated myosin light chain phosphorylation in mouse platelets. J. Cell. Biol. 144, 745–754 (1999).

    Article  CAS  Google Scholar 

  29. Essler, M., Staddon, J. M., Weber, P. C. & Aepfelbacher, M. Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of Rho/Rho kinase signaling. J. Immunol. 164, 6543–6549 (2000).

    Article  CAS  Google Scholar 

  30. Ishizaki, T. et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol. Pharmacol. 57, 976–983 (2000).

    CAS  Google Scholar 

  31. Sekine, A., Fujiwara, M. & Narumiya, S. Asparagine residue in the rho gene product is the modification site for botulinum ADP–ribosyltransferase. J. Biol. Chem. 264, 8602–8605 (1989).

    CAS  Google Scholar 

  32. Ridley, A. J. & Hall, A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389–399 (1992).

    Article  CAS  Google Scholar 

  33. Nagahara, H. et al. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nature Med. 4, 1449–1452 (1998).

    Article  CAS  Google Scholar 

  34. Hirose, M. et al. Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells. J. Cell. Biol. 141, 1625–1636 (1998).

    Article  CAS  Google Scholar 

  35. Hall, A. Rho GTPases and the actin cytoskeleton. Science 279, 509–514 (1998).

    Article  CAS  Google Scholar 

  36. McCarthy, N. J., Whyte, M. K., Gilbert, C. S. & Evan, G. I. Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J. Cell. Biol. 136, 215–227 (1997).

    Article  CAS  Google Scholar 

  37. Katoh, H. et al. Constitutively active α12, Gα13, and Gαq induce Rho-dependent neurite retraction through different signaling pathways. J. Biol. Chem. 273, 28700–28707 (1998).

    Article  CAS  Google Scholar 

  38. Rao, J. Y. et al. Alterations of the actin polymerization status as an apoptotic morphological effector in HL-60 cells. J. Cell Biochem. 75, 686–697 (1999).

    Article  CAS  Google Scholar 

  39. Ohashi, K. et al. Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop. J. Biol. Chem. 275, 3577–35782 (2000).

    Article  CAS  Google Scholar 

  40. Ratcliffe, M. J., Smales, C. & Staddon, J. M. Dephosphorylation of the catenins p120 and p100 in endothelial cells in response to inflammatory stimuli. Biochem. J. 338, 471–478 (1999).

    Article  CAS  Google Scholar 

  41. Zapata, J. M., Takahashi, R., Salvesen, G. S. & Reed, J. C. Granzyme release and caspase activation in activated human T-lymphocytes. J. Biol. Chem. 273, 6916–6920 (1998).

    Article  CAS  Google Scholar 

  42. Chardin, P. et al. The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. EMBO J. 8, 1087–1092 (1989).

    Article  CAS  Google Scholar 

  43. Lang, P. et al. Characterization of a monoclonal antibody specific for the Ras-related GTP-binding protein Rho A. Biochem. Biophys. Res. Commun. 196, 1522–1528 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Zugaza for comments and suggestions. This work was supported by INSERM and by grants from the Association pour la Recherche sur le Cancer. M.S. is supported by a fellowship from ARERS.

Author information

Authors and Affiliations

  1. INSERM U461, Faculté de Pharmacie, Châtenay-Malabry, 92296, France

    Michaël Sebbagh, Claire Renvoizé, Jocelyne Hamelin, Nicole Riché, Jacques Bertoglio & Jacqueline Bréard

Authors
  1. Michaël Sebbagh
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Claire Renvoizé
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Jocelyne Hamelin
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Nicole Riché
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Jacques Bertoglio
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Jacqueline Bréard
    View author publications

    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence to Jacqueline Bréard.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sebbagh, M., Renvoizé, C., Hamelin, J. et al. Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat Cell Biol 3, 346–352 (2001). https://doi.org/10.1038/35070019

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35070019

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








ApplySandwichStrip

pFad - (p)hone/(F)rame/(a)nonymizer/(d)eclutterfier!      Saves Data!


--- a PPN by Garber Painting Akron. With Image Size Reduction included!

Fetched URL: https://doi.org/10.1038%2F35070019

Alternative Proxies:

Alternative Proxy

pFad Proxy

pFad v3 Proxy

pFad v4 Proxy