Skip to main content
SpringerLink
Log in

Overexpression of ROCK in human breast cancer cells: Evidence that ROCK activity mediates intracellular membrane traffic of lysosomes

  • Article
  • Published:
Pathology Oncology Research

Abstract

Small GTPase Rho and its downstream effectors, ROCK family of Rho-associated serine-threonine kinases, are thought to participate in cell morphology, motility, and tumor progression through regulating the rearrangement of actin cytoskeleton. Here we present evidence that transfection of human breast cancer cells with cDNA encoding a dominant active mutant of ROCK causes dispersal of lysosomal vesicles throughout the cytoplasm without perturbing the machinery of the endocytic pathway. The intracellular distribution of lysosomes and endocytosed transferrin, an early endosomal marker, were further assessed by confocal immunofluorescence microscopy. In the active ROCK transfected cells the lysosomal proteins, cathepsin D, LIMPII, and LAMP1, were found throughout the cytoplasm in dispersed small vesicles, which were not accessible to the endocytosed Texas Red-labeled transferrin. 3D-image analysis of lysosomal distribution in the active ROCK-transfectants revealed abundant punctate signals in the peripheral region of the basal plasma membrane. Cells expressing vector alone did not exhibit these alterations. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, induced LIMPII-positive/transferrin negative large vacuoles in the perinuclear region, and disappearence of the dispersed small vesicular structures. To our knowledge, this is the first evidence that increasing ROCK expression contributes to selective cellular dispersion of lysosomes in invasive breast cancer cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Paterson HF, Self AJ, Garrett MD et al: Microinjection of recombinant p21rho induces rapid changes in cell morphology. J Cell Biol 111: 1001–1007, 1990.

    Article  PubMed  CAS  Google Scholar 

  2. Perona R, Esteve P, Jimene, B et al: Tumorigenic activity of rho genes from Aphysia California. Oncogene 8: 1285–1292, 1993.

    PubMed  CAS  Google Scholar 

  3. Kishi K, Sasaki T, Kuroda S et al: Regulation of cytoplasmic division of Xenopus Embryo by rhop21 and its inhibitory GDP/GTP exchange protein (rhoGDI). J Cell Biol 120: 1187–1195, 1993.

    Article  PubMed  CAS  Google Scholar 

  4. Takaishi K, Kikuchi A, Kuroda S et al; Involvement of rhop21 and its inhibitory GDP/GTP exchange protein (rhoGDI) in cell motility. Mol Cell Biol 13: 72–79, 1993

    PubMed  CAS  Google Scholar 

  5. Hall, A. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Biol 10: 31–54, 1994

    Article  PubMed  CAS  Google Scholar 

  6. Prendergast GC, Khosravi-Far R, Solski PA, et al: Critical role of Rho in cell transformation by oncogenic Ras. Oncogene 10: 2289–2296, 1995

    PubMed  CAS  Google Scholar 

  7. Fritz G, Just I, Kaina B: Rho GTPases are over-expressed in human tumors. Int J Cancer 81: 682–687, 1999

    Article  PubMed  CAS  Google Scholar 

  8. Yoshioka K, Nakamori S, Itoh K: Overexpression of small GTP-binding protein RhoA promotes invasion of tumor cells. Cancer Res 59: 2004–2010, 1999

    PubMed  CAS  Google Scholar 

  9. Yoshioka K, Matsumura F, Akedo H, Itoh K: Small GTP-binding protein Rho stimulates the actomyosin system, leading to invasion of tumor cells. J Biol Chem 273: 5146–5154, 1998.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  11. Itoh K, Yoshioka K, Akedo H et al: An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nature Medicine 5: 221–225, 1999

    Article  PubMed  CAS  Google Scholar 

  12. Sloane BF, Moin K, Sameni M et al: Membrane association of cathepsin B can be induced by transfection of human breast epithelial cells with c-Ha-Ras oncogene. J Cell Sci 107: 373–384, 1994

    PubMed  CAS  Google Scholar 

  13. Sameni M, Elliott E, Ziegler G et al: Cathepsin B and D are localized at the surface of human breast cancer cells. Pathol Oncol Res 1:43–53, 1995

    Article  PubMed  CAS  Google Scholar 

  14. Taniguchi S, Nishimura Y, Takahashi, T et al: Augmented excretion of procathepsin L of a fos-transferred highly metastatic cell line. Biochem Biophys Res Commun 168: 520–526, 1990

    Article  PubMed  CAS  Google Scholar 

  15. Kageshita T, Yoshii A, Kimura T et al: Biochemical and immunohistochemical analysis of cathepsins B, H, L and D in human melanocytic tumours. Arch Dermatol Res 287: 266–272, 1995

    Article  PubMed  CAS  Google Scholar 

  16. Albini A, Graf J, Kitten GT et al: 17β-Estradiol regulates and v-Ha-ras transfection constitutively enhances MCF7 breast cancer cell interactions with basement membrane. Proc Natl Acad Sci US 83: 8182–8186, 1986

    Article  CAS  Google Scholar 

  17. Ichikawa T, Kyprianou N, Isaacs JT: Genetic instability and the acquisition of metastatic ability by rat mammary cancer cells following v-H-ras oncogene transfection. Cancer Res 50: 6349–6357, 1990.

    PubMed  CAS  Google Scholar 

  18. Nishimura Y, Sameni M, Sloane BF: Malignant transformation alters intracellular trafficking of lysosomal cathepsin D in human breast epithelial cells. Pathol Oncol Res 4: 283–296, 1998

    Article  PubMed  CAS  Google Scholar 

  19. Nishimura Y, Itoh K, Yoshioka, K et al: Small guanosine triphosphatase Rho/Rho-associated kinase as a novel regulator of intracellular redistribution of lysosomes in invasive tumor cells. Cell Tissue Res 342: 341–351, 2000.

    Article  Google Scholar 

  20. Uehata M, Ishizaki T, Satoh H et al: A key role for pl60ROCK-mediated Ca++sensitization of smooth muscle in hypertension. Nature 389: 990–994, 1997.

    Article  PubMed  CAS  Google Scholar 

  21. Matsui T, Amano M, Xamamoto T et al: Rho-associated kinase, a novel serine threonine kinase, as a putative target for the small GTP binding protein Rho. EMBO J 15: 2208–2216, 1996

    PubMed  CAS  Google Scholar 

  22. Nakagawa O, Fujisawa K, Ishizaki et al: ROCK-I and ROCK-II; two isoforms of Rho-associated coiled-coil forming protein serine/threonine kinase in mice. FEBS Lett 392: 189–193, 1996

    Article  PubMed  CAS  Google Scholar 

  23. Amano M, Chihara K, Kimur, Y et al: Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 275: 1308–1311, 1997.

    Article  PubMed  CAS  Google Scholar 

  24. Ishizaki T, Naito M, Fujisaw, K et al: pl60ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesion. FEBS Lett 404: 118–124, 1997

    Article  PubMed  CAS  Google Scholar 

  25. Nishimura Y, Itoh K, Xoshioka K, et al: A role for small GTPase RhoA in regulating intracellular membrane traffic of lysosomes in invasive rat hepatoma cells. Histochem J 34: 189–213, 2002.

    Article  PubMed  CAS  Google Scholar 

  26. Lippincott-Schwart J, Fambrough DM: Lysosomal membrane dynamics: structure and interorganellar movement of a major lysosomal membrane glycoprotein. J Cell Biol 102: 1593–1605, 1986.

    Article  Google Scholar 

  27. Griffiths G, Hoflack B, Simon, K et al: The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell 52: 329–341, 1998

    Article  Google Scholar 

  28. Kornfeld S, Mellman I: The biogenesis of lysosomes. Ann Rev Cell Biol 5: 483–525, 1989.

    PubMed  CAS  Google Scholar 

  29. Okazaki I, Himeno M, Ezaki J et al: Purification and characterization of an 85 kDa sialoglycoprotein in rat liver. J Biochem 111: 763–769, 1992.

    PubMed  CAS  Google Scholar 

  30. Sandoval IV, Arredondo JJ, Alcalde J et al: The residues Leu(Ile)475-Ile(Leu)476, contained in the extended carboxyl cytoplasmic tail, are critical for targeting of the resident lysosomal membrane protein LIMPII to lysosomes. J Biol Chem 269: 6622–6631, 1994

    PubMed  CAS  Google Scholar 

  31. Tabuchi N, Akasaki K, Tsuji H: Two acidic amino acid residues, Asp(470) and Glu(471), contained in the carboxyl cytoplasmic tail of a major lysosomal membrane protein, LGP85/LIMPII, are important for its accumulation in secondary lysosomes. Biochem Biophys Res Commun 270: 557–563, 2000.

    Article  PubMed  CAS  Google Scholar 

  32. Soule HD, Vazguez J, Long et al.: A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst 51: 1409–1416, 1973.

    PubMed  CAS  Google Scholar 

  33. Nishimura Y, Higaki M, Kato K: Identification of a precursor form of cathepsin D in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro. Biochem Biophys Res Commun 148: 335–343, 1987

    Article  PubMed  CAS  Google Scholar 

  34. Nishimura Y, Kawabata T, Kato K: Identification of latent procathepsins B and L in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro. Arch Biochem Biophys 261: 64–71, 1988

    Article  PubMed  CAS  Google Scholar 

  35. Matteoni R, Kreis TE: Translocation and clustering of endosomes and lysosomes depends on microtubules. J Cell Biol 105: 1253–1265, 1987

    Article  PubMed  CAS  Google Scholar 

  36. Mu FT, Callaghan JM, Steele-Mortimer O et al: EEAl, an early endosome-associated protein. EEAl is a conserved alpha-helical peripheral membrane protein flanked by cysteine “fingers” and contains a calmodulin-binding IQ motif. J Biol Chem 270: 13503–13511, 1995

    Article  PubMed  CAS  Google Scholar 

  37. Stack JH, Emr SD: Genetic and biochemical studies of protein sorting to the yeast vacuole. Curr Biol 5: 641–646, 1993.

    CAS  Google Scholar 

  38. Brown WJ Emr SD, Plutner H, Balch WE: Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J Cell Biol 130: 797–805, 1995

    Article  Google Scholar 

  39. Davidson H: Wortmannin causes mis-targeting of procathepsin D. Evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes. J Cell Biol 130: 797–805, 1995

    Article  PubMed  CAS  Google Scholar 

  40. Reaves B, Bright NA, Mullock B, Luzio JP: The effect of wortmannin on the localization of lysosomal type I integral membrane glycoproteins suggests a role for phosphoinositide 3-kinase activity in regulating membrane traffic late in the endocytic pathway. J Cell Sci 109: 749–762, 1996

    PubMed  CAS  Google Scholar 

  41. Pfeffer SR: GTP-binding proteins in intracellular transport. Trends Cell Biol 2: 41–46, 1992

    Article  PubMed  CAS  Google Scholar 

  42. Zerial M, Stenmark H: Rab GTPases in vesicular transport. Curr Opin Cell Biol 5: 613–620, 1993

    Article  PubMed  CAS  Google Scholar 

  43. Chavrier P, Goud B: The role of ARF and Rab GTPases in membrane transport. Curr Opin Cell Biol, 11: 466–475, 1999

    Article  PubMed  CAS  Google Scholar 

  44. Pfeffer SR: Transport-vesicle targeting: tethers before SNAREs. Nature Cell Biol 1: E17-E22, 1999.

    Article  PubMed  CAS  Google Scholar 

  45. Bucci C, Thomsen P, Nicoziani P et al: Rab7: A key to lysosome biogenesis. Mol Biol Cell 11: 467–480, 2000

    PubMed  CAS  Google Scholar 

  46. Press B, Feng Y, Hoflack B, Windinger-Nes A: Mutant Rab7 causes the accumulation of cathepsin D and cation-independent mannose 6-phosphate receptor in an early endocytic compartment. J Cell Biol 140: 1075–1089, 1998.

    Article  PubMed  CAS  Google Scholar 

  47. Hirose M, Ishizaki T, Watanabe N et al: Molecular dissection of the Rho-associated kinase (pl60ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells. J Cell Biol 141: 1625–1636, 1998

    Article  PubMed  CAS  Google Scholar 

  48. Hollenbeck PJ, Swanson JA: Radial extension of macrophage tubular lysosomes supported by kinesin. Nature 346: 864–866, 1990

    Article  PubMed  CAS  Google Scholar 

  49. Nakata T, Hirokawa N: Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport. J Cell Biol 131: 1039–1053, 1995

    Article  PubMed  CAS  Google Scholar 

  50. Hotta K, Tanak K, Mino A, et al: Interaction of the Rho family small G proteins with kinectin, an anchoring protein of kinesin motor. Biochem Biophys Res Commun 225: 69–74, 1996

    Article  PubMed  CAS  Google Scholar 

  51. Vignal E, Blangy A, Martin M, et al: Kinectin is a key effector of RhoG microtubule-dependent cellular activity. Mol Cell Biol 21:8022–8034, 2001

    Article  PubMed  CAS  Google Scholar 

  52. Timar J, Tang D, Bazaz R, et al: PKC mediates 12-(S)-HETE-induced cytoskeletal rearrangement in B16a melanoma cells. Cell Motil Cytoskel 26: 49–65, 1993

    Article  CAS  Google Scholar 

  53. Timar J, Bazaz R, Kimler V, et al: Immunomorphological characterization and effects of 12-(S)-HETE on a dynamic intracellular pool of the αIIbβ3-integrin in melanoma cells. J Cell Sci 108: 2175–2186, 1995

    PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Kazuo Tokuda

    Present address: Fuji Gotemba Laboratories, Chugai Pharmaceutical Co, Ltd, Gotemba, Shizuoka, Japan

Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, 812-8582, Fukuoka, Japan

    Yukio Nishimura, Kazuo Tokuda & Masaru Himeno

  2. Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan

    Kazuyuki Itoh & Kiyoko Yoshioka

Authors
  1. Yukio Nishimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kazuyuki Itoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kiyoko Yoshioka
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kazuo Tokuda
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Masaru Himeno
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yukio Nishimura.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nishimura, Y., Itoh, K., Yoshioka, K. et al. Overexpression of ROCK in human breast cancer cells: Evidence that ROCK activity mediates intracellular membrane traffic of lysosomes. Pathol. Oncol. Res. 9, 83–95 (2003). https://doi.org/10.1007/BF03033750

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03033750

Keywords

Search

Navigation