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:

LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2

Oncogene volume 33, pages 3571–3582 (2014)Cite this article

Subjects

Abstract

Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2ΔEx2) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2ΔEx2 MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2ΔEx2 MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2ΔEx2 MSCs. The decreased viability of Nf2ΔEx2 MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

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
Figure 9

Similar content being viewed by others

References

  1. Rouleau GA, Merel P, Lutchman M, Sanson M, Zucman J, Marineau C et al. Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2. Nature 1993; 363: 515–521.

    Article  CAS  PubMed  Google Scholar 

  2. Asthagiri AR, Parry DM, Butman JA, Kim HJ, Tsilou ET, Zhuang Z et al. Neurofibromatosis type 2. Lancet 2009; 373: 1974–1986.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Baser ME, De Rienzo A, Altomare D, Balsara BR, Hedrick NM, Gutmann DH et al. Neurofibromatosis 2 and malignant mesothelioma. Neurology 2002; 59: 290–291.

    Article  CAS  PubMed  Google Scholar 

  4. Plotkin SR, Stemmer-Rachamimov AO, Barker FG 2nd, Halpin C, Padera TP, Tyrrell A et al. Hearing improvement after bevacizumab in patients with neurofibromatosis type 2. N Engl J Med 2009; 361: 358–367.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kalamarides M, Acosta MT, Babovic-Vuksanovic D, Carpen O, Cichowski K, Evans DG et al. Neurofibromatosis 2011: a report of the Children’s Tumor Foundation annual meeting. Acta Neuropathol 2012; 123: 369–380.

    Article  PubMed  Google Scholar 

  6. Li W, Cooper J, Karajannis MA, Giancotti FG . Merlin: a tumour suppressor with functions at the cell cortex and in the nucleus. EMBO Rep 2012; 13: 204–215.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Stamenkovic I, Yu Q . Merlin, a ‘magic’ linker between extracellular cues and intracellular signaling pathways that regulate cell motility, proliferation, and survival. Curr Protein Pept Sci 2010; 11: 471–484.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pelton PD, Sherman LS, Rizvi TA, Marchionni MA, Wood P, Friedman RA et al. Ruffling membrane, stress fiber, cell spreading and proliferation abnormalities in human schwannoma cells. Oncogene 1998; 17: 2195–2209.

    Article  CAS  PubMed  Google Scholar 

  9. Kissil JL, Johnson KC, Eckman MS, Jacks T . Merlin phosphorylation by p21-activated kinase 2 and effects of phosphorylation on merlin localization. J Biol Chem 2002; 277: 10394–10399.

    Article  CAS  PubMed  Google Scholar 

  10. Shaw RJ, Paez JG, Curto M, Yaktine A, Pruitt WM, Saotome I et al. The Nf2 tumor suppressor, merlin, functions in Rac-dependent signaling. Dev Cell 2001; 1: 63–72.

    Article  CAS  PubMed  Google Scholar 

  11. Xiao GH, Beeser A, Chernoff J, Testa JR . p21-Activated kinase links Rac/Cdc42 signaling to merlin. J Biol Chem 2002; 277: 883–886.

    Article  CAS  PubMed  Google Scholar 

  12. Kissil JL, Wilker EW, Johnson KC, Eckman MS, Yaffe MB, Jacks T . Merlin, the product of the Nf2 tumor suppressor gene, is an inhibitor of the p21-activated kinase, Pak1. Mol Cell 2003; 12: 841–849.

    Article  CAS  PubMed  Google Scholar 

  13. Nakai Y, Zheng Y, MacCollin M, Ratner N . Temporal control of Rac in Schwann cell-axon interaction is disrupted in NF2-mutant schwannoma cells. J Neurosci 2006; 26: 3390–3395.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Thaxton C, Lopera J, Bott M, Fernandez-Valle C . Neuregulin and laminin stimulate phosphorylation of the NF2 tumor suppressor in Schwann cells by distinct protein kinase A and p21-activated kinase-dependent pathways. Oncogene 2008; 27: 2705–2715.

    Article  CAS  PubMed  Google Scholar 

  15. Yi C, Wilker EW, Yaffe MB, Stemmer-Rachamimov A, Kissil JL . Validation of the p21-activated kinases as targets for inhibition in neurofibromatosis type 2. Cancer Res 2008; 68: 7932–7937.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Manetti F . LIM kinases are attractive targets with many macromolecular partners and only a few small molecule regulators. Med Res Rev 2011; 32: 968–998.

    Article  PubMed  Google Scholar 

  17. Yang N, Higuchi O, Ohashi K, Nagata K, Wada A, Kangawa K et al. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 1998; 393: 809–812.

    Article  CAS  PubMed  Google Scholar 

  18. Goyal P, Pandey D, Behring A, Siess W . Inhibition of nuclear import of LIMK2 in endothelial cells by protein kinase C-dependent phosphorylation at Ser-283. J Biol Chem 2005; 280: 27569–27577.

    Article  CAS  PubMed  Google Scholar 

  19. Scott RW, Olson MF . LIM kinases: function, regulation and association with human disease. J Mol Med (Berl) 2007; 85: 555–568.

    Article  CAS  Google Scholar 

  20. Yokoo T, Toyoshima H, Miura M, Wang Y, Iida KT, Suzuki H et al. p57Kip2 regulates actin dynamics by binding and translocating LIM-kinase 1 to the nucleus. J Biol Chem 2003; 278: 52919–52923.

    Article  CAS  PubMed  Google Scholar 

  21. Amano T, Kaji N, Ohashi K, Mizuno K . Mitosis-specific activation of LIM motif-containing protein kinase and roles of cofilin phosphorylation and dephosphorylation in mitosis. J Biol Chem 2002; 277: 22093–22102.

    Article  CAS  PubMed  Google Scholar 

  22. Kaji N, Muramoto A, Mizuno K . LIM kinase-mediated cofilin phosphorylation during mitosis is required for precise spindle positioning. J Biol Chem 2008; 283: 4983–4992.

    Article  CAS  PubMed  Google Scholar 

  23. Sumi T, Hashigasako A, Matsumoto K, Nakamura T . Different activity regulation and subcellular localization of LIMK1 and LIMK2 during cell cycle transition. Exp Cell Res 2006; 312: 1021–1030.

    Article  CAS  PubMed  Google Scholar 

  24. Po’uha ST, Shum MS, Goebel A, Bernard O, Kavallaris M . LIM-kinase 2, a regulator of actin dynamics, is involved in mitotic spindle integrity and sensitivity to microtubule-destabilizing drugs. Oncogene 2010; 29: 597–607.

    Article  PubMed  Google Scholar 

  25. Nikonova AS, Astsaturov I, Serebriiskii IG, Dunbrack RL Jr., Golemis EA . Aurora A kinase (AURKA) in normal and pathological cell division. Cell Mol Life Sci 2013; 70: 661–687.

    Article  CAS  PubMed  Google Scholar 

  26. Barr AR, Gergely F . Aurora-A: the maker and breaker of spindle poles. J Cell Sci 2007; 120: 2987–2996.

    Article  CAS  PubMed  Google Scholar 

  27. Ritchey L, Ottman R, Roumanos M, Chakrabarti R . A functional cooperativity between Aurora A kinase and LIM kinase1: implication in the mitotic process. Cell Cycle 2012; 11: 296–309.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Johnson EO, Chang KH, Ghosh S, Venkatesh C, Giger K, Low PS et al. LIMK2 is a crucial regulator and effector of Aurora-A-kinase-mediated malignancy. J Cell Sci 2012; 125: 1204–1216.

    Article  CAS  PubMed  Google Scholar 

  29. Bagheri-Yarmand R, Mazumdar A, Sahin AA, Kumar R . LIM kinase 1 increases tumor metastasis of human breast cancer cells via regulation of the urokinase-type plasminogen activator system. Int J Cancer 2006; 118: 2703–2710.

    Article  CAS  PubMed  Google Scholar 

  30. Horita Y, Ohashi K, Mukai M, Inoue M, Mizuno K . Suppression of the invasive capacity of rat ascites hepatoma cells by knockdown of Slingshot or LIM kinase. J Biol Chem 2008; 283: 6013–6021.

    Article  CAS  PubMed  Google Scholar 

  31. Okamoto I, Pirker C, Bilban M, Berger W, Losert D, Marosi C et al. Seven novel and stable translocations associated with oncogenic gene expression in malignant melanoma. Neoplasia 2005; 7: 303–311.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. McConnell BV, Koto K, Gutierrez-Hartmann A . Nuclear and cytoplasmic LIMK1 enhances human breast cancer progression. Mol Cancer 2011; 10: 75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Manetti F . Recent findings confirm LIM domain kinases as emerging target candidates for cancer therapy. Curr Cancer Drug Targets 2012; 12: 543–560.

    Article  CAS  PubMed  Google Scholar 

  34. Giovannini M, Robanus-Maandag E, van der Valk M, Niwa-Kawakita M, Abramowski V, Goutebroze L et al. Conditional biallelic Nf2 mutation in the mouse promotes manifestations of human neurofibromatosis type 2. Genes Dev 2000; 14: 1617–1630.

    CAS  PubMed Central  PubMed  Google Scholar 

  35. Ross-Macdonald P, de Silva H, Guo Q, Xiao H, Hung CY, Penhallow B et al. Identification of a nonkinase target mediating cytotoxicity of novel kinase inhibitors. Mol Cancer Ther 2008; 11: 3490–3498.

    Article  Google Scholar 

  36. Hung G, Li X, Faudoa R, Xeu Z, Kluwe L, Rhim JS et al. Establishment and characterization of a schwannoma cell line from a patient with neurofibromatosis 2. Int J Oncol 2002; 20: 475–482.

    CAS  PubMed  Google Scholar 

  37. Arber S, Barbayannis FA, Hanser H, Schneider C, Stanyon CA, Bernard O et al. Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 1998; 393: 805–809.

    Article  CAS  PubMed  Google Scholar 

  38. Bashour AM, Meng JJ, Ip W, MacCollin M, Ratner N . The neurofibromatosis type 2 gene product, merlin, reverses the F-actin cytoskeletal defects in primary human schwannoma cells. Mol Cell Biol 2002; 22: 1150–1157.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sparrow N, Manetti ME, Bott M, Fabianac T, Petrilli A, Bates ML et al. The actin-severing protein cofilin is downstream of neuregulin signaling and is essential for Schwann cell myelination. J Neurosci 2012; 32: 5284–5297.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Cote MC, Lavoie JR, Houle F, Poirier A, Rousseau S, Huot J . Regulation of vascular endothelial growth factor-induced endothelial cell migration by LIM kinase 1-mediated phosphorylation of annexin 1. J Biol Chem 2010; 285: 8013–8021.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Thaxton C, Bott M, Walker B, Sparrow NA, Lambert S, Fernandez-Valle C . Schwannomin/merlin promotes Schwann cell elongation and influences myelin segment length. Mol Cell Neurosci 2011; 47: 1–9.

    Article  CAS  PubMed  Google Scholar 

  42. Iacovelli J, Lopera J, Bott M, Baldwin E, Khaled A, Uddin N et al. Serum and forskolin cooperate to promote G1 progression in Schwann cells by differentially regulating cyclin D1, cyclin E1, and p27Kip expression. Glia 2007; 55: 1638–1647.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr Patrick Wood, The Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL, USA, for vials of cultured human Schwann cells, Dr Maria Elisa Manetti for the Halo-tag NF2 plasmid, Nicklaus Sparrow and Marga Bott for their creation of the Nf2ΔEx2 MSCs and Rashell Hallford for animal husbandry. This work was supported in part by a DHHS/NIH award to CF-V (5R01DC10189), and AP is the recipient of a Young Investigator Award from the Children’s Tumor Foundation.

Author information

Authors and Affiliations

  1. Department of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL, USA

    A Petrilli, A Copik, M Posadas & C Fernández-Valle

  2. Center for Childhood Cancer, Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA

    L-S Chang

  3. Department of Pediatrics, Ohio State University College of Medicine, Columbus, OH, USA

    L-S Chang

  4. Department of Otolaryngology, Ohio State University College of Medicine, Columbus, OH, USA

    D B Welling

  5. Division of Clinical and Translational Research, House Research Institute, Los Angeles, CA, USA

    M Giovannini

Authors
  1. A Petrilli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A Copik
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M Posadas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L-S Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D B Welling
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M Giovannini
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. C Fernández-Valle
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C Fernández-Valle.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Petrilli, A., Copik, A., Posadas, M. et al. LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2. Oncogene 33, 3571–3582 (2014). https://doi.org/10.1038/onc.2013.320

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2013.320

Keywords

This article is cited by

Search

Advanced search

Quick links