Abstract
LIM-kinase 2 (LIMK2) belongs to the LIMK family of proteins, which comprises LIMK1 and LIMK2. Both proteins regulate actin polymerization through phosphorylation and inactivation of the actin depolymerizing factor cofilin. In this study, we show that the level of LIMK2 protein is increased in neuroblastoma, BE(2)-C cells, selected for resistance to microtubule-destabilizing agents, vincristine and colchicine. However, the level of phosphorylated LIMK1 and LIMK2 was similar in the resistant and parental BE(2)-C cells. In contrast, the level of phospho-cofilin was greatly increased in the drug-resistant cells. Downregulation of LIMK2 expression increases sensitivity of neuroblastoma SH–EP cells to vincristine and vinblastine but not to microtubule-stabilizing agents, while it's overexpression increased its resistance to vincristine. Its vincristine-induced mitotic arrest was moderately inhibited in the LIMK2 knockdown cells, suggesting that the increased drug sensitivity is through an alternative mechanism other then mitotic arrest and apoptosis. Moreover, downregulation of LIMK2 expression induces formation of abnormal mitotic spindles, an effect enhanced in the presence of microtubule-destabilizing agents. LIMK2 is important for normal mitotic spindle formation and altered LIMK2 expression mediates sensitivity to microtubule destabilizing agents. These findings suggest that inhibition of LIMK2 activity may be used for the treatment of tumors resistant to microtubule-destabilizing drugs.
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References
Acevedo K, Li R, Soo P, Suryadinata R, Sarcevic B, Valova VA et al. (2007). The phosphorylation of p25/TPPP by LIM kinase 1 inhibits its ability to assemble microtubules. Exp Cell Res 313: 4091–4106.
Acevedo K, Moussi N, Li R, Soo P, Bernard O . (2006). LIM kinase 2 is widely expressed in all tissues. J Histochem Cytochem 54: 487–501.
Amano T, Tanabe K, Eto T, Narumiya S, Mizuno K . (2001). LIM-kinase 2 induces formation of stress fibres, focal adhesions and membrane blebs, dependent on its activation by Rho-associated kinase-catalysed phosphorylation at threonine-505. Biochem J 354: 149–159.
Ambach A, Saunus J, Konstandin M, Wesselborg S, Meuer SC, Samstag Y . (2000). The serine phosphatases PP1 and PP2A associate with and activate the actin-binding protein cofilin in human T lymphocytes. Eur J Immunol 30: 3422–3431.
Arber S, Barbayannis FA, Hanser H, Schneider C, Stanyon CA, Bernard O et al. (1998). Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393: 805–809.
Bhalla KN . (2003). Microtubule-targeted anticancer agents and apoptosis. Oncogene 22: 9075–9086.
Chakrabarti R, Jones JL, Oelschlager DK, Tapia T, Tousson A, Grizzle WE . (2007). Phosphorylated LIM kinases colocalize with gamma-tubulin in centrosomes during early stages of mitosis. Cell Cycle 6: 2944–2952.
Croft DR, Olson MF . (2006). The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms. Mol Cell Biol 26: 4612–4627.
Dan C, Kelly A, Bernard O, Minden A . (2001). Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin. J Biol Chem 276: 32115–32121.
Dan S, Tsunoda T, Kitahara O, Yanagawa R, Zembutsu H, Katagiri T et al. (2002). An integrated database of chemosensitivity to 55 anticancer drugs and gene expression profiles of 39 human cancer cell lines. Cancer Res 62: 1139–1147.
Davila M, Jhala D, Ghosh D, Grizzle WE, Chakrabarti R . (2007). Expression of LIM kinase 1 is associated with reversible G1/S phase arrest, chromosomal instability and prostate cancer. Mol Cancer 6: 40.
Deschesnes RG, Patenaude A, Rousseau JLC, Fortin JS, Ricard C, Cote M-F et al. (2007). Microtubule-destabilizing agents induce focal adhesion structure disorganization and anoikis in cancer cells. J Pharmacol Exp Ther 320: 853–864.
Don S, Verrills NM, Liaw TY, Liu ML, Norris MD, Haber M et al. (2004). Neuronal-associated microtubule proteins class III beta-tubulin and MAP2c in neuroblastoma: role in resistance to microtubule-targeted drugs. Mol Cancer Ther 3: 1137–1146.
Edwards DC, Sanders LC, Bokoch GM, Gill GN . (1999). Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol 1: 253–259.
Eiseler T, Doppler H, Yan IK, Kitatani K, Mizuno K, Storz P . (2009). Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot. Nat Cell Biol 11: 545–556.
Gohla A, Birkenfeld J, Bokoch GM . (2005). Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamics. Nat Cell Biol 7: 21–29.
Gorovoy M, Niu J, Bernard O, Profirovic J, Minshall R, Neamu R et al. (2005). LIM kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells. J Biol Chem 280: 26533–26542.
Groth-Pedersen L, Ostenfeld MS, Hoyer-Hansen M, Nylandsted J, Jaattela M . (2007). Vincristine induces dramatic lysosomal changes and sensitizes cancer cells to lysosome-destabilizing siramesine. Cancer Res 67: 2217–2225.
Jordan MA, Thrower D, Wilson L . (1991). Mechanism of inhibition of cell proliferation by vinca alkaloids. Cancer Res 51: 2212–2222.
Kaji N, Muramoto A, Mizuno K . (2008). LIM kinase-mediated cofilin phosphorylation during mitosis is required for precise spindle positioning. J Biol Chem 283: 4983–4992.
Kobayashi M, Nishita M, Mishima T, Ohashi K, Mizuno K . (2006). MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration. Embo J 25: 713–726.
LaQuaglia MP, Kopp EB, Spengler BA, Meyers MB, Biedler JL . (1991). Multidrug resistance in human neuroblastoma cells. J Pediatr Surg 26: 1107–1112.
Mizuno K, Okano I, Ohashi K, Nunoue K, Kuma K, Miyata T et al. (1994). Identification of a human cDNA encoding a novel protein kinase with two repeats of the LIM/double zinc finger motif. Oncogene 9: 1605–1612.
Niwa R, Nagata-Ohashi K, Takeichi M, Mizuno K, Uemura T . (2002). Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin. Cell 108: 233–246.
Nunoue K, Ohashi K, Okano I, Mizuno K . (1995). LIMK-1 and LIMK-2, two members of a LIM motif-containing protein kinase family. Oncogene 11: 701–710.
Okano I, Hiraoka J, Otera H, Nunoue K, Ohashi K, Iwashita S et al. (1995). Identification and characterization of a novel family of serine/threonine kinases containing two N-terminal LIM motifs. J Biol Chem 270: 31321–31330.
Peterburs P, Heering J, Link G, Pfizenmaier K, Olayioye MA, Hausser A . (2009). Protein kinase D regulates cell migration by direct phosphorylation of the cofilin phosphatase slingshot 1 like. Cancer Res 69: 5634–5638.
Sarmiere PD, Bamburg JR . (2004). Regulation of the neuronal actin cytoskeleton by ADF/cofilin. J Neurobiol 58: 103–117.
Soosairajah J, Maiti S, Wiggan O, Sarmiere P, Moussi N, Sarcevic B et al. (2005). Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin. Embo J 24: 473–486.
Sumi T, Hashigasako A, Matsumoto K, Nakamura T . (2006). Different activity regulation and subcellular localization of LIMK1 and LIMK2 during cell cycle transition. Exp Cell Res 312: 1021–1030.
Sumi T, Matsumoto K, Nakamura T . (2001). Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase. J Biol Chem 276: 670–676.
Sumi T, Matsumoto K, Takai Y, Nakamura T . (1999). Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2. J Cell Biol 147: 1519–1532.
Verrills NM, Flemming CL, Liu M, Ivery MT, Cobon GS, Norris MD et al. (2003). Microtubule alterations and mutations induced by desoxyepothilone B: implications for drug-target interactions. Chem Biol 10: 597–607.
Verrills NM, Kavallaris M . (2005). Improving the targeting of tubulin-binding agents: lessons from drug resistance studies. Curr Pharm Des 11: 1719–1733.
Verrills NM, Liem NL, Liaw TY, Hood BD, Lock RB, Kavallaris M . (2006a). Proteomic analysis reveals a novel role for the actin cytoskeleton in vincristine resistant childhood leukemia—an in vivo study. Proteomics 6: 1681–1694.
Verrills NM, Po'uha ST, Liu ML, Liaw TY, Larsen MR, Ivery MT et al. (2006b). Alterations in gamma-actin and tubulin-targeted drug resistance in childhood leukemia. J Natl Cancer Inst 98: 1363–1374.
Yang N, Higuchi O, Ohashi K, Nagata K, Wada A, Kangawa K et al. (1998). Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 393: 809–812.
Acknowledgements
We thank Professor Kensaku Mizuno, Tohoku University, Japan, for providing the LIMK2 plasmid and Professor Peter Gunning, University of New South Wales, Sydney, Australia, for γ-actin Ab. This work was supported by the Children's Cancer Institute Australia for Medical Research, which is affiliated with the University of New South Wales and Sydney Children's Hospital, and by grants from the New South Wales Cancer Council (MK). MK is supported by a NHMRC Senior Research Fellowship, STP was supported by an Endeavour International Postgraduate Research Scholarship and MSYS is supported by an Australian Postgraduate Award. OB is a NHMRC Principal Research Fellow.
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Po'uha, S., Shum, M., Goebel, A. et al. LIM-kinase 2, a regulator of actin dynamics, is involved in mitotic spindle integrity and sensitivity to microtubule-destabilizing drugs. Oncogene 29, 597–607 (2010). https://doi.org/10.1038/onc.2009.367
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DOI: https://doi.org/10.1038/onc.2009.367
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