Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Actin Dysfunction Induces Cell Cycle Delay at G2/M with Sustained ERK and RSK Activation in IMR-90 Normal Human Fibroblasts

  • Shrestha, Deepmala (Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University) ;
  • Choi, Daeun (Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University) ;
  • Song, Kiwon (Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University)
  • Received : 2017.10.24
  • Accepted : 2018.02.12
  • Published : 2018.05.31
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Abstract

The actin cytoskeleton plays a key role in the entry of mitosis as well as in cytokinesis. In a previous study, we showed that actin disruption delays mitotic entry at G2/M by sustained activation of extracellular signal-related kinase 1/2 (ERK1/2) in primary cells but not in transformed cancer cell lines. Here, we examined the mechanism of cell cycle delay at G2/M by actin dysfunction in IMR-90 normal human fibroblasts. We observed that de-polymerization of actin with cytochalasin D (CD) constitutively activated ribosomal S6 kinase (RSK) and induced inhibitory phosphorylation of Cdc2 (Tyr 15) in IMR-90 cells. In the presence of an actin defect in IMR-90 cells, activating phosphorylation of Wee1 kinase (Ser 642) and inhibitory phosphorylation of Cdc25C (Ser 216) was also maintained. However, when kinase-dead RSK (DN-RSK) was overexpressed, we observed sustained activation of ERK1/2, but no delay in the G2/M transition, demonstrating that RSK functions downstream of ERK in cell cycle delay by actin dysfunction. In DN-RSK overexpressing IMR-90 cells treated with CD, phosphorylation of Cdc25C (Ser 216) was blocked and phosphorylation of Cdc2 (Tyr 15) was decreased, but the phosphorylation of Wee1 (Ser 642) was maintained, demonstrating that RSK directly controls phosphorylation of Cdc25C (Ser 216), but not the activity of Wee1. These results strongly suggest that actin dysfunction in primary cells activates ERK1/2 to inhibit Cdc2, delaying the cell cycle at G2/M by activating downstream RSK, which phosphorylates and blocks Cdc25C, and by directly activating Wee1.

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References

  1. Alphine, A.E., Stewart, S.A., Assoian, R.K., and Juliano, R.L. (2001). Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1. J. Cell Biol. 153, 273-282. https://doi.org/10.1083/jcb.153.2.273
  2. Asano, E., Maeda, M., Hasegawa, H., Ito, S., Hyodo, T., Yuan, H., Takahashi, M., Hamaguchi, M., and Senga, T. (2011). Role of palladin phosphorylation by extracellular signal-regulated kinase in cell migration. PloS One 6, e29338. https://doi.org/10.1371/journal.pone.0029338
  3. Bulavin, D.V., Higashimoto Y., Demidenko, Z.N., Meek, S., Graves, P., Phillips, C., Zhao, H., Moody, S.A., Appella, E., Piwnica-Worms, H., Fornace A.J., et al. (2003). Dual phosphorylation controls Cdc25 phosphatases and mitotic entry. Nat. Cell Biol. 5, 545-551. https://doi.org/10.1038/ncb994
  4. Carreno, S., Kouranti, I., Glusman, E.S., Fuller, M.T., Echard, A., and Payre, F. (2008). Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells. J. Cell Biol. 180, 739-746. https://doi.org/10.1083/jcb.200709161
  5. Chun, J., Chau, A.S., Maingat, F.G., Edmonds, S.D., Ostergaard, H.L., and Shibuya, E.K. (2005). Phosphorylation of Cdc25C by p90RSK contributes to the G2 cell cycle arrest in Xenopus Cycling Egg extracts. Cell Cycle 4, 148-154. https://doi.org/10.4161/cc.4.1.1323
  6. Cukier, I.H., Li, Y., and Lee, J.M. (2007). Cyclin B1/Cdk1 binds and phosphorylates Filamin A and regulates its ability to cross-link actin. FEBS Lett. 581, 1661-1672. https://doi.org/10.1016/j.febslet.2007.03.041
  7. Fujibuchi, T., Abe, Y., Takeuchi, T., Imai, Y., Kamei, Y., Murase, R., Ueda, N., Shigemoto, K., Yamamoto, H., Kito, K., et al. (2005). AIP1/WDR1 supports mitotic cell rounding. Biochem. Biophys. Res. Commun. 327, 268-275. https://doi.org/10.1016/j.bbrc.2004.11.156
  8. Kaji, N., Muramoto, A., and Mizuno, K. (2008). LIM-kinase-mediated cofilin phosphorylation during mitosis is required for precise spindle positioning. J. Biol. Chem. 283, 4983-4992. https://doi.org/10.1074/jbc.M708644200
  9. Kunda, P., Baum, B. (2009). The actin cytoskeleton in spindle assembly and positioning. Trends Cell. Biol. 19, 174-179. https://doi.org/10.1016/j.tcb.2009.01.006
  10. Kunda, P., Pelling, A.E., Liu, T., and Baum, B. (2008). Moesin controls cortical rigidity, cell rounding, and spindle morphogenesis during mitosis. Curr. Biol. 18, 91-101. https://doi.org/10.1016/j.cub.2007.12.051
  11. Lancaster, O.M., and Baum, B. (2014). Shaping up to divide: coordinating actin and microtubule cytoskeletal remodelling during mitosis. Semin. Cell Dev. Biol. 34, 109-115. https://doi.org/10.1016/j.semcdb.2014.02.015
  12. Lee, K., and Song, K. (2007). Actin dysfunction activates ERK1/2 and delays entry into mitosis in mammalian cells. Cell Cycle 6, 1487-1495.
  13. Leinweber, B.D., Leavis, P.C., Grabarek, Z., Wang, C.L., and Morgan, K.G. (1999). Extracellular regulated kinase (ERK) interaction with actin and the calponin homology (CH) domain of actin-binding proteins. Biochem. J. 344, 117-123. https://doi.org/10.1042/bj3440117
  14. Maddox, A.S., and Burridge, K. (2003). RhoA is required for cortical retraction and rigidity during mitotic cell rounding. J. Cell Biol. 160, 255-265. https://doi.org/10.1083/jcb.200207130
  15. Martin-Encabo, S., Santos, E., and Guerrero, C. (2007). C3G mediated suppression of malignant transformation involves activation of PP2A phosphatases at the subcortical actin cytoskeleton. Exp Cell Res. 313, 3881-3891. https://doi.org/10.1016/j.yexcr.2007.07.036
  16. Nam, H.J., Lee, I.J., Jang, S., Bae, C.D., Kwak, S.J., and Lee, J.H. (2014). p90 ribosomal S6 kinase 1 (RSK1) isoenzyme specifically regulates cytokinesis progression. Cell Signal. 26, 208-219. https://doi.org/10.1016/j.cellsig.2013.11.014
  17. Ohta, Y., and Hartwig, J.H. (1996). Phosphorylation of actin-binding protein 280 by growth factors is mediated by p90 ribosomal protein S6 kinase. J. Biol. Chem. 271, 11858-11864. https://doi.org/10.1074/jbc.271.20.11858
  18. Peng, C.Y., Graves, P.R., Thomas, R.S., Wu, Z., Shaw, A.S., and Piwnica-Worms, H. (1997). Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science 277, 1501-1505. https://doi.org/10.1126/science.277.5331.1501
  19. Perry, J.A., and Kornbluth, S. (2007). Cdc25 and Wee1: analogous opposites? Cell Div. 2, 12. https://doi.org/10.1186/1747-1028-2-12
  20. Rajeshkumar, N.V., De Oliveira, E., Ottenhof, N., Watters, J., Brooks, D., Demuth, T., Shumway, S.D., Mizuarai, S., Hirai, H., Maitra, A., et al. (2011). MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts. Clin. Cancer Res. 17, 2799-2806. https://doi.org/10.1158/1078-0432.CCR-10-2580
  21. Rosenblatt, J., Cramer, L.P., Baum, B., and McGee, K.M. (2004). Myosin II dependent cortical movement is required for centrosome separation and positioning during mitotic spindle assembly. Cell 117, 361-372. https://doi.org/10.1016/S0092-8674(04)00341-1
  22. Sanchez, Y., Wong, C., Thomas, R.S., Richman, R., Wu, Z., Piwnica-Worms, H., and Elledge, S.J. (1997). Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science 277, 1497-1501. https://doi.org/10.1126/science.277.5331.1497
  23. Shechter, D., Dormann, H.L., Allis, C.D., and Hake, S.B. (2007). Extraction, purification and analysis of histones. Nat. Protoc. 2, 1445-1457. https://doi.org/10.1038/nprot.2007.202
  24. Telles, E., Gurjar, M., Ganti, K., Gupta, D., and Dalala, S.N. (2011). Filamin A stimulates Cdc25C function and promotes entry into mitosis. Cell Cycle 10, 776-782. https://doi.org/10.4161/cc.10.5.14954
  25. Thery, M., Racine, V., Pepin, A., Piel, M., Chen, Y., Sibarita, J.B., and Bornens, M. (2005). The extracellular matrix guides the orientation of the cell division axis. Nat. Cell Biol. 7, 947-953. https://doi.org/10.1038/ncb1307
  26. Toyoshima, F., and Nishida, E. (2007). Integrin-mediated adhesion orients the spindle parallel to the substratum in an EB1- and myosin X-dependent. EMBO J. 26, 1487-1498. https://doi.org/10.1038/sj.emboj.7601599
  27. Walter, S.A., Guadagno, T.M., and Ferrell, J.E. (1997). Induction of a G2-phase arrest in Xenopus egg extracts by activation of p42 mitogen-activated protein kinase. Mol. Biol. Cell. 8, 2157-2169. https://doi.org/10.1091/mbc.8.11.2157
  28. Watanabe, N., Broome, M, and Hunter, T. (1995). Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle. EMBO J. 14, 1878-1891.
  29. Woo, M.S., Ohta, Y., Rabinovitz, I., Stossel, T.P., and Blenis, J. (2004). Ribosomal S6 kinase (RSK) regulates phosphorylation of filamin A on an important regulatory site. Mol. Cell Biol. 24, 3025-3035. https://doi.org/10.1128/MCB.24.7.3025-3035.2004

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