Investigation of MEK activity in COS7 cells entering mitosis

Shi,Huaiping; Zhang,Tianying; Yi,Yongqing; Luo,Jun

doi:10.3892/mmr.2014.2590

Investigation of MEK activity in COS7 cells entering mitosis

Authors:
- Huaiping Shi
- Tianying Zhang
- Yongqing Yi
- Jun Luo
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Affiliations: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P.R. China
Published online on: September 22, 2014 https://doi.org/10.3892/mmr.2014.2590
Pages: 3163-3168

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Abstract

Although the mitogen-activated protein kinase (MAPK) pathway has been extensively investigated, numerous events remain unclear. In the present study, we examined mitogen-activated protein kinase kinase (MEK) expression from interphase to mitosis. Following nocodazole treatment, COS7 cells gradually became round as early as 4 h after treatment. Cyclin B1 expression gradually increased from 4 to 24 h in the presence of nocodazole. When cells were treated with nocodazole for 4 h, the level of epidermal growth factor (EGF)-mediated MEK phosphorylation did not significantly change between nocodazole-untreated and -treated (4 h) cells (P>0.05). However, EGF-mediated MEK phosphorylation was significantly inhibited upon treatment with nocodazole for 8 and 24 h compared to nocodazole-untreated cells (P<0.05). MEK phosphorylation levels were comparable between 1, 5, 10 and 50 ng/ml EGF treatments. Phorbol 12-myristic 13-acetate (PMA) did not activate MEK in mitotic cells. Following treatment of COS7 cells at the interphase with AG1478 or U0126, MEK phosphorylation was blocked. In addition, the investigation of the expression of proteins downstream of MEK demonstrated that EGF does not significantly affect the phosphorylation level of extracellular-signal-regulated kinase (ERK), ribosomal protein S6 kinase (RSK) and Elk in mitotic cells (P>0.05). The results showed that MEK expression is gradually inhibited from cell interphase to mitosis, and that MEK downstream signaling is affected by this inhibition, which probably reflects the requirements of cell physiology during mitosis.

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1	Falchook GS, Lewis KD, Infante JR, et al: Activity of the oral MEK inhibitor trametinib in patients with advanced melanoma: a phase 1 dose-escalation trial. Lancet Oncol. 13:782–789. 2012. View Article : Google Scholar : PubMed/NCBI
2	Lavoie JN, L’Allemain G, Brunet A, Muller R and Pouyssegur J: Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J Biol Chem. 271:20608–20616. 1996. View Article : Google Scholar : PubMed/NCBI
3	Weber JD, Raben DM, Phillips PJ and Baldassare JJ: Sustained activation of extracellular-signal-regulated kinase 1 (ERK1) is required for the continued expression of cyclin D1 in G1 phase. Biochem J. 326:61–68. 1997.
4	Kreiner T and Moore HP: Membrane traffic between secretory compartments is differentially affected during mitosis. Cell Regul. 1:415–424. 1990.PubMed/NCBI
5	Berlin RD, Oliver JM and Walter RJ: Surface functions during mitosis I: phagocytosis, pinocytosis and mobility of surface-bound Con A. Cell. 15:327–341. 1978. View Article : Google Scholar : PubMed/NCBI
6	Berlin RD and Oliver JM: Surface functions during mitosis. II Quantitation of pinocytosis and kinetic characterization of the mitotic cycle with a new fluorescence technique. J Cell Biol. 85:660–671. 1980. View Article : Google Scholar
7	Sager PR, Brown PA and Berlin RD: Analysis of transferrin recycling in mitotic and interphase HeLa cells by quantitative fluorescence microscopy. Cell. 39:275–282. 1984. View Article : Google Scholar : PubMed/NCBI
8	Warren G, Featherstone C, Griffiths G and Burke B: Newly synthesized G protein of vesicular stomatitis virus is not transported to the cell surface during mitosis. J Cell Biol. 97:1623–1628. 1983. View Article : Google Scholar
9	Kiyokawa N, Lee EK, Karunagaran D, Lin SY and Hung MC: Mitosis-specific negative regulation of epidermal growth factor receptor, triggered by a decrease in ligand binding and dimerization, can be overcome by overexpression of receptor. J Biol Chem. 272:18656–18665. 1997. View Article : Google Scholar
10	Gomez-Cambronero J: p42-MAP kinase is activated in EGF-stimulated interphase but not in metaphase-arrested HeLa cells. FEBS Lett. 443:126–130. 1999. View Article : Google Scholar : PubMed/NCBI
11	Klein S, Kaszkin M, Barth H and Kinzel V: Signal transduction through epidermal growth factor receptor is altered in HeLa monolayer cells during mitosis. Biochem J. 322:937–946. 1997.PubMed/NCBI
12	Newberry EP and Pike LJ: Cell-cycle-dependent modulation of EGF-receptor-mediated signaling. Biochem Biophys Res Commun. 208:253–259. 1995. View Article : Google Scholar : PubMed/NCBI
13	Choi C and Helfman DM: The Ras-ERK pathway modulates cytoskeleton organization, cell motility and lung metastasis signature genes in MDA-MB-231 LM2. Oncogene. Sep 2–2013.(Epub ahead of print).
14	Cha DS, Datla US, Hollis SE, Kimble J and Lee MH: The Ras-ERK MAPK regulatory network controls dedifferentiation in Caenorhabditis elegans germline. Biochim Biophys Acta. 1823:1847–1855. 2012. View Article : Google Scholar : PubMed/NCBI
15	Yoon S and Seger R: The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors. 24:21–44. 2006. View Article : Google Scholar : PubMed/NCBI
16	Hayne C, Xiang X and Luo Z: MEK inhibition and phosphorylation of serine 4 on B23 are two coincident events in mitosis. Biochem Biophys Res Commun. 321:675–680. 2004. View Article : Google Scholar : PubMed/NCBI
17	Laird AD, Morrison DK and Shalloway D: Characterization of Raf-1 activation in mitosis. J Biol Chem. 274:4430–4439. 1999. View Article : Google Scholar : PubMed/NCBI
18	Ziogas A, Lorenz IC, Moelling K and Radziwill G: Mitotic Raf-1 is stimulated independently of Ras and is active in the cytoplasm. J Biol Chem. 273:24108–24114. 1998. View Article : Google Scholar : PubMed/NCBI
19	Dangi S and Shapiro P: Cdc2-mediated inhibition of epidermal growth factor activation of the extracellular signal-regulated kinase pathway during mitosis. J Biol Chem. 280:24524–24531. 2005. View Article : Google Scholar : PubMed/NCBI
20	Liu L, Shi H, Chen X and Wang Z: Regulation of EGF-stimulated EGF receptor endocytosis during M phase. Traffic. 12:201–217. 2011. View Article : Google Scholar : PubMed/NCBI
21	Margadant C, Cremers L, Sonnenberg A and Boonstra J: MAPK uncouples cell cycle progression from cell spreading and cytoskeletal organization in cycling cells. Cell Mol Life Sci. 70:293–307. 2013. View Article : Google Scholar : PubMed/NCBI
22	Shi H and Shen L: The alteration of extracellular signal-regulated kinase (ERK) activity of COS7 cells from interphase to mitosis. Cell Mol Biol (Noisy-le-grand). 56(Suppl): OL1427–OL1433. 2010.PubMed/NCBI
23	Anderson D, Koch CA, Grey L, Ellis C, Moran MF and Pawson T: Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors. Science. 250:979–982. 1990. View Article : Google Scholar : PubMed/NCBI
24	Moran MF, Koch CA, Anderson D, et al: Src homology region 2 domains direct protein-protein interactions in signal transduction. Proc Natl Acad Sci USA. 87:8622–8626. 1990. View Article : Google Scholar : PubMed/NCBI
25	Koch CA, Anderson D, Moran MF, Ellis C and Pawson T: SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 252:668–674. 1991. View Article : Google Scholar
26	Pawson T: Protein modules and signalling networks. Nature. 373:573–580. 1995. View Article : Google Scholar : PubMed/NCBI
27	Zhao Y, Thomas HD, Batey MA, et al: Preclinical evaluation of a potent novel DNA-dependent protein kinase inhibitor NU7441. Cancer Res. 66:5354–5362. 2006. View Article : Google Scholar : PubMed/NCBI
28	Ma DM, Ji YJ, Yang F, Liu W, Wan Z and Li RY: Effects of U0126 on growth and activation of mitogen-activated protein kinases in Aspergillus fumigatus. Chin Med J (Engl). 126:220–225. 2013.PubMed/NCBI
29	Newton R, Cambridge L, Hart LA, Stevens DA, Lindsay MA and Barnes PJ: The MAP kinase inhibitors, PD098059, UO126 and SB203580, inhibit IL-1beta-dependent PGE(2) release via mechanistically distinct processes. Br J Pharmacol. 130:1353–1361. 2000. View Article : Google Scholar : PubMed/NCBI
30	Nam HJ, Kim S, Lee MW, et al: The ERK-RSK1 activation by growth factors at G2 phase delays cell cycle progression and reduces mitotic aberrations. Cell Signal. 20:1349–1358. 2008. View Article : Google Scholar : PubMed/NCBI
31	Rao VN and Reddy ES: elk-1 proteins interact with MAP kinases. Oncogene. 9:1855–1860. 1994.PubMed/NCBI