Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins

Shi,Huaiping; Zhang,Tianying; Yi,Yongqing; Ma,Yue

doi:10.3892/or.2016.4696

Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins

Authors:
- Huaiping Shi
- Tianying Zhang
- Yongqing Yi
- Yue Ma
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Affiliations: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P.R. China
Published online on: March 21, 2016 https://doi.org/10.3892/or.2016.4696
Pages: 3593-3599

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Abstract

Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.

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1	Cha DS, Datla US, Hollis SE, Kimble J and Lee MH: The Ras-ERK MAPK regulatory network controls dedifferen-tiation in Caenorhabditis elegans germline. Biochim Biophys Acta. 1823:1847–1855. 2012. View Article : Google Scholar : PubMed/NCBI
2	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. 33:3668–3676. 2014. View Article : Google Scholar
3	Nishida Y: Function of Raf/MAP kinase cascade in the regulation of cellular proliferation and differentiation. Tanpakushitsu Kakusan Koso. 41(Suppl 12): S1673–S1679. 1996.
4	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
5	Batzer AG, Rotin D, Ureña JM, Skolnik EY and Schlessinger J: Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol Cell Biol. 14:5192–5201. 1994. View Article : Google Scholar : PubMed/NCBI
6	Sasaoka T, Langlois WJ, Leitner JW, Draznin B and Olefsky JM: The signaling pathway coupling epidermal growth factor receptors to activation of p21^ras. J Biol Chem. 269:32621–32625. 1994.PubMed/NCBI
7	Kolch W: Meaningful relationships: The regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J. 351:289–305. 2000. View Article : Google Scholar : PubMed/NCBI
8	Roberts PJ and Der CJ: Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene. 26:3291–3310. 2007. View Article : Google Scholar : PubMed/NCBI
9	Higashi N, Kunimoto H, Kaneko S, Sasaki T, Ishii M, Kojima H and Nakajima K: Cytoplasmic c-Fos induced by the YXXQ-derived STAT3 signal requires the co-operative MEK/ERK signal for its nuclear translocation. Genes Cells. 9:233–242. 2004. View Article : Google Scholar : PubMed/NCBI
10	Manimala NJ, Frost CD, Lane ML, Higuera M, Beg R and Vesely DL: Cardiac hormones target nuclear oncogenes c-Fos and c-Jun in carcinoma cells. Eur J Clin Invest. 43:1156–1162. 2013.PubMed/NCBI
11	Lavoie JN, L'Allemain G, Brunet A, Müller R and Pouysségur J: Cyclin D1 expression is regulated positively by the p42/p44^MAPK and negatively by the p38/HOG^MAPK pathway. J Biol Chem. 271:20608–20616. 1996. View Article : Google Scholar : PubMed/NCBI
12	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. View Article : Google Scholar : PubMed/NCBI
13	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
14	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 : PubMed/NCBI
15	Kreiner T and Moore HP: Membrane traffic between secretory compartments is differentially affected during mitosis. Cell Regul. 1:415–424. 1990.PubMed/NCBI
16	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
17	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 : PubMed/NCBI
18	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
19	Margadant C, Cremers L, Sonnenberg A and Boonstra J: MAPK uncouples cell cycle progression from cell spreading and cyto-skeletal organization in cycling cells. Cell Mol Life Sci. 70:293–307. 2013. View Article : Google Scholar :
20	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 : PubMed/NCBI
21	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
22	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. View Article : Google Scholar : PubMed/NCBI
23	Laird AD, Taylor SJ, Oberst M and Shalloway D: Raf-1 is activated during mitosis. J Biol Chem. 270:26742–26745. 1995. View Article : Google Scholar : PubMed/NCBI
24	Herrmann C, Martin GA and Wittinghofer A: Quantitative analysis of the complex between p21^ras and the Ras-binding domain of the human Raf-1 protein kinase. J Biol Chem. 270:2901–2905. 1995. View Article : Google Scholar : PubMed/NCBI
25	Cochet C, Kashles O, Chambaz EM, Borrello I, King CR and Schlessinger J: Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent. J Biol Chem. 263:3290–3295. 1988.PubMed/NCBI
26	Gamett DC, Pearson G, Cerione RA and Friedberg I: Secondary dimerization between members of the epidermal growth factor receptor family. J Biol Chem. 272:12052–12056. 1997. View Article : Google Scholar : PubMed/NCBI
27	Hyde R, Corkins ME, Somers GA and Hart AC: PKC-1 acts with the ERK MAPK signaling pathway to regulate Caenorhabditis elegans mechanosensory response. Genes Brain Behav. 10:286–298. 2011. View Article : Google Scholar
28	Couet J, Sargiacomo M and Lisanti MP: Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinase activities. J Biol Chem. 272:30429–30438. 1997. View Article : Google Scholar : PubMed/NCBI
29	Mineo C, Gill GN and Anderson RG: Regulated migration of epidermal growth factor receptor from caveolae. J Biol Chem. 274:30636–30643. 1999. View Article : Google Scholar : PubMed/NCBI
30	Smart EJ, Ying YS, Mineo C and Anderson RG: A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc Natl Acad Sci USA. 92:10104–10108. 1995. View Article : Google Scholar : PubMed/NCBI
31	Yamabhai M and Anderson RG: Second cysteine-rich region of epidermal growth factor receptor contains targeting information for caveolae/rafts. J Biol Chem. 277:24843–24846. 2002. View Article : Google Scholar : PubMed/NCBI
32	Carpenter G: Receptors for epidermal growth factor and other polypeptide mitogens. Annu Rev Biochem. 56:881–914. 1987. View Article : Google Scholar : PubMed/NCBI
33	Fielding AB, Willox AK, Okeke E and Royle SJ: Clathrin-mediated endocytosis is inhibited during mitosis. Proc Natl Acad Sci USA. 109:6572–6577. 2012. View Article : Google Scholar : PubMed/NCBI
34	Aguilar RC and Wendland B: Endocytosis of membrane receptors: Two pathways are better than one. Proc Natl Acad Sci USA. 102:2679–2680. 2005. View Article : Google Scholar : PubMed/NCBI
35	Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW and Burgess AW: Epidermal growth factor receptor: Mechanisms of activation and signalling. Exp Cell Res. 284:31–53. 2003. View Article : Google Scholar : PubMed/NCBI
36	Pawson T: Specificity in signal transduction: From phosphot-yrosine-SH2 domain interactions to complex cellular systems. Cell. 116:191–203. 2004. View Article : Google Scholar : PubMed/NCBI
37	Morrison DK and Cutler RE Jr: The complexity of Raf-1 regulation. Curr Opin Cell Biol. 9:174–179. 1997. View Article : Google Scholar : PubMed/NCBI
38	Vojtek AB, Hollenberg SM and Cooper JA: Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 74:205–214. 1993. View Article : Google Scholar : PubMed/NCBI
39	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
40	Dent P, Haser W, Haystead TA, Vincent LA, Roberts TM and Sturgill TW: Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. Science. 257:1404–1407. 1992. View Article : Google Scholar : PubMed/NCBI
41	Howe LR, Leevers SJ, Gómez N, Nakielny S, Cohen P and Marshall CJ: Activation of the MAP kinase pathway by the protein kinase raf. Cell. 71:335–342. 1992. View Article : Google Scholar : PubMed/NCBI
42	Kyriakis JM, App H, Zhang XF, Banerjee P, Brautigan DL, Rapp UR and Avruch J: Raf-1 activates MAP kinase-kinase. Nature. 358:417–421. 1992. View Article : Google Scholar : PubMed/NCBI
43	Lange-Carter CA, Pleiman CM, Gardner AM, Blumer KJ and Johnson GL: A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science. 260:315–319. 1993. View Article : Google Scholar : PubMed/NCBI
44	Moodie SA, Willumsen BM, Weber MJ and Wolfman A: Complexes of Ras. GTP with Raf-1 and mitogen-activated protein kinase kinase. Science. 260:1658–1661. 1993. View Article : Google Scholar : PubMed/NCBI
45	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