The extracellular-signal-regulated-kinase (ERK) signaling pathway is essential for vertebrate development and is frequently deregulated in human and zebrafish tumors. Previously, we cloned and characterized the zebrafish MAPK gene family and showed that ERK2 is crucial for cell migration and early zebrafish embryogenesis. To further study ERK2 function we generated constitutively active mutant forms of the ERK proteins by introducing conserved point mutations. We validated the enhanced protein activity in vitro by transfection of constructs into zebrafish fibroblast (zf4) cells and demonstrated elevated phosphorylation levels of downstream targets P90RSK and CREB, by and specifically. In vivo validation was performed by ectopic expression of corresponding mRNAs in the transgenic zebrafish FGF-ERK2 reporter fish line Tg(Dusp6:d2EGFP). Both mutant ERK2 isoforms induced elevated transgene expression compared to , confirming increased kinase activity in vivo. Phospho-kinomic analysis on peptide microarrays was performed to identify new targets in embryos injected with FGF8 or mRNAs. We detected both FGF8 specific and common signalling targets. Interestingly, with both mRNAs we found increased phosphorylation levels of CDK1, which is critical for proper G2/M phase transition and mitotic entry in proliferation control. These results corroborate that constitutive activation of the ERK2 pathway leads to enhanced, possibly oncogenic, proliferation. 1. Introduction The mitogen-activated protein (MAP) kinases extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are crucial components of the regulatory machinery underlying normal and malignant cell proliferation. The rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/MAPK kinase (MEK1/2)/ERK1/2 oncogenic pathway is induced by various growth factors and forms a convergence point of multiple signaling pathways to control essential cellular processes including migration, differentiation, growth, and survival [1–5]. Approximately, 30% of all human cancers display evidence of enhanced activation of the RAS/RAF/MEK MAPK pathway [6]. This pathway has been extensively studied in relation to tumor formation and this was greatly facilitated by the availability of constitutively active forms of RAS, RAF, and MEK [7, 8]. Expression of oncogenic RAS as well as constitutive active forms of RAF, and MEK can lead to induced tumorigenic transformation of NIH 3T3 cells [9, 10]. In addition, in vivo studies with transgenic mice models, expressing constitutive active MEK in the heart, lens chondrocytes, or skin,
References
[1]
J. A. McCubrey, L. S. Steelman, W. H. Chappell et al., “Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance,” Biochimica et Biophysica Acta, vol. 1773, no. 8, pp. 1263–1284, 2007.
[2]
P. J. Roberts and C. J. Der, “Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer,” Oncogene, vol. 26, no. 22, pp. 3291–3310, 2007.
[3]
S. Yoon and R. Seger, “The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions,” Growth Factors, vol. 24, no. 1, pp. 21–44, 2006.
[4]
A. Zebisch, A. P. Czernilofsky, G. Keri, J. Smigelskaite, H. Sill, and J. Troppmair, “Signaling through RAS-RAF-MEK-ERK: from basics to bedside,” Current Medicinal Chemistry, vol. 14, no. 5, pp. 601–623, 2007.
[5]
Z. Wei and H. T. Liu, “MAPK signal pathways in the regulation of cell proliferation in mammalian cells,” Cell Research, vol. 12, no. 1, pp. 9–18, 2002.
[6]
R. Hoshino, Y. Chatani, T. Yamori et al., “Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors,” Oncogene, vol. 18, no. 3, pp. 813–822, 1999.
[7]
N. Askari, R. Diskin, M. Avitzour, G. Yaakov, O. Livnah, and D. Engelberg, “MAP-quest: could we produce constitutively active variants of MAP kinases?” Molecular and Cellular Endocrinology, vol. 252, no. 1-2, pp. 231–240, 2006.
[8]
S. J. Mansour, W. T. Matten, A. S. Hermann et al., “Transformation of mammalian cells by constitutively active MAP kinase kinase,” Science, vol. 265, no. 5174, pp. 966–970, 1994.
[9]
S. Cowley, H. Paterson, P. Kemp, and C. J. Marshall, “Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells,” Cell, vol. 77, no. 6, pp. 841–852, 1994.
[10]
W. Kolch, G. Heidecker, P. Lloyd, and U. R. Rapp, “Raf-1 protein kinase is required for growth of induced NIH/3T3 cells,” Nature, vol. 349, no. 6308, pp. 426–428, 1991.
[11]
F. A. Scholl, P. A. Dumesic, and P. A. Khavari, “Effects of active MEK1 expression in vivo,” Cancer Letters, vol. 230, no. 1, pp. 1–5, 2005.
[12]
M. Karasarides, A. Chiloeches, R. Hayward et al., “B-RAF is a therapeutic target in melanoma,” Oncogene, vol. 23, no. 37, pp. 6292–6298, 2004.
[13]
E. E. Patton, H. R. Widlund, J. L. Kutok et al., “BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma,” Current Biology, vol. 15, no. 3, pp. 249–254, 2005.
[14]
E. E. Patton and L. I. Zon, “Taking human cancer genes to the fish: a transgenic model of melanoma in zebrafish,” Zebrafish, vol. 1, no. 4, pp. 363–368, 2005.
[15]
J. F. Amatruda and E. E. Patton, “Genetic models of cancer in zebrafish,” International Review of Cell and Molecular Biology, vol. 271, pp. 1–34, 2008.
[16]
J. F. Amatruda, J. L. Shepard, H. M. Stern, and L. I. Zon, “Zebrafish as a cancer model system,” Cancer Cell, vol. 1, no. 3, pp. 229–231, 2002.
[17]
W. Goessling, T. E. North, and L. I. Zon, “New waves of discovery: modeling cancer in zebrafish,” Journal of Clinical Oncology, vol. 25, no. 17, pp. 2473–2479, 2007.
[18]
H. L. Siew, L. W. Yi, V. B. Vega et al., “Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression,” Nature Biotechnology, vol. 24, no. 1, pp. 73–75, 2006.
[19]
K. Stoletov and R. Klemke, “Catch of the day: zebrafish as a human cancer model,” Oncogene, vol. 27, no. 33, pp. 4509–4520, 2008.
[20]
D. E. Abbott, L.-M. Postovit, E. A. Seftor, N. V. Margaryan, R. E. B. Seftor, and M. J. C. Hendrix, “Exploiting the convergence of embryonic and tumorigenic signaling pathways to develop new therapeutic targets,” Stem Cell Reviews, vol. 3, no. 1, pp. 68–78, 2007.
[21]
M. J. C. Hendrix, E. A. Seftor, R. E. B. Seftor, J. Kasemeier-Kulesa, P. M. Kulesa, and L.-M. Postovit, “Reprogramming metastatic tumour cells with embryonic microenvironments,” Nature Reviews Cancer, vol. 7, no. 4, pp. 246–255, 2007.
[22]
A. Moustakas and C.-H. Heldin, “Signaling networks guiding epithelial-mesenchymal transitions during embryogenesis and cancer progression,” Cancer Science, vol. 98, no. 10, pp. 1512–1520, 2007.
[23]
S. F. G. Krens, S. He, G. E. M. Lamers et al., “Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation,” Developmental Biology, vol. 319, no. 2, pp. 370–383, 2008.
[24]
M. A. Emrick, A. N. Hoofnagle, A. S. Miller, L. F. Ten Eyck, and N. G. Ahn, “Constitutive activation of extracellular signal-regulated kinase 2 by synergistic point mutations,” Journal of Biological Chemistry, vol. 276, no. 49, pp. 46469–46479, 2001.
[25]
J. P. Hall, V. Cherkasova, E. Elion, M. C. Gustin, and E. Winter, “The osmoregulatory pathway represses mating pathway activity in Saccharomyces cerevisiae: isolation of a FUS3 mutant that is insensitive to the repression mechanism,” Molecular and Cellular Biology, vol. 16, no. 12, pp. 6715–6723, 1996.
[26]
D. Brunner, N. Oellers, J. Szabad, W. H. Biggs III, S. Lawrence Zipursky, and E. Hafen, “A gain-of-function mutation in Drosophila MAP kinase activates multiple receptor tyrosine kinase signaling pathways,” Cell, vol. 76, no. 5, pp. 875–888, 1994.
[27]
M. Tsang, S. Maegawa, A. Kiang, R. Habas, E. Weinberg, and I. B. Dawid, “A role for MKP3 in axial patterning of the zebrafish embryo,” Development, vol. 131, no. 12, pp. 2769–2779, 2004.
[28]
H. A. Harrington, M. Komorowski, M. Beguerisse-Diaz, G. M. Ratto, and M. P. Stumpf, “Mathematical modeling reveals the functional implications of the different nuclear shuttling rates of Erk1 and Erk2,” Physical Biology, vol. 9, Article ID 036001, 2012.
[29]
H. Shankaran, D. L. Ippolito, W. B. Chrisler et al., “Rapid and sustained nuclear-cytoplasmic ERK oscillations induced by epidermal growth factor,” Molecular Systems Biology, vol. 5, article 332, 2009.
[30]
G. A. Molina, S. C. Watkins, and M. Tsang, “Generation of FGF reporter transgenic zebrafish and their utility in chemical screens,” BMC Developmental Biology, vol. 7, article 62, 2007.
[31]
R. A. MacCorkle and T.-H. Tan, “Mitogen-activated protein kinases in cell-cycle control,” Cell Biochemistry and Biophysics, vol. 43, no. 3, pp. 451–461, 2005.
[32]
K. Coulonval, H. Kooken, and P. P. Roger, “Coupling of T161 and T14 phosphorylations protects cyclin B-CDK1 from premature activation,” Molecular Biology of the Cell, vol. 22, no. 21, pp. 3971–3985, 2011.
[33]
W. Krek and E. A. Nigg, “Cell cycle regulation of vertebrate p34cdc2 activity: identification of Thr161 as an essential in vivo phosphorylation site,” The New Biologist, vol. 4, no. 4, pp. 323–329, 1992.
