%0 Journal Article
%T Generation of Constitutive Active ERK Mutants as Tools for Cancer Research in Zebrafish
%A Hanan Rian
%A S. F. Gabriel Krens
%A Herman P. Spaink
%A B. Ewa Snaar-Jagalska
%J ISRN Cell Biology
%D 2013
%R 10.1155/2013/867613
%X 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¨C5]. 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,
%U http://www.hindawi.com/journals/isrn.cell.biology/2013/867613/