Table of Contents
ISRN Cell Biology
Volume 2013 (2013), Article ID 867613, 11 pages
http://dx.doi.org/10.1155/2013/867613
Research Article

Generation of Constitutive Active ERK Mutants as Tools for Cancer Research in Zebrafish

Institute of Biology, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands

Received 26 August 2013; Accepted 26 September 2013

Academic Editors: J.-F. Bodart, R. Hurta, and P. Storz

Copyright © 2013 Hanan Rian et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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.