Characterizing the role and dynamics of the Vaccinia-Related Kinase 1 in C. elegans and human cells

Resumen del póster presentado en el European Worm Meeting: Berlin C. elegans Meeting, celebrado en Berlín del 3 al 5 de mayo de 2014.Phosphorylation of proteins is an important regulatory mechanism that controls numerous biological processes. Vaccinia-Related Kinase 1 (VRK1) is a conserved protein kinase that is related to cell proliferation and survival. In mammals, loss of VRK1 leads to sterility and may cause neurological disorders. VRK1 is known to phosphorylate chromatin proteins, such as histone H2A and BAF as well as the transcription factors p53, c-Jun and ATF2. In Caenorhabditis elegans VRK-1 plays critical roles in development of the vulva and uterus, as well as in germ cell proliferation. In order to characterize the dynamics of VRK1 we have generated VRK1 singlecopy transgenic C. elegans strains and human cell lines. We report here that, like previously described in C. elegans embryos, human VRK1 is nuclear during interphase and is associated with condensed chromosomes in mitosis. We have mapped a short localization domain of both, C. elegans and human VRK1. Moreover, site-directed mutagenesis identified a novel, conserved motif responsible for chromatin localization during mitosis. Fluorescence Recovery After Photobleaching (FRAP) analysis suggests transient association of VRK1 with chromatin. Identical kinetics were observed in interphase and mitosis, suggesting VRK1 may interact with the same chromatin protein(-s) throughout the cell cycle. To identify interaction partners we expressed and purified VRK1 from human cells followed by high-resolution mass spectrometry. Our novel C. elegans transgenic strains show expression of VRK-1 not only in previously reported cells (neurons, hypodermal cells and vulva precursor cells), but also in the anchor cell (AC), that plays an essential role in vulval development. During C. elegans vulval development the AC fuses with uterine cells to form the utse syncytium. Defects in utse formation cause in adults a protruding vulva phenotype. Using tissue-specific knockdown and rescue strategies, we show that the AC fails to fuse in vrk-1 mutants, most likely due to the loss of VRK-1 from uterine tissue, which is characterized by defects in proliferation and differentiation.Peer Reviewe