Interaction partners of Vaccinia-Related Kinase 1, an ubiquitous kinase implicated in development of C. elegans reproductive organs

Abstract

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 BAF and histones H2A and H3 as well as the transcription factors p53, c-Jun, ATF2 and CREB. To characterize the dynamics of VRK1 we have generated VRK1 single-copy transgenic Caenorhabditis elegans strains and human cell lines. We have mapped a short localization domain of both, C. elegans and human VRK1 that is sufficient to localize the protein to condensed chromosomes in mitosis. Our novel C. elegans transgenic strains, in contrast to previous observations, show expression of VRK-1 in all cell types including proliferating and post-mitotic cells. During C. elegans vulval development, a specialized gonadal cell, called the anchor cell (AC), fuses with uterine cells to form the utse syncytium. Defects in utse formation cause in adults a protruding vulva phenotype. Our data show that VRK-1 is required for AC fusion with neighboring cells to form the utse syncytium. To identify interaction partners we expressed and purified VRK1 from human cells followed by high-resolution mass spectrometry. Among top candidate proteins are several nuclear envelope proteins, including the LEM-domain protein emerin, which was subsequently confirmed by Y2H assays. In addition, we have developed a bicistronic system to study protein-protein interactions by Bimolecular Fluorescence Complementation (BiFC) analysis. This system enables us to test for VRK1 in vivo interactions at physiological expression levels but the system can be adapted to any other protein pair.