Genetic and cell biological analyses of the role of the VRK1 protein kinase in cell proliferation in vitro and in vivo

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. VRK1 is the seminal member of the VRK family and has been identified as a key regulator of cell cycle dynamics. The cell cycle is orchestrated by a profound combination of factors. Errors at any point in the replication and faithful segregation of the chromosomes can be catastrophic and can lead to many disease states, including cancer. Genetic analyses of mutant alleles of the Drosophila melanogaster and Caenorhabditis elegans VRK1 orthologs have revealed phenotypes ranging from embryonic lethality to mitotic and meiotic defects. Recently, VRK1 was implicated as part of a "gene-expression signature" correlated with poor clinical outcome in breast cancer patients. Herein I describe my investigations of the role of VRK1 in vivo and in culture.;I have focused my studies on VRK1 function within human cell lines derived from normal and cancerous mammary epithelia. I have shown that stable shRNA-mediated depletion of VRK1 slows proliferation in both transformed and non-transformed human mammary epithelial cells (MCF10-a, MCF7, MDA-MB-231). Cell cycle-analysis following VRK1 depletion in culture is consistent with VRK1 participating in both G1/S and G2/M transitions in a cell type dependent manner. VRK1 substrates are involved in regulation of both G1/S and G2/M phases of the cell cycle. In reference to the latter, our laboratory has demonstrated that the small dimeric protein BAF (barrier-to-autointegration factor) is a highly efficient substrate for VRK1. BAF is an abundant, highly conserved protein that binds to dsDNA, chromatin and proteins within the nuclear lamina. BAF plays an integral role in mitotic nuclear assembly, nuclear organization and cell cycle progression. VRK1-mediated phosphorylation of BAF ablates its DNA-binding activity and reduces its interaction with the LEM domain containing proteins of the nuclear envelope. For the first time, I have shown that depletion of VRK1 leads to an in vivo reduction of BAF phosphorylation, most strongly during mitosis. Within mammary epithelial cells the interruption of the VRK1/BAF signaling axis exacerbated a slowed proliferation phenotype and led to an increase in the frequency of cells with aberrant nuclear envelope morphology.;Stable overexpression of a FLAG-tagged VRK1 transgene lacked the ability to impart a growth advantage to cells cultured under normal growth conditions. However, when MDA-MB-231 cells were cultured under serum starved conditions, stable overexpression of VRK1 appeared to increase survival signaling and impart a growth advantage. I have compared the impact of VRK1 depletion on the tumorigenicity of luciferase-expressing MDA-MB-231 cells in an orthotopic xenograft model of breast cancer. The tumors depleted of VRK1 were reduced in size by over 50%, on average; metastasis was also reduced to those organs to which breast cancer has a metastatic proclivity. These are the first studies to show a causative role for VRK1 in the progression of cancer.;The laboratory has also generated a line of mice containing a gene-trap insertion in the Vrk1 locus. Herein we describe that the expression of full-length transcripts is reduced by 85% in homozygous mice. Male VRK1-deficient mice are infertile due to a progressive loss of proliferating spermatogonia. The spermatogenic 'niche' of the testes appears to be intact and therefore the infertile trait appears to be cell intrinsic. I also developed a mouse strain in which the Vrk3 (vaccinia-related kinase 3) locus was genetically interrupted by gene-trap insertion. Vrk3-null mice developed normally, exhibited a standard life-span and were fertile. Crossing the VRK3-null mice with the Vrk1 gene-trapped mice enabled us to demonstrate that the infertility of the Vrk1 hypomorphic mice was unaffected by the Vrk3-null background.;VRK1 has many substrates that operate within all parts of the cell cycle and its overexpression has been associated with multiple cancers. These studies emphasize that overexpression and under expression of VRK1 appear to be a seeding point for cellular abnormality that can contribute to disease.