Human VRK1 is an early response gene and its loss causes a block in cell cycle progression

Abstract

Background: In mammalian cells regulatory proteins controlling the cell cycle are necessary due to the requirements of living in a heterogeneous environment of cell-interactions and growth factors. VRK1 is a novel serine-threonine kinase that phosphorylates several transcription factors and is associated with proliferation phenotypes.

Methodology/principal findings: In this report VRK1 has been identified as regulated in the cell cycle. VRK1 gene expression is activated by the addition of serum to starved cells, indicating it is required for the exit of G0 phase and entry in G1; a response that parallels the re-expression of MYC, FOS and CCND1 (cyclin D1) genes, suggesting that VRK1 is an early-response gene. VRK1 gene expression is also shutdown by serum withdrawal. The human VRK1 gene promoter cloned in a luciferase reporter responds similarly to serum. In response to serum, the level of VRK1 protein expression has a positive correlation with cell proliferation markers such as phosphorylated-Rb or PCNA, and is inversely correlated with cell cycle inhibitors such as p27. The elimination of VRK1 by siRNA results in a G1 block in cell division, and in loss of phosphorylated-Rb, cyclin D1, and other proliferation markers. Elimination of VRK1 by siRNA induces a reduction of cell proliferation. VRK1 colocalizes with p63 in proliferating areas of squamous epithelium, and identifies a subpopulation in the basal layer.

Conclusions/significance: VRK1 is an immediate early response gene required for entry in G1, and due to its implication in normal cell proliferation and division, might be a new target for development of inhibitors of cellular proliferation.

Figure 1. VRK1 levels in different growth conditions of human fibroblasts.

(A). Flow cytometry profile of WS1 cell cycle in non-synchronized (left) and serum starved cells (right). (B). Nuclear localization of VRK1 and morphology of WS1 cells in non-synchronized and starved WS1 cells. Endogenous VRK1 protein was detected with a specific polyclonal antibody (VE1) in the cell nuclei. The size bar represents 50 µm. (C). Level of VRK1 endogenous protein determined in an immunoblot. (D). Change in VRK1 gene expression by qRT-PCR upon withdrawal or addition of serum to the cell culture. These experiments were independently performed three times.

Figure 2. Changes in VRK1 RNA level upon serum withdrawal or readdition to the culture.

(A). Quantification of the VRK1 gene expression in WS1 fibroblasts at different time points upon serum withdrawal (top) and serum readdition to the cell culture (bottom) which was previously serum-deprived for five days. These experiments were independently performed three times. (B). Regulation of the human proximal promoter of human VRK1 by serum. WS1 cells were transfected with plasmid pGL2-B-VRK1(−1028+52). The luciferase activity was determined under different experimental conditions. Control activity is that of cells in the continuous presence of serum. The result is the mean of three experiments.

Figure 3. Cell cycle gene expression induced by removal or serum readdition.

(A). Expression of MYC, FOS and VRK1 genes after serum readdition to the culture. Before serum readdition cells were maintained for forty hours in 0.1% FBS. (B). Sequential expression of five cyclin genes after serum readdition to starved WS1 cells. (C). Timing of Rb phosphorylation after serum readdition to the starved WS1 cell culture detected with a specific anti p-Rb antibody. These experiments were independently performed three times.

Figure 4. Time course of changes in cell cycle related proteins level after serum withdrawal and serum readdition to WS1 cells.

The proteins were determined in an immunoblot with specific antibodies for each protein, VRK1, p27, PCNA and phospho-Rb. The level of actin was used as a loading control. At the bottom is shown the quantification of the level of each protein. In the case of VRK1 it is also shown the quantification by RT-PCR of its RNA (dashed line) at the same time points. These experiments were independently performed three times.

Figure 5. Stability of endogenous or transfected VRK1 protein.

(A) The variation in levels of endogenous VRK1 and p53 proteins in HeLa cells was followed at different time points after addition of cycloheximide. The p53 protein drops in less than 30 minutes as expected, but there is no detectable change in VRK1 protein even after twelve hours suggesting it has a very high stability. (B). HEK293T cells were transfected with plasmids expressing wild-type human VRK1 or its kinase-dead K179E mutant. The stability of both proteins was followed at different time points after cycloheximide addition. Cells were transfected with plasmids pCEFL-HA-VRK1 or pCEFL-HA-VRK1(K179E) as previously described (Vega et al., 2004). These experiments were independently performed three times.

Figure 6. Effect of siVRK1 on proteins related with cell cycle progression.

Levels of proteins in non-transfected, transfected with siRNA control, or with siRNA specific for VRK1 were determined in after 120 hours post transfection. The proteins were determined with the corresponding antibody indicated in the methods section, and identified in the corresponding immunoblot. These experiments were independently performed five times.

Figure 7. Effect of siRNA on endogenous VRK1 and cell proliferation.

(A). Proliferation of WS1 cells that were transfected with siRNA control or siRNA specific for VRK1. The total cell number in the dish was determined at different time points (top). The endogenous protein was detected in an immunoblot (bottom) (B) Photograph of the WS1 cell cultures treated with siRNA control (top) or siVRK1 (bottom). The proliferation is much slower in cells transfected with the specific siVRK1. These experiments were independently performed three times. (C). Effect on VRK1 RNA levels in WS1 cells transfected with either siControl or siVRK1. NTC: non transfected cells.

Figure 8. Localization of VRK1 and p63 in the squamous epithelium of the human pharynx.

(A). Immunohistochemistry detection of p63 and VRK1 proteins in the pharynx. The arrows indicate cells (3 out of 56) in the basal layer with higher levels of VRK1. The p63 was detected with a monoclonal antibody. VRK1 was detected with the VC1 polyclonal antibody. (B). Immunofluorescence of p63 and VRK1 in the human pharynx epithelium. The arrows indicate cells (3 out of 57) in the basal layer that have higher levels of VRK1.