The DNA damage effector Chk1 kinase regulates Cdc14B nucleolar shuttling during cell cycle progression

Abstract

Chk1 is a critical effector of DNA damage checkpoints necessary for the maintenance of chromosome integrity during cell cycle progression. Here we report, that Chk1 co-localized with the nucleolar marker, fibrillarin in response to radiation-induced DNA damage in human cells. Interestingly, in vitro studies using GST pull down assays identified the dual-specificity serine/threonine nucleolar phosphatase Cdc14B as a Chk1 substrate. Furthermore, Chk1, but not a kinase-dead Chk1 control, was shown to phosphorylate Cdc14B using an in vitro kinase assay. Co-immunoprecipitation experiments using exogenous Cdc14B transfected into human cells confirmed the interaction of Cdc14B and Chk1 during cell cycle. In addition, reduction of Chk1 levels via siRNA or UCN-01 treatment demonstrated that Chk1 activation following DNA damage was required for Cdc14B export from the nucleolus. These studies have revealed a novel interplay between Chk1 kinase and Cdc14B phosphatase involving radiation-induced nucleolar shuttling to facilitate error-free cell cycle progression and prevent genomic instability.

Figure 1

Chk1 translocates to the nucleoli in response to irradiation. (A) Merged images at 40× magnification of irradiated HeLa cells stained with Chk1 (green), fibrillarin (red) and DNA (DAPI-blue) show translocated Chk1 foci in the nucleoli and colocalization with fibrillarin in a time-dependent manner from 0–90 min. (B) Merged images at 20× magnification of irradiated HeLa cells stained with Chk1 (green), fibrillarin (red) and DNA (DAPI-blue) show Chk1 colocalized with fibrillarin at 90 min in siControl-treated HeLa cells and not in siChk1-treated HeLa cells. Enlarged inset shows colocalization of Chk1 with fibrillarin (yellow arrows) at 90 min in irradiated siControl HeLa cells and not in irradiated siChk1 HeLa cells (yellow arrows). (C) Immunoblotting (IB) with Chk1 antibody confirms reduction of Chk1 levels in siChk1-transfected HeLa cells as compared to siControl-transfected HeLa cells.

Figure 2

Chk1 interacts with and phosphorylates Cdc14B in vitro. (A) GST pull down assay shows GST-Cdc14B interacts with Chk1 in vitro and not a GST-luciferase control. (B) In vitro kinase assay shows active Chk1, but not kinase dead Chk1 (Chk1KD) phosphorylates Cdc14B. (C) Immunoprecipitation (IP) with polyclonal anti-Chk1 antibody and immunoblotting (IB) with monoclonal anti-Flag antibody (anti-mFlag) show that Chk1 interacts with Flag-Cdc14B in transfected HeLa cells and not the control anti-IgG.

Figure 3

Sub-nuclear shuttling of Cdc14B in response to radiation-induced DNA damage. (A) Merged images at 40× magnification of irradiated HeLa cells stained for Flag-Cdc14B (green), Fibrillarin (red) and DNA (DAPI-blue) show sub-nuclear shuttling and co-localization of Flag-Cdc14B foci with fibrillarin in a time-dependent manner from 0–90 min. (B) Quantitation indicates a significant decrease in the percentage of transfected HeLa cells showing Flag-Cdc14B foci in the nucleoli at 30 and 60 min post-irradiation, compared to 0 and 90 min. (C) Immunoblotting (IB) with an anti-Flag antibody confirms expression of Flag-Cdc14B in exogenously transfected HeLa cells compared to vector-transfected HeLa cells. Alpha-tubulin was used as loading control.

Figure 4

Reduced Chk1 levels prevent sub-nuclear shuttling of Cdc14B after radiation-induced DNA damage. (A) Merged images at 40× magnification of transfected siRNA-treated HeLa cells stained for Flag-Cdc14B (red), fibrillarin (green) and DNA (DAPI-blue) show a reduction in sub-nuclear shuttling and colocalization of Flag-Cdc14B foci with fibrillarin in siChk1 HeLa cells as compared to siControl HeLa cells within 30 min post-radiation. The enlarged inset shows siControl- and siChk1-treated HeLa cells (yellow arrows) in the absence and presence Flag-Cdc14B foci colocalized with fibrillarin in the nucleoli 30 min post-radiation, respectively. (B) Quantitation indicates a significant increase of percentage of transfected HeLa cells showing Flag-Cdc14B foci in the nucleoli at 30 min post-irradiation in siChk1 HeLa cells as compared to siControl HeLa cells. (C) Merged images at 40× magnification of transfected UCN-01 treated HeLa cells stained for Flag-Cdc14B (red), fibrillarin (green) and DNA (DAPI-blue) show the reduction in sub-nuclear shuttling and colocalization of Flag-Cdc14B foci with fibrillarin after UCN-01 treatment as compared to DMSO control within 30 min post-radiation. The enlarged inset shows DMSO and UCN-01 treated HeLa cells (yellow arrows) in the absence and presence of Flag-Cdc14B foci colocalized with fibrillarin in the nucleoli 30 min post-radiation, respectively. (D) Quantitation indicates a significant increase of percentage of transfected HeLa cells showing Flag-Cdc14B foci in the nucleoli at 30 min post-irradiation in UCN-01 treated-HeLa cells, as compared to DMSO treated-HeLa cells.