Homeodomain-interacting protein kinase 1 modulates Daxx localization, phosphorylation, and transcriptional activity

Abstract

We describe an interaction between homeodomain-interacting protein kinase 1 (HIPK1) and Daxx, two transcriptional regulators important in transducing growth-regulatory signals. We demonstrate that HIPK1 is ubiquitously expressed in mice and humans and localizes predominantly to the nucleus. Daxx normally resides within the nucleus in promyelocytic leukemia protein (PML) oncogenic domains (PODs), where it physically interacts with PML. Under certain circumstances, Daxx is relocalized from PODs to chromatin, where it then acts as a transcriptional repressor through an association with histone deacetylase (HDAC1). We propose two novel mechanisms for regulating the activity of Daxx, both mediated by HIPK1. First, HIPK1 physically interacts with Daxx in cells and consequently relocalizes Daxx from PODs. Daxx relocalization disrupts its interaction with PML and augments its interaction with HDAC1, likely influencing Daxx activity. Although the relocalization of Daxx from PODs is phosphorylation independent, an active HIPK1 kinase domain is required, suggesting that HIPK1 autophosphorylation is important in this interaction. Second, HIPK1 phosphorylates Daxx on Ser 669, and phosphorylation of this site is important in modulating the ability of Daxx to function as a transcriptional repressor. Mutation of Daxx Ser 669 to Ala results in increased repression in three of four transcriptional reporters, suggesting that phosphorylation by HIPK1 diminishes Daxx transcriptional repression of specific promoters. Taken together, our results indicate that HIPK1 and Daxx collaborate in regulating transcription.

FIG. 1.

HIPK1 is ubiquitously expressed in mouse and human tissues and localizes to nuclear speckles. (A) Mouse multiple-tissue Northern blot probed for HIPK1 (upper panel) and GAPDH (lower panel) as a control for RNA loading. Numbers indicate the relative migration of RNA molecular size standards. (B) Human multiple-tissue Northern blot probed for HIPK1 (upper panel) and β-actin (lower panel) as a control for RNA loading. Numbers indicate the relative migration of RNA molecular size standards. (C) Human RNA master blot probed for HIPK1. The adjacent diagram indicates the RNA or DNA source for each spot. (D) Representative immunofluorescent images of a HIPK1-Myc fusion expressed in NIH 3T3 cells and stained with anti-HIPK1 antibody (green) and anti-Myc antibody (red). Overlapping localization is shown in the merged image (yellow). DNA (blue) was stained with Hoechst 33258. The yellow arrow indicates the nucleus of the HIPK1-Myc-transfected cell.

FIG. 2.

HIPK1 interacts with Daxx in vivo and in vitro. (A) Endogenous HIPK1 was immunoprecipitated (IP) with anti-HIPK1 or a control antibody from 293 cell nuclear lysates. The immunoprecipitates were Western blotted, and the blots were probed with anti-HIPK1 antibody (upper panel) or anti-Daxx antibody (lower panel). (B) Daxx was immunoprecipitated with anti-Daxx antibody from 293 cells expressing an empty vector (pβ), pCAGGS/Daxx (Daxx), pβ/HIPK1 (HIPK1), or pCAGGS/Daxx and pβ/HIPK1 together (Daxx/HIPK1). Daxx immunoprecipitates were Western blotted, and the blots were probed with anti-HIPK1 antibody. (C) ³⁵S-radiolabeled HIPK1 and MEK1 proteins (left panel, autoradiogram) were incubated with GST or Daxx-GST fusion proteins (center panel, colloidal blue-stained gel), and the pulled-down protein aggregates were evaluated by SDS-PAGE (right panel, autoradiogram). Numbers indicate the relative migration of protein molecular size standards (in kilodaltons).

FIG. 3.

HIPK1 phosphorylates Daxx in vivo and in vitro. (A) Daxx was immunoprecipitated with anti-Myc antibody from 293 cells expressing pCAGGS/Daxx with an empty vector (pβ), pβ/HIPK1 (HIPK1), or pβ/K219A (K219A). Daxx immunoprecipitates were lambda phosphatase treated (+ Phosphatase) or mock treated (− Phosphatase) and Western blotted. The blot was probed with anti-Daxx antibody. Daxx* indicates the upper-migrating or hyperphosphorylated bands, and Daxx indicates the lower-migrating or basal phosphorylation bands in the non-phosphatase-treated samples. (B) HIPK1 was immunoprecipitated with anti-HIPK1 antibody from 293 cells expressing an empty vector (pβ), pβ/HIPK1 (HIPK1), or pβ/K219A (K219A). HIPK1 immunoprecipitates were used as the enzyme in an in vitro kinase reaction with myelin basic protein (MBP) or immunoprecipitated Daxx obtained from pCAGGS/Daxx expressed in 293 cells. HIPK1 but not pβ or K219A underwent autophosphorylation (upper panel) and phosphorylated myelin basic protein (middle panel) and Daxx (lower panel).

FIG. 4.

HIPK1 relocalizes Daxx from PODs, disrupts the Daxx/PML interaction, and enhances the association between Daxx and HDAC1. (A) Representative immunofluorescent images of HIPK1-Flag and K219A-Flag fusions expressed in NIH 3T3 cells and stained with anti-Flag antibody (green) and anti-Daxx antibody (red). The yellow arrows indicate the transfected cells. (B) Western blot from 293 cells expressing pβ, pβ/HIPK1 (HIPK1), or pβ/K219A (K219A) probed with anti-HIPK1 to ensure equal HIPK1 and K219A transfection efficiencies. (C) Daxx was immunoprecipitated (IP) with anti-Daxx antibody from 293 cells expressing an empty vector (pβ), pβ/HIPK1 (HIPK1), or pβ/K219A (K219A). Daxx immunoprecipitates were Western blotted, and the blots were probed with anti-Daxx antibody (upper panel), anti-PML antibody (middle panel), or anti-HDAC1 antibody (lower panel). (D) Representative immunofluorescent images of HIPK1-Flag and K219A-Flag fusions expressed in U2OS cells with pSG5/PML and stained with anti-Flag antibody (green) and anti-PML antibody (red). Overlapping localization is shown in the merged images (yellow).

FIG. 5.

HIPK1 phosphorylates Daxx on Ser 669. Daxx was immunoprecipitated with anti-Myc antibody from 293 cells expressing pCAGGS/Daxx (wild type), pCAGGS/Daxx-S502/669A (S502/669A), pCAGGS/Daxx-S502A (S502A), or pCAGGS/Daxx-S669A (S669A) with an empty vector (pβ), pβ/HIPK1 (HIPK1), or pβ/K219A (K219A). The immunoprecipitates were Western blotted, and the blot was probed with anti-Myc antibody. Daxx* indicates the upper-migrating or hyperphosphorylated bands, and Daxx indicates the lower-migrating or basally phosphorylated bands.

FIG. 6.

HIPK1-Daxx interaction, not phosphorylation of Ser 669, is important in Daxx relocalization. Representative immunofluorescent images of HIPK1-Flag and K219A fusions expressed in NIH 3T3 cells with pCAGGS/Daxx (wild type) or pCAGGS/Daxx-S669A (S669A) and stained with anti-HIPK1 antibody (green) and anti-Myc antibody (red). (B) Western blots from 293 cells expressing pβ/HIPK1 (HIPK1) or pβ/K219A (K219A) alone or in combination with pCAGGS/Daxx (HIPK1/Daxx and K219A/Daxx, respectively). The blots were probed with anti-HIPK1 (upper panel) or anti-Daxx (lower panel) to ensure equal HIPK1, K219A, and Daxx transfection efficiencies. (C) Daxx and HIPK1 were immunoprecipitated (IP) with anti-Daxx and anti-HIPK1 antibodies, respectively, from 293 cells expressing pβ/HIPK1 (HIPK1) or pβ/K219A (K219A) alone or in combination with pCAGGS/Daxx (HIPK1/Daxx and K219A/Daxx, respectively). Daxx and HIPK1 immunoprecipitates were Western blotted, and the blots were probed with anti-HIPK1 (upper panel) or anti-Daxx (lower panel), respectively.

FIG. 7.

Phosphorylation of Ser 669 modulates Daxx activity. 293 cells were transfected with the CRE-luciferase (A), E2F1-luciferase (B), Met-luciferase (C), or Sp1-Luciferase (D) reporter construct with an empty vector (pβ), pCAGGS/Daxx (wild type), or pCAGGS/Daxx-S669A (S669A) and pCMVβ. Data are presented as relative luciferase activity, determined by normalizing the absolute luciferase values for transfection efficiency by measuring the β-galactosidase activity. In each graph, the ordinate is divided into two scales.