The homeodomain transcription factor Hoxa2 interacts with and promotes the proteasomal degradation of the E3 ubiquitin protein ligase RCHY1

Abstract

Hox proteins are conserved homeodomain transcription factors known to be crucial regulators of animal development. As transcription factors, the functions and modes of action (co-factors, target genes) of Hox proteins have been very well studied in a multitude of animal models. However, a handful of reports established that Hox proteins may display molecular activities distinct from gene transcription regulation. Here, we reveal that Hoxa2 interacts with 20S proteasome subunits and RCHY1 (also known as PIRH2), an E3 ubiquitin ligase that targets p53 for degradation. We further show that Hoxa2 promotes proteasome-dependent degradation of RCHY1 in an ubiquitin-independent manner. Correlatively, Hoxa2 alters the RCHY1-mediated ubiquitination of p53 and promotes p53 stabilization. Together, our data establish that Hoxa2 can regulate the proteasomal degradation of RCHY1 and stabilization of p53.

Figure 1. Hoxa2 interacts with RCHY1, PSMA3 and PSMB2.

(A) Yeast two-hybrid analyses with Hoxa2 as prey (AD-Hoxa2) and PSMA3, PSMB2 or RCHY1 as bait (DB-PSMA3, DB-PSMB2, DB-RCHY1). Yeast transformed with expression vectors for GAL4-DB and GAL4-AD fusion proteins were mated on complete medium (YPD) and transferred on synthetic dropout medium plates lacking histidine, leucine and tryptophan (-L-W-H) to select diploids in which the GAL1-HIS3 reporter is activated as a consequence of hybrid proteins interaction. Negative control plates were composed of synthetic dropout medium containing cycloheximide and lacking histidine and leucine (-L+W-H+C) and positive controls for matings were transferred on synthetic dropout medium plates lacking leucine and tryptophan (-L-W). (B) Co-precipitation assays. HEK293T cells were co-transfected with expression vectors for FLAG- and GST-tagged Hoxa1 as a positive control, for FLAG-tagged Hoxa2 alone as a negative control and for FLAG-tagged and GST-tagged PSMA3 or GST-tagged PSMB2. Forty-eight hours after transfection, cell lysates were subjected to western blotting analyses to detect protein expression (beta-actin used as a protein load control). Protein interactions were then verified by co-precipitation on glutathione beads directed toward the GST tag. Eluted proteins were analysed by western blotting using M2 antibody to detect the presence of FLAG-tagged Hoxa2 (CoP). (C) Similar co-precipitation assays using MG132 or DMSO treated HEK293T cells reveal FLAG-tagged Hoxa2 and GST-tagged RCHY1 interaction upon proteasome inhibition.

Figure 2. Hoxa2 induces proteasome-dependent ubiquitin-independent RCHY1 degradation and colocalizes with RCHY1 in the nucleus.

(A) HEK293T cells were co-transfected with expression vectors for Hoxa2 and FLAG-tagged RCHY1, and treated with MG132 proteasome inhibitor, or DMSO. Cell lysates were loaded on SDS-PAGE for proteins separation and western blotting (beta-actin used as a protein load control). (B) FLAG-Hoxa2 and GST-RCHY1 colocalize in the nucleus. HEK293T cells were co-transfected with FLAG-tagged Hoxa2 and GST-tagged RCHY1, treated with MG132 proteasome inhibitor, or DMSO, and subjected to immunocytochemistry with anti-FLAG M2 antibody (green) and anti-GST antibody (red). Nuclei were stained with DAPI (blue). (C) Ubiquitination assays for FLAG-tagged RCHY1. HEK293T cells were co-transfected with indicated expression vectors and treated with MG132. Cells were then lysed and 6His-ubiquitin conjugated proteins were purified using Ni-NTA beads. Purified proteins and cell lysates were analysed by western blotting using M2 antibody to detect ubiquitinated forms of FLAG-tagged RCHY1. Lysate samples were loaded on a SDS-PAGE to verify protein levels prior to purification (Input; beta-actin was used as a protein load control). Lane numbering under the gels identifies cell samples. I: input sample; P: Ni-NTA purified sample.

Figure 3. The integrity of the Hoxa2 homeodomain is required for the Hoxa2-induced RCHY1 decay.

(A) Co-precipitation assays involving mutant forms of Hoxa2. HEK293T cells were cotransfected with expression vectors for FLAG-tagged Hoxa2 (FLAG-Hoxa2wt), FLAG-tagged Hoxa2^KQN-RAA (FLAG-Hoxa2KQN-RAA), FLAG-tagged Hoxa2^WM-AA (FLAG-Hoxa2WM-AA), GST-tagged RCHY1 and GST proteins, and treated with the proteasome inhibitor MG132. Forty-eight hours after transfection, cell lysates were subjected to western blotting (input) and protein interactions were verified by co-precipitation on glutathione beads directed toward the GST tag. Eluted proteins were analysed by western blotting using the M2 anti-FLAG antibody (CoP). (B) Amino acid substitutions in the Hoxa2 homeodomain abolish the Hoxa2-mediated degradation of RCHY1. HEK293T cells were transfected with expression vectors for FLAG-tagged RCHY1 (FLAG-RCHY1) and GST-tagged Hoxa2 (GST-Hoxa2wt, GST-Hoxa2KQN-RAA, GST-Hoxa2WM-AA) proteins. Cells were then treated for proteasome inhibition (MG132) and compared to untreated controls (DMSO) for the decay of FLAG-RCHY1 revealed by western blot detection. Detection of beta-actin was used as a protein load control.

Figure 4. Hoxa1 does not promote RCHY1 degradation.

(A) Co-precipitation assays involving Hoxa1. HEK293T cells were cotransfected with expression vectors for FLAG-tagged Hoxa1 (FLAG-Hoxa1), GST-tagged RCHY1 and GST proteins, and treated with the proteasome inhibitor MG132. Forty-eight hours after transfection, cell lysates were subjected to western blotting (input) and protein interactions were verified by co-precipitation on glutathione beads directed toward the GST tag. Eluted proteins were analysed by western blotting using the M2 anti-FLAG antibody (CoP). (B) Hoxa1 does not promote the degradation of RCHY1. HEK293T cells were transfected with expression vectors for FLAG-tagged RCHY1 (FLAG-RCHY1) and GST-tagged Hoxa1 (GST-Hoxa1) proteins. Cells were then treated for proteasome inhibition (MG132) and compared to untreated controls (DMSO) for the decay of FLAG-RCHY1 revealed by western blot detection. Detection of beta-actin was used as a protein load control.

Figure 5. Hoxa2 inhibits RCHY1-dependent ubiquitination of p53 and stabilizes p53.

(A) Ubiquitination assays for p53. HEK293T cells were co-transfected with indicated plasmids and treated with MG132. Cells were lysed and Ni-NTA beads were used to pull down 6His-ubiquitin-conjugated proteins. Proteins were separated by SDS–PAGE and western blotting was performed using an anti-p53 antibody to detect ubiquitinated forms of p53. Lysate samples were loaded on a SDS-PAGE to verify protein levels prior to purification (Input; beta-actin was used as a protein load control). Lane numbering under the gels identifies cell samples. I: input sample; P: Ni-NTA purified sample. (B) p53 protein stabilization by Hoxa2. PA1 cells were transfected with indicated plasmids and treated with MG132, or DMSO as control. Cells were lysed, proteins were separated by SDS-PAGE and western blot detection was performed with indicated antibodies.