Phosphorylation by p38MAPK and recruitment of SUG-1 are required for RA-induced RAR gamma degradation and transactivation

Abstract

The nuclear retinoic acid receptor RAR gamma 2 undergoes proteasome-dependent degradation upon ligand binding. Here we provide evidence that the domains that signal proteasome-mediated degradation overlap with those that activate transcription, i.e. the activation domains AF-1 and AF-2. The AF-1 domain signals RAR gamma 2 degradation through its phosphorylation by p38MAPK in response to RA. The AF-2 domain acts via the recruitment of SUG-1, which belongs to the 19S regulatory subunit of the 26S proteasome. Blocking RAR gamma 2 degradation through inhibition of either the p38MAPK pathway or the 26S proteasome function impairs its RA-induced transactivation activity. Thus, the turnover of RAR gamma 2 is linked to transactivation.

Fig. 1. RA-induced RARγ2 degradation and transactivation are reversed by the proteasome inhibitor MG132 and require engagement of the receptor at a RARE. (A) COS-1 cells cotransfected with the DR5-tk-CAT reporter construct and the expression vectors for mRARγ2 (WT or ΔC) and RXRα, were treated for 48 h with vehicle or 1 × 10^–6 M RA. When mentioned, MG132 was added 15 h before harvesting. In lanes 15 and 16 the DR5 element of the CAT reporter gene was mutated and in lanes 17 and 18 the DR5 element was deleted. WCEs were immunoblotted with RPγ(F) (upper panels) or actin antibodies (lower panels). (B) COS-1 cells were cotransfected with the DR5-tk-CAT reporter construct and the expression vector for mRARγ2, in the absence (–) or presence (+) of RXRα as indicated. In lane 5 the DR5 responsive element was mutated. The cells were treated as in (A) and analyzed for CAT activity. The results are the mean ± SD of three independent experiments.

Fig. 2. RA-induced RARγ2 degradation and transcription are also reversed by MG132 in F9 cells. (A) F9 WT cells were treated for 48 h with vehicle or 1 × 10^–7 M RA. When mentioned, MG132 was added 15 h before harvesting. Whole cell extracts (WCEs) were resolved by SDS–10% PAGE and immunoblotted with RPγ(F), RPRXα(A), or actin antibodies. (B) F9 WT cells were treated for 48 h as in (A), as indicated. Transcripts for collagen IV, laminin B1, Stra4, HNF1β, HNF3α and RARγ2 were analyzed by quantitative RT–PCR. The presented values are the mean ± SD of three individual experiments and correspond to the fold-induction relative to the amount of transcripts present in vehicle-treated cells which was given an arbitrary value of 1. (C) RXRα^–/– F9 cells were treated as in (A) and WCEs were immunoblotted with RPγ(F) or actin antibodies. (D) F9 cells that were either WT (lanes 1–4) or expressing RARγΔH12 (lanes 5–8) or RARγS66/68A (lanes 9–12) were treated as in (A) and WCEs were immunoblotted with RPγ(F) or actin antibodies.

Fig. 3. Both the AF-1 and AF-2 activation domains contribute to the RA-induced degradation of RARγ2. (A) Schematic representation (not to scale) of mRARγ2 with the DBD and the functional AF-1 and AF-2 domains, which lie in the A/B and E regions, respectively. The target sequence for phosphorylation by proline-directed kinases in the B region is shown and the corresponding serine residues, which have been mutated to alanine (S66 and S68), are indicated. (B) COS-1 cells were cotransfected with the DR5-tk-CAT reporter construct and the expression vector for mRARγ2 either WT, ΔF, ΔH12, ΔAB, ΔA, ΔB or S66/68A and treated with vehicle or 1 × 10^–6 M RA as indicated. When mentioned, MG132 was added 15 h before harvesting. Equal amounts of WCEs, as estimated by immunoblotting with actin antibodies (data not shown) were resolved by SDS–10% PAGE and immunoblotted with RPγ(F) or Ab5γ(D) in the case of RARγ2ΔF. (C) Cells transfected as in (B) were analyzed for CAT activity. The results are the mean ± SD of three independent experiments.

Fig. 4. Disruption of the ubiquitin-activating enzyme (UBA) function abrogates RA-induced RARγ2 degradation and transactivation. The temperature-sensitive UBA mutant ts85 cell line was transfected with the DR5-tk-CAT reporter gene and the expression vector for mRARγ2, treated with vehicle or 1 × 10^–6 M RA and incubated at permissive (30°C) or restrictive (37°C) temperature for 24 h before harvesting. Extracts were immunoblotted with RPγ(F) and actin antibodies (A) and analyzed for CAT activity (B). The results, which correspond to the fold-induction relative to the CAT activity in vehicle-treated cells, are the mean ± SD of three independent experiments.

Fig. 5. RA increases the amount of RARγ2 phosphorylated in its AF-1 domain, subsequent to the activation of p38MAPK. (A) COS-1 cells plated in 10 cm Petri dishes and cotransfected with the DR5-tk-CAT reporter construct and the expression vector for mRARγ2 either WT (lanes 1–4) or S66/68A (lanes 5 and 6) were treated with vehicle (lane 1) or 1 × 10^–6 M RA (lane 2). In lanes 3 and 4, RA was combined with SB203580 (10 µM) or PD98059 (5 µM), respectively. Lanes 7 and 8 correspond to F9 WT cells, treated or not with RA (1 × 10^–7 M). Cells were labelled with [³²P]orthophosphate and WCEs were immunoprecipitated with mAb2γ(mF). Immunoprecipitates containing equal amounts of RARγ2 were resolved by SDS–10% PAGE, electrotransferred onto nitrocellulose (NC) filters, autoradiographed [³²P] and immunoprobed with RPγ(F) by western blotting (WB). (B) Two-dimensional tryptic phosphopeptide mapping of ³²P-labelled immunoprecipitated RARγ2WT (panels 1–3) and RARγ2S66/68A (panel 4). (C) RA activates p38MAPK. Transfected COS-1 cells (lanes 1 and 2) and F9 WT cells (lanes 3 and 4), were treated for 24 h with vehicle or RA as indicated. Then the cells were lysed and immunoprecipitated with a p38MAPK rabbit polyclonal antibody immobilized on Protein A– Sepharose beads. The immunoprecipitates were immunoblotted with antibodies recognizing specifically p38MAPK or its phosphorylated form, P-p38MAPK. (D) Phosphorylation of ATF-2 upon activation of p38MAPK. F9 WT cells were treated for 48 h with vehicle (lane 1), 1 × 10^–7 M RA (lane 2), 10 µM SB203580 (lane 3), or 5 µM PD98058 (lane 5). In lanes 4 and 6, RA was combined with SB203580 or PD98058. WCEs were immunoprecipitated with a Phospho-p38MAPK rabbit polyclonal antibody immobilized on Protein A–Sepharose beads, washed and processed for phosphorylation of the 40 kDa ATF-2 fusion protein (5 µg) in kinase buffer (25 mM HEPES, 25 mM MgCl₂, 25 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na₃V0₄ and 20 µM ATP) for 30 min at room temperature. The reaction was terminated upon addition of the SDS sample buffer, and the phospho (P-ATF-2) and non-phospho forms of ATF-2 were detected by immunoblotting with specific antibodies.

Fig. 6. Phosphorylation by p38MAPK is required for RA-induced RARγ2 degradation and transactivation. (A) COS-1 cells cotransfected with the DR5-tk-CAT reporter construct and the expression vector for mRARγ2 were treated for 48 h with vehicle, RA, SB203580 or PD98058, either alone or in combination, as indicated. WCEs were resolved by SDS–10% PAGE and immunoblotted with RPγ(F) (upper panels) or actin antibodies (lower panels). (B) F9 WT cells were treated for 48 h with vehicle, 1 × 10^–7 M RA, 10 µM SB203580 or 5 µM PD98059 either alone or in association as indicated. Transcripts for laminin B1, HNF3α, HNF1β and RARγ2 were analyzed by quantitative RT–PCR. The presented results are an average of at least three independent experiments which agreed within ±15%. The values correspond to the fold-induction relative to the amount of transcripts present in vehicle-treated cells.

Fig. 7. Overexpression of SUG-1 interferes with RA-induced RARγ2 degradation and transactivation. (A) COS-1 cells were cotransfected with the DR5-tk-CAT reporter construct and the expression vector for mRARγ2, without (lanes 1 and 2) or with increasing amounts (0.1, 0.2 and 0.5 µg) of an expression vector for SUG-1 [either WT (lanes 3–5), or mutated (lanes 8–10)] and treated for 48 h with vehicle (lanes 1 and 6) or 1 × 10^–6 M RA (lanes 2–5 and 7–10). Equal amounts of WCEs were immunoblotted with RPγ(F), SUG-1 antibodies or actin antibodies. (B) COS-1 cells were cotransfected and RA-treated as in (A). After 48 h, extracts were analyzed for CAT activity. Lanes 1 and 2 correspond to the activity of the reporter gene in the absence of cotransfected RARγ2. The results correspond to the fold-induction relative to the CAT activity in the absence of RA. They are the mean ± SD of three independent experiments. (C) COS-1 cells were cotransfected with the DR5-tk-CAT reporter construct and the expression vector for mRARγ2 with or without the vectors (0.5 µg) for SUG-1WT, TIF2 and SRC-1 as indicated. Cells were treated for 48 h with vehicle or 1 × 10^–6 M RA and processed as in (A). (D) COS-1 cells were cotransfected with the (17mer)×5-TATA-CAT reporter construct and the expression vector for Gal-VP16 with or without the SUG-1 vector. WCEs were immunoblotted with antibodies recognizing the DNA binding domain of GAL4 or β-actin. In lane 2, cells were treated with MG132, 15 h before harvesting. (E) COS-1 cells were treated for 48 h with vehicle or with RA. In lane 2, cells were treated with MG132, 15 h before harvesting. In lane 4, cells were transfected with the SUG-1 expression vector and RA-treated. WCEs were immunoblotted with antibodies recognizing cyclin D1 or β-actin.

Fig. 8. RA-induced degradation of RARγ2 by the ubiquitin–proteasome pathway. Upon ligand binding, RARγ2/RXR heterodimers bound to a RAR response element (step 1) recruit coactivators that decompact chromatin and allow the recruitment of the general transcription machinery at the promoter. Then RARγ2 becomes phosphorylated at Ser66 or Ser68 by the cdk7 subunit of TFIIH (step 2). Assembly of the transcription initiation complex leads to transcription initiation. After 24 h of RA treatment, p38MAPK is activated (step 3), resulting in the increase of the phosphorylation of both serine residues located in the AF-1 domain (step 4). This acts as a signal for ubiquitylation (step 5) and subsequent recognition and degradation (step 6) by proteasomal SUG-1 bound to the AF-2 domain.