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Cullin 3 Mediates SRC-3 Ubiquitination and Degradation to Control the Retinoic Acid Response

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Cullin 3 Mediates SRC-3 Ubiquitination and Degradation to Control the Retinoic Acid Response Cullin 3 mediates SRC-3 ubiquitination and degradation to control the retinoic acid response Christine Ferrya, Samia Gaouara, Benoit Fischerb, Marcel Boeglinc, Nicodeme Pauld, Eric Samaruta, Aleksandr Piskunova, Gabriella Pankotai-Bodoa, Laurent Brinob, and Cecile Rochette-Eglya,1 aDepartment of Functional Genomics and Cancer, bHigh Throughput Screening Facility, dBioinformatics Platform; and cImaging Platform, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche Médicale U964, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7104, Université de Strasbourg, BP 10142, 67404 Illkirch Cedex, France Edited by Johan Auwerx, Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland, and accepted by the Editorial Board November 1, 2011 (received for review February 16, 2011) SRC-3 is an important coactivator of nuclear receptors including the tinated SRC-3 accumulated in the presence of the proteasome retinoic acid (RA) receptor α. Most of SRC-3 functions are facilitated inhibitor MG132 (Fig. 1B). Ubiquitination was also enhanced in by changes in the posttranslational code of the protein that involves response to RA, either in the absence or presence of MG132 mainly phosphorylation and ubiquitination. We recently reported (Fig. 1B). that SRC-3 is degraded by the proteasome in response to RA. Here, Then we aimed at investigating which E3-ubiquitin ligase is by using an RNAi E3-ubiquitin ligase entry screen, we identified CUL- involved in the RA-induced ubiquitination and degradation of 3 and RBX1 as components of the E3 ubiquitin ligase involved in the SRC-3. We performed a high-throughput screen based on the RA-induced ubiquitination and subsequent degradation of SRC-3. use of a siRNA thematic library to create transient gene knock- down in MCF7 cells. The screen was based on the immunoflu- We also show that the RA-induced ubiquitination of SRC-3 depends fi on its prior phosphorylation at serine 860 that promotes binding of orescence analysis of SRC-3 with speci c antibodies. Through the CUL-3–based E3 ligase in the nucleus. Finally, phosphorylation, combining the imaging of cells in microtiter plates with powerful image analysis algorithms, the screen determines whether si- ubiquitination, and degradation of SRC-3 cooperate to control the fi dynamics of transcription. In all, this process participates to the anti- lencing of a speci c E3 ligase reverses the RA-induced degra- dation of SRC-3. proliferative effect of RA. First, the technique was validated by checking that the signal disappears upon knockdown of SRC-3 with specific siRNAs (Fig. etinoic acid (RA) influences cell differentiation, prolifer- 1C). Then kinetic experiments performed after RA addition Ration, and apoptosis through modifications in the expression indicated that SRC-3 degradation occurs within 3–5 h (Fig. 1D). of target genes. The transcription of RA target genes is a highly This degradation process was reversed by siRNAs targeting coordinated process that requires a well-defined cross-talk subunits of the 20S core proteasome (PSMB1 and PSMB2) or among RA nuclear receptors (RARs), basal transcription ma- the SUG-1 subunit of the 19S subcomplex (Fig. 1E) corrobo- chinery, and several transcriptional coregulators including the rating that it involves the 26S proteasome. p160 family of coactivators (SRC-1, SRC-2, and SRC-3) (1). For For the screen, we used a library of 111 siRNAs with four each transcriptional component, there is a fine-tuned code of different siRNAs per target (Dataset S1). Upon statistical anal- posttranslational modifications that control their activity, part- ysis of SRC-3 nuclear intensities displayed in the transfected ners’ association/dissociation, localization, and turnover (2, 3). cells, we determined two lists of candidate genes, differing by the This regulation is especially true for the coactivator SRC-3, level of selection stringency (α = 1.5 and α = 2 for maximum which is a key regulator of nuclear receptors, metabolic ho- stringency). Seven potential hits validated by at least two siRNAs meostasis, and cell proliferation. Indeed, much of its function is (α = 2) were found, among which CUL-3 and RBX1 were highly facilitated through changes in the posttranslational code of the significant (P values) and validated by 3 and 4 siRNAs, re- protein including phosphorylation and several types of post- spectively (Dataset S1 and Fig. 1F). However, the screen did not translational modifications (2, 4, 5). identify with high confidence any of the other E3 ligases (CUL-1- In response to RA, SRC-3 binds to RARs and then recruits skp1-Fbw7α and E6AP) previously reported to regulate SRC-3 a battery of coregulatory proteins such as chromatin remodelers degradation (4, 8) (Fig. 1F). Finally, CUL-3 silencing also re- and modifiers that act in a coordinated and combinatorial versed the increase in SRC-3 ubiquitination observed at 2.5 h manner to decompact chromatin and direct the transcriptional after RA addition (before degradation) (Fig. 1G). In conclusion, machinery to the promoter. Recently, we demonstrated that, in our screen indicates that the RA-induced ubiquitination and response to RA, SRC-3 is degraded by the proteasome (6, 7). degradation of SRC-3 involves a cullin-RING Ligase (CRL) However, the underlying mechanism of SRC-3 degradation and assembled with CUL-3 (CRL3). its link with the transcription of RA target genes was still unclear. Here, in a high-throughput screen based on the use of a siRNA SRC-3 Is Phosphorylated at S860 Before Degradation. CRLs consist thematic library and chemical transfection to create transient of three core components: a cullin scaffold protein, a RING gene knockdown in MCF7 cells, we identified cullin 3 (CUL-3) domain protein (RBX1) that recruits the E2 conjugating enzyme, and the Ring protein RBX1 as components of the E3 ligase and a substrate-adaptor protein that binds target proteins (9). complex involved in SRC-3 ubiquitination and degradation. We Given that CRL3s recognize substrates containing serine-rich also show that SRC-3 degradation is involved in the transcription of RAR target genes and in the antiproliferative action of RA, CELL BIOLOGY “ ” through a phosphorylation-dependent ubiquitination code. Author contributions: C.R.-E. designed research; C.F., S.G., M.B., E.S., A.P., and G.P.-B. performed research; C.F., B.F., M.B., N.P., and A.P. contributed new reagents/analytic Results tools; L.B. analyzed data; and C.R.-E. wrote the paper. CUL-3–Based E3 Ligase Controls RA-Induced Degradation of SRC-3. The authors declare no conflict of interest. Given that in human MCF7 breast cancer cells, SRC-3 is degraded This article is a PNAS Direct Submission. J.A. is a guest editor invited by the Editorial in response to RA by the 26S proteasome (Fig. 1A) (6), we Board. addressed whether this process is regulated by ubiquitination. 1To whom correspondence should be addressed. E-mail: [email protected]. In immunoprecipitation experiments, SRC-3 was constitutively This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ubiquitinated in agreement with other reports (4) and ubiqui- 1073/pnas.1102572108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1102572108 PNAS | December 20, 2011 | vol. 108 | no. 51 | 20603–20608 Downloaded by guest on September 27, 2021 overexpressing SRC-3 WT (Fig. 2D). No signal was obtained with SRC-3 (S860A) (Fig. 2D, lanes 4–6), validating the speci- ficity of the phosphospecific antibodies. Collectively these results indicate that SRC-3 becomes phosphorylated at S860 in response to RA. Phosphorylation at S860 Is the Signal for SRC-3 Ubiquitination/ Degradation and for CUL-3 Binding. Next, we analyzed whether S860 phosphorylation controls the ability of SRC-3 to be ubiq- uitinated and degraded in response to RA. Immunoprecipitation experiments were performed with MCF7 cells and our antibodies recognizing specifically SRC-3 phosphorylated at S860. The amount of phosphorylated SRC-3 increased in response to RA as well as the amount of ubiquitinated SRC-3 (Fig. 2E), and this effect was inhibited upon knockdown of CUL-3 (Fig. 2F). Fi- nally, in contrast to SRC-3 WT, the S860A mutant was not de- graded after RA addition (Fig. 2G). Thus, ubiquitination by CRL3 and degradation concern SRC-3 phosphorylated at S860. Then we compared the ability of FLAG-SRC-3 to coimmu- noprecipitate with HA-CUL-3 in transfected COS cells. SRC- 3WT but not SRC-3 (S860A) was pulled down with CUL-3 in response to RA (Fig. 2H). Altogether these results indicate that the RA-induced ubiquitination and degradation of SRC-3 re- quire a priming phosphorylation at S860 that controls the bind- ing of CUL-3 complexes. CUL-3 Migrates to the Nucleus in Response to RA. Then we analyzed the intracellular distribution of CUL-3 and SRC-3 in MCF7 cells by immunofluorescence and confocal analysis (Fig. 3A). In the ab- sence of RA, SRC-3 was present essentially in nuclei (Fig. 3A, a) and CUL-3 in the cytoplasm (Fig. 3A, c). After RA treatment, CUL-3 accumulated at the perinuclear surface and in nuclei where it colocalized with SRC-3 (Fig. 3A, d, f,andh). That CUL- 3 migrates to nuclei was corroborated by immunoblotting of nu- Fig. 1. Screening of the E3 ligase involved in the RA-induced degradation clear extracts (Fig. 3B). Most interestingly, the amount of SRC-3 and ubiquitination of SRC-3. (A and B) Extracts from MCF7 cells treated or phosphorylated at S860 also increased in nuclei (Fig. 4A). not with RA (0.1 μM) and MG132 (4 μM) were analyzed by immunoblotting Next, to explore further the interaction between CUL-3 and for SRC-3 degradation and for SRC-3 ubiquitination after immunoprecipi- SRC-3 phosphorylated at S860, we used a proximity ligation fl tation.
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