SMRT Derepression by the IκB Kinase α Jamie E. Hoberg, Fan Yeung, Marty W. Mayo  Molecular Cell  Volume 16, Issue 2, Pages 245-255 (October 2004) DOI: 10.1016/j.molcel.2004.10.010

Figure 1 Cellular Attachment Stimulates NF-κB for Survival (A) Attachment stimulates NF-κB DNA binding activity in EMSA. The Oct-1 probe serves as a loading control. Supershift (SS) complexes indicate NF-κB/DNA complex composed of RelA/p65 and p50 in DU145 nuclear extracts. The p50/p65, p50/p50, and the SS complexes are shown. (B) DU145 cells expressing the NF-κB-responsive luciferase reporter (3× κB-Luc) or the mutant luciferase reporter (3× mut κB-Luc) were attached to laminin over the time course indicated, and luciferase activities were determined. Data represent the mean ± SD of three independent experiments performed in triplicate. (C) RNase protection assays were performed on DU145 cells expressing either the SR-IκBα or the GFP control proteins. Detection of the mutant IκBα transcript confirms the expression of the SR-IκBα. (D) DU145 cells expressing either the SR-IκBα protein or the vector control (CMV) were plated on laminin and analyzed for induction of apoptosis by measuring nucleosome release and caspase-3 activity. Data represent three independent experiments performed in duplicate. Molecular Cell 2004 16, 245-255DOI: (10.1016/j.molcel.2004.10.010)

Figure 2 Inverse Correlation between Chromatin Bound SMRT and IKKα (A) IKKα and IKKβ translocate to the nucleus after attachment to laminin. Cytosolic (C) and nuclear (N) protein extracts isolated from DU145 cells after attachment were analyzed by immunoblot. (B) Laminin attachment stimulates IKK activity as shown by in vitro kinase assay with a phosphospecific IκBα (S32) antibody. Immunoprecipitated IKKα and total GST-IκBα proteins are shown. (C and D) Inverse correlation between chromatin bound IKKα and SMRT was observed in ChIP analysis with DU145 cells after attachment. The GAPDH served as a negative control. Molecular Cell 2004 16, 245-255DOI: (10.1016/j.molcel.2004.10.010)

Figure 3 IKKα Regulates Chromatin-Associated SMRT Levels (A) siRNAs directed to either IKKα or IKKβ knockdown IKK protein expression in DU145 cells as shown by Western blot analysis. (B) SMRT derepression is regulated by IKKα. ChIP analysis of DU145 cells expressing siRNAs to IKKα, IKKβ, or control after attachment. RT-PCR analysis demonstrates that siRNA to IKKα and IKKβ disrupts cIAP-2 and IL-8 gene expression. (C) p50 is required for SMRT/HDAC3 occupancy. ChIP analysis of DU145 cells expressing siRNA to p50 demonstrates the importance of p50 for the recruitment of SMRT and HDAC3 to the cIAP-2 and IL-8 promoters. siRNAs to p50 specifically knock down protein expression. (D) IKKα inhibits SMRT-mediated deacetylation of RelA/p65. Acetylation assays were performed as described in Experimental Procedures. Acetylated RelA/p65 and IgG heavy chain proteins are shown. Immunoprecipitated RelA/p65 proteins were detected with α-FLAG antibody. (E) IKKα relieves SMRT-mediated repression of RelA/p65. DU145 cells were cotransfected with plasmids encoding Gal4-p65 fusion protein, the 4× Gal4-luciferase reporter, and with expression vectors encoding either SMRT or vector control (pCMV). Additionally, cells were cotransfected with expression vectors encoding activated Akt, IKKα, or dominant negative (DN) IKKα proteins. Molecular Cell 2004 16, 245-255DOI: (10.1016/j.molcel.2004.10.010)

Figure 4 IKKα Directly Phosphorylates SMRT to Stimulate Nuclear Export (A) IKKα phosphorylates SMRT in vitro. Illustration of the SMRT protein showing the repressor domains, the nuclear receptor interacting domains I and II, and the GST-SMRT fusion protein constructs. The potential IKKα phosphorylation sites are identified by in vitro kinase assays. Autophosphorylated IKKα and radiolabeled GST-SMRT proteins are shown. GST-SMRT protein levels are shown by Coomassie stain (bottom). (B) Table summarizing IKKα-mediated localization of FLAG-SMRT and mutant SMRT proteins. (C) Requirement of S2028 and S2410 for IKKα-mediated export of SMRT. DU145 cells expressing either FLAG-tagged SMRT or mutant SMRT(S2028,2410A) proteins were analyzed for cytosolic (C) and nuclear (N) proteins after expression of HA-IKKα (+) or the vector control (−). RNA Pol II and p105 served as nuclear and cytoplasmic loading controls. (D) Laminin attachment stimulates IKKα-mediated nuclear export of SMRT. The protein level of FLAG-SMRT or mutant SMRT(S2028,2410A) in DU145 cells at T0 (unattached) or T30 (attached) are shown in Western blot. Polyhema plates were used as a negative control for attachment. Molecular Cell 2004 16, 245-255DOI: (10.1016/j.molcel.2004.10.010)

Figure 5 IKKα Phosphorylates SMRT on Chromatin to Stimulate Nuclear Export (A) IKKα in vitro kinase assays were performed to demonstrate the selectivity of the phosphospecific SMRT(S2410) antibody. (B) Endogenous SMRT becomes phosphorylated at S2410 in vivo. Whole-cell extracts, isolated from unstimulated cells (No Add) or cells after TNFα or laminin attachment, were immunoblotted with the SMRT(pS2410) antibody. siRNA knockdown demonstrates the importance of IKKα for phosphorylating SMRT in response to TNFα or attachment. Total SMRT protein shown as loading control. (C) IKKα phosphorylates SMRT at S2410 on chromatin. ChIP analysis of the cIAP-2 promoter was performed on DU145 cells after attachment. (D) SMRT(S2410) phosphorylation is associated with proteasome-dependent degradation of SMRT. Cytosolic (C) and nuclear (N) proteins were isolated from DU145 cells after the attachment in either the absence (−) or presence (+) of MG132. RNA Pol II and p105 served as nuclear and cytoplasmic loading controls. Molecular Cell 2004 16, 245-255DOI: (10.1016/j.molcel.2004.10.010)

Figure 6 SMRT Phosphorylation by IKKα Is Required for Derepression, NF-κB Transcription, and Survival (A) ChIP analysis indicated the recruitment of E2 and E3 ubiquitin ligase complex and the 14-3-3 chaperone after attachment in DU145. (B) Phosphorylation of SMRT(S2410) by IKKα is required for 14-3-3ϵ binding as shown in GST pull-down assay. [S35]-SMRT and Coomassie blue-stained GST-14-3-3 proteins are shown. (C) Phosphorylation of SMRT at S2028 and S2410 is critical for derepression as shown in ChIP analysis with HEK 293T cells transiently expressing either wild-type SMRT or mutant SMRT(S2028,2410A). (D) Expression of mutant SMRT blocks NF-κB transcription. RNase protection assays demonstrate that HEK 293T cells overexpressing the mutant SMRT proteins display reduced cIAP-2 and IL-8 expression, as compared to cells expressing wild-type SMRT protein. (E and F) Loss of cell viability and induction of apoptosis were observed after expression of the mutant SMRT protein. (E) DU145 cells expressing either GFP-SMRT or mutant GFP-SMRT(S2028,2410A) proteins were counted for cell viability. The percent cell viability was determined by counting CFP-positive DU145 cells upon attachment. (F) DU145 cells expressing either GFP-SMRT or mutant SMRT were attached to laminin (T30 or T90 min) and analyzed for GFP fluorescence or active caspase-3 protein expression with a rhodamine-conjugated secondary antibody. Molecular Cell 2004 16, 245-255DOI: (10.1016/j.molcel.2004.10.010)

Figure 7 IKKα Regulates SMRT Derepression Classic NF-κB-regulated genes, like cIAP-2 and IL-8, are susceptible to SMRT-mediated repression where p50:p50 homodimers recruit SMRT and HDAC3 activities. After stimulation, IKKα phosphorylates SMRT on chromatin to recruit TBL1/TBLR1 and Ubc5 proteins. Secondly, IKKα-induced phosphorylation of SMRT is associated with the recruitment of 14-3-3ϵ and Ubc5 binding (T30), which is required for nuclear export and proteasome-dependent targeting of SMRT. Full derepression is required before the RelA/p65:p50 heterodimer and HATs are recruited to chromatin, fulfilling the exchange of corepressor for coactivator complexes. Molecular Cell 2004 16, 245-255DOI: (10.1016/j.molcel.2004.10.010)