Volume 12, Issue 2, Pages 413-423 (August 2003) TORCs  Michael D. Conkright, Gianluca Canettieri, Robert Screaton, Ernesto Guzman, Loren Miraglia, John B. Hogenesch, Marc Montminy  Molecular Cell  Volume 12, Issue 2, Pages 413-423 (August 2003) DOI: 10.1016/j.molcel.2003.08.013

Figure 1 Identification of a Family of Transducers of Regulated CREB Activity (TORCs) (A) Left, transient assay of EVX-1 reporter cotransfected into HEK293T cells with TORC1 and TORC2 expression vectors. Right, effect of TORC1 and TORC2 on reporters containing consensus sequences for AP-1, heat shock (HSE), serum response (SRE), NF-κB, and glucocorticoid receptor (GRE) response elements relative to control (minimal promoter) or CRE-containing vectors. (B) Left, effects of TORCs and PKA on induction of wild-type and CRE mutant EVX-1 promoters. Middle, effect of cotransfected dominant-negative A-CREB expression vector on induction of the EVX-1 reporter by TORC1 and TORC2 in HEK293T cells. Right, TORCs act synergistically with cAMP agonist to regulate CRE reporter activity. Transient assay of the EVX-1 promoter in HEK293T cells cotransfected with either TORC1, TORC2, P300, or CBP expression vector. Treatment with forskolin indicated. (C) TORC potentiation of CREB activity is promoter context dependent. Effect of TORC1 and TORC2 overexpression on TATA-less (gray bars; MAGOH, SENP2, YWHAH, BNIP3L, CDC37) and TATA-containing (black bars; CDX4, FLT, NR4A2, SST) promoters, all of which contain promoter proximal CREs, in HEK293T cells. Right, effects of TORC1, TORC2, and PKA overexpression on wild-type CDC37 and mutant CDC37+TATA promoters in HEK293T cells. Molecular Cell 2003 12, 413-423DOI: (10.1016/j.molcel.2003.08.013)

Figure 2 TORC Is Required for Activation of cAMP-Responsive Genes via CREB (A) Transient assay of EVX-1 luciferase reporter in CREB−/− MEFs transfected with empty vector, wild-type, or phosphorylation defective (Ser133Ala) CREB expression vector. Cotransfection with TORC1 plasmid indicated. (B) Effect of TORC1 on endogenous expression of the TATA-containing NR4A2 gene. Levels of NR4A2 induction were determined by quantitative PCR analysis of RNA from HEK293T cells transfected with TORC1 expression vector. Cotransfection with dominant-negative A-CREB expression vector indicated. Effect of forskolin treatment shown. (C) Effect of CREB and TORC knockdown on cAMP-dependent reporter activity. Transient assay of EVX-1 luciferase reporter in HEK293T cells cotransfected with short hairpin RNAi vectors for CREB and TORC2 shown. Control cells were cotransfected with nonspecific RNAi vector. Treatment with forskolin indicated. (D) Effect of CREB and TORC2 knockdown on endogenous NR4A2 expression. HEK293T cells were transfected with CREB or TORC2 RNAi vectors, and levels of NR4A2 mRNA were determined by quantitative RT-PCR analysis after 72 hr. (E) Western blot assay of endogenous TORC protein levels in HEK293T cells transfected with control (nonspecific), TORC1, or TORC2 RNAi vectors as indicated. Molecular Cell 2003 12, 413-423DOI: (10.1016/j.molcel.2003.08.013)

Figure 3 CREB Interacts with TORCs via Its bZIP DNA Binding/Dimerization Domain (A) Left, coimmunoprecipitation assay of Flag-tagged TORC1 and HA-tagged CREB in transfected HEK293T cells. Anti-HA tag Western blot assay was performed on immunoprecipitates of TORC1 prepared with anti-Flag beads. To control for nonspecific binding to beads, extracts were preincubated with excess Flag peptide prior to immunoprecipitation with anti-Flag beads (IP+Flag peptide). 5% input CREB and immunoprecipitated HA-CREB bands shown. Center, Western blot assay of endogenous TORC detected in immunoprecipitates of FLAG-tagged CREB (CREB Ab) from HEK293 stable line expressing Flag-tagged CREB protein. Effect of excess FLAG peptide addition to anti-FLAG beads prior to immunoprecipitation shown. Input levels (5%) of TORC also indicated. Right, Western blot assay of CREB levels in whole-cell extracts of parent HEK293 and FL-CREB stable cells. CREB and Flag-CREB bands indicated. (B) Top, GST pull-down assay of full-length 35S-labeled TORC1 with GST resins containing either full-length CREB-related CREMα protein, CREB ΔbZIP (aa 1–283), Q1 domain (aa 1–100), the KID domain (aa 100–160), Q2 domain (aa 160–283), the bZIP domain (aa 284–341), or GST resin alone shown. Bottom, transient assay of GAL4 luciferase activity in HEK293T cells transfected with GAL4 CREB polypeptides containing the GAL4 DNA binding domain fused to either full-length CREB, to the CREB transactivation domain (aa 1–283), or to the bZIP domain alone (aa 285–341). Cells were cotransfected with TORC1 expression vector or control empty vector and treated with forskolin or vehicle as indicated. (C) Effect of TORC1 on CREB DNA binding activities in HEK293T cells. Gel mobility shift assay of nuclear extracts from HEK293T cells prior to (left) or following (right) transfection with increasing amounts of empty vector or TORC1 expression plasmid. Nuclear extracts were incubated with 32P-labeled CRE oligo corresponding to the SST promoter, and complexes were resolved by nondenaturing gel electrophoresis. Effect of anti-Flag or anti-CREB antisera on mobility of CREB:CRE complexes shown. Addition of 100-fold molar excess of CRE competitor oligo to binding reaction indicated. CRE binding complexes I and II marked. (D) Chromatin immunoprecipitation assay of HEK293T cells using anti-TORC antiserum or nonspecific IgG. PCR amplification of NR4A2 promoter fragment from TORC and IgG immunoprecipitates shown. Decrescendo bar indicates decreasing amounts of input DNA (10, 1, 0.1%) to show linearity of the assay. Molecular Cell 2003 12, 413-423DOI: (10.1016/j.molcel.2003.08.013)

Figure 4 TORCs Enhance the Association of CREB with TAFII130 (A) Left, transient assay of EVX-1 reporter in HEK293T cells cotransfected with wild-type and C-terminal deletion mutants of TORC1. Amino acid endpoints for each TORC1 polypeptide indicated. Right, activity of GAL4 luciferase reporter in cells cotransfected with wild-type and mutant GAL4-TORC1 expression vectors. (B) Effect of TORC1 and TORC2 overexpression on interaction of CREB with TAFII130. Coimmunoprecipitation assay of Flag-tagged TAFII130 and HA-tagged CREB in HEK293T cells cotransfected with TORC1, TORC2, or control expression vectors. Recovery of CREB from TAFII130 immunoprecipitates determined by Western blot assay with anti-HA tag antiserum. 5% input HA-CREB in each sample shown. (C) Coimmunoprecipitation assay of Flag-tagged TORC1 and HA-tagged TAFII130 in transfected HEK293T cells. Anti-HA tag Western blot assay was performed on immunoprecipitates of TORC1 prepared with anti-Flag beads. To control for nonspecific binding to beads, extracts were preincubated with excess Flag peptide prior to immunoprecipitation with anti-Flag beads (IP+Flag peptide). 5% input TAFII130 and immunoprecipitated HA-TAFII130 bands shown. (D) Western blot of phospho(Ser133) CREB levels in HEK293T cells after stimulation with forskolin (10 μM, 30 min) or following cotransfection with TORC1, TORC2, or empty expression vector. Comparable amounts of total CREB in each sample were verified by Western blot assay with nondiscriminating CREB antiserum (data not shown). Molecular Cell 2003 12, 413-423DOI: (10.1016/j.molcel.2003.08.013)

Figure 5 TORCs Bind to CREB via an N-Terminal Tetrameric Coiled-Coil Domain (A) Glutaraldehyde crosslinking assay of 35S-labeled TORC (left) and CREB (full-length and ΔbZIP) (right) polypeptides in vitro. Samples were incubated with .005% glutaraldehyde for 30 min at room temperature, and crosslinked products were resolved by SDS-PAGE. Arrow points to monomeric 50 kDa TORC (1–435) polypeptide (left). Asterisks placed alongside crosslinked TORC and CREB products. (B) GST pull-down assay of C-terminally truncated 35S-labeled TORC1 polypeptides (full length, 1–629, 1–435) using GST CREB bZIP or control GST resins. 5% input labeled TORC polypeptides shown. Bottom, DNA is not required for binding of CREB bZIP to N-terminal coiled-coil domain of TORC1. Effect of ethidium bromide addition on binding of TORC (aa 1–56) peptide to CREB bZIP by GST pull-down assay. Molecular Cell 2003 12, 413-423DOI: (10.1016/j.molcel.2003.08.013)

Figure 6 The N-Terminal CREB Binding Domain of TORC1 Is Translocated onto the Mastermind Coactivator MAML2 in Mucoepidermoid Carcinomas (A) Sequence alignment of mammalian, Fugu, Drosophila, and C. elegans TORC coiled-coil domains with homologous regions highlighted. Translocation of TORC1 onto MAML2 in TORC1/MAML2 fusion protein indicated. (B) Coimmunoprecipitation assay of Flag-tagged TORC1/MAML2 fusion protein and HA-tagged CREB in transfected HEK293T cells. Anti-HA tag Western blot assay was performed on immunoprecipitates of TORC1/MAML2 fusion protein prepared with anti-Flag beads. 5% input CREB and immunoprecipitated HA-CREB shown. (C) TORC1 (MECT1)/MAML2 oncogene containing N-terminal CREB binding domain of TORC1 fused to the Notch coactivator Mastermind-like 2 (MAML2) stimulates CREB activity. Transient assay of EVX-1 luciferase reporter in HEK293T cells cotransfected with TORC1/MAML2, MAML2, or full-length TORC1 expression vectors. Effect of dominant-negative A-CREB expression vector indicated. (D) TORC1/MAML2 oncogene stimulates the Notch target gene HES-1 via a CREB-dependent mechanism. Transient assay of a HES-1 (−467–+46) luciferase reporter plasmid in HepG2 cells transfected with either TORC1/MAML2, MAML2, or TORC2 expression vector as indicated. Effect of dominant-negative A-CREB plasmid on HES-1 reporter activity shown. Molecular Cell 2003 12, 413-423DOI: (10.1016/j.molcel.2003.08.013)

Figure 7 Distinct Domains of CREB Interact with Different Coactivators to Regulate Cellular Gene Expression Phosphorylation of CREB at Ser133 promotes recruitment of CBP and consequent acetylation of nucleosomal histones. The tetrameric TORCs bind the bZIP domain and potentiate CREB activity by enhancing recruitment of TAFII130 to the Q2 domain of CREB. Molecular Cell 2003 12, 413-423DOI: (10.1016/j.molcel.2003.08.013)