Tunable regulation of CREB DNA binding activity couples genotoxic stress response and metabolism

Abstract

cAMP response element binding protein (CREB) is a key regulator of glucose metabolism and synaptic plasticity that is canonically regulated through recruitment of transcriptional coactivators. Here we show that phosphorylation of CREB on a conserved cluster of Ser residues (the ATM/CK cluster) by the DNA damage-activated protein kinase ataxia-telangiectasia-mutated (ATM) and casein kinase1 (CK1) and casein kinase2 (CK2) positively and negatively regulates CREB-mediated transcription in a signal dependent manner. In response to genotoxic stress, phosphorylation of the ATM/CK cluster inhibited CREB-mediated gene expression, DNA binding activity and chromatin occupancy proportional to the number of modified Ser residues. Paradoxically, substoichiometric, ATM-independent, phosphorylation of the ATM/CK cluster potentiated bursts in CREB-mediated transcription by promoting recruitment of the CREB coactivator, cAMP-regulated transcriptional coactivators (CRTC2). Livers from mice expressing a non-phosphorylatable CREB allele failed to attenuate gluconeogenic genes in response to DNA damage or fully activate the same genes in response to glucagon. We propose that phosphorylation-dependent regulation of DNA binding activity evolved as a tunable mechanism to control CREB transcriptional output and promote metabolic homeostasis in response to rapidly changing environmental conditions.

Figure 1.

cAMP-induced phosphorylation of the CREB ATM/CK cluster. (A) Schematic depiction of the CREB ATM/CK phosphorylation cluster. (B) CREB^+/+ MEFs were pretreated with KU-55933 (KU) for 30 min and then the cells were treated with Fsk for 90 min. Cell extracts were immunoblotted with α-pCREB-108/111/114, α-CREB or α-HSP90 antibodies. (C) CREB^+/+ MEFs were pretreated with either H-89 (PKA inhibitor), KU-55933 (ATM inhibitor) or 20 Gy IR followed by Fsk for 30 or 90 min. Cell extracts were analyzed using α-CREB, α-pCREB-108/111/114 or α-HSP90 antibodies. (D) Phosphorylation of CREB was analyzed by western blotting using cell lysates prepared from HEK 293T cells transfected with plasmids encoding CREB^WT, KCREB, CREB^Q321A (CRTC2 binding mutant) or Gal4-CREB. The transfected cells were treated with Fsk or 2 nM CLM for 60 min. CREB expression and phosphorylation were determined by immunoblotting with indicated antibodies. Arrowheads (lane 1–9) indicate presumed Sumo-CREB detected by CREB antibodies. (E) Recombinant CREB was incubated with CK1 and ATP with the indicated amount of CRE oligonucleotide. CREB phosphorylation was analyzed by western blotting using α-CREB or α-pCREB-121 antibodies. (F) Recombinant CREB was phosphorylated with CK1 in the presence or absence of CRE or scrambled DNA (Scr) for the indicated times. CREB phosphorylation was analyzed by western blotting using α-CREB or α-pCREB-121 antibodies.

Figure 2.

ATM/CK cluster positively and negatively regulates CREB transcriptional activity. (A and B) A Ser-111 to Ala knock-in mutation abolishes ATM/CK cluster phosphorylation in response to DNA damage (A) and Fsk (B). In panel (A) ex vivo cultured splenocytes were sham treated or exposed to 10 Gy IR and cell extracts were prepared 1 h later for western analysis using the indicated antibodies. In panel (B) CREB^+/+ and CREB^S111A MEFs were treated with 10 μM Fsk and the cells were collected at the indicated times. Cell extracts were immunoblotted with α-pCREB-108/111/114, α-pCREB-133 or α-CREB antibodies. (C) CREB^S111A protein exhibited increased affinity for the CBP KIX domain. GST-KIX pull-downs were performed using spleen extracts from CREB^+/+ or CREB^S111A mice before or after 10 Gy irradiation. GST-KIX binding activity was measured by densitometric analysis. n = 4. (D) DNA damage-resistant gene expression in CREB^S111A MEFs. CREB^+/+ and CREB^S111A MEFs were treated as in (E) and levels of indicated genes determined by qPCR. Each bar represents averaged results for three biological replicates, assayed three times each. (E) Fsk-inducible genes in CREB^+/+ and CREB^S111A MEFs determined by microarray analysis. (F) Defective cAMP-induced expression of CREM in CREB^S111A MEFs. CREB^+/+ and CREB^S111A MEFs were treated with Fsk for 90 min and CREM mRNA expression analyzed by qPCR. Each bar represents averaged results, n = 24. Data information: in (C–F), data are presented as mean ± SEM. *P < 0.05, **P < 0.02 (Student's t-test).

Figure 3.

Impact of DNA damage and CREB S111 phosphorylation on coactivator recruitment and DNA binding activity. (A) ChIP analysis of CREB, CBP and CRTC2 over the CREM promoter in CREB^+/+ and CREB^S111A MEFs. Cells were treated with Fsk for 90 min, irradiated with 20 Gy IR for 120 min (20 Gy) or treated with 20 Gy IR 30 min prior to Fsk (20 Gy-Fsk) and processed for ChIP-qPCR using the indicated antibodies. Y-axis represents the CREM promoter occupancy. Each bar represents averaged results, n = 3. Error bars indicate SEM. *P < 0.05 (Student's t-test). (B) DNA damage inhibits CREB–CRTC2–DNA ternary complex formation in an S111 phosphorylation-dependent manner. HEK 293T cells transfected with CREB^WT or CREB^3A were treated with 2 nM CLM for 1 h and cell lysates incubated with GST-CRTC2^CBD and either CRE-containing or scrambled oligonucleotides in the presence of GSH-agarose beads. Immobilized CREB–CRTC2–DNA ternary complexes were washed under indicated buffer conditions analyzed by western blotting with α-CREB and α-GST antibodies. (C) Immortalized CREB^+/+ and CREB^S111A MEFs were treated with Fsk for 90 min, treated with 2 nM CLM for 120 min (CLM), or treated with CLM for 30 min prior to Fsk (CLM + Fsk). CREB–CRE complexes were analyzed by electrophoretic mobility shift assay (EMSA). (D) DNA-binding activity was analyzed by EMSA using cell lysates prepared from HEK 293T cells transfected with empty vector, or plasmids encoding CREB^WT, CREB^3A (100A, 111A and 121A), CREB^Q112D or DNA-binding defective KCREB. The transfected cells were irradiated with 50 Gy IR for 1 h and CREB expression analyzed by immunoblotting. (E) CRE binding activity was measured by densitometric analysis. *P < 0.05 (Student's t-test). (F) DNA damage caused dissociation of CREB from bulk chromatin. HeLa cells were irradiated with 10 Gy IR, collected at the indicated times, and fractionated into soluble (Sol) or chromatin (Chr) fractions prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresisand immunoblotting with α-CREB or α-MCM3 antibodies. (G) CREB chromatin dissociation is ATM dependent. HeLa cells were pretreated with DMSO or ATM inhibitor (KU-55933) for 30 min, and then irradiated with 10 Gy IR for 1 h. Soluble (Sol) or chromatin (Chr) fractions were analyzed using α-CREB, α-HSP90, α-MCM3 or α-TDP-43 antibodies. (H) The S111A mutation inhibits IR-dependent CREB chromatin eviction. Chromatin fractionation of CREB^+/+ and CREB^S111A MEFs was carried out 1 h after irradiation of 10 Gy IR.

Figure 4.

Phosphorylation of the ATM/CK cluster inhibits DNA binding and CREB–CRTC2–DNA ternary complex formation in graded fashion. (A) ChIP analysis of CREB phosphosite mutants. HeLa cells transfected with FLAG-tagged CREB^WT, CREB^S114A, CREB^S117A, CREB^S121A or CREB^3A were treated with 2 nM CLM for 1 h and subjected to ChIP-qPCR using α-FLAG antibodies and primers spanning the CREB binding site in the CREM/ICER promoter. *P < 0.05 (Student's t-test). (B) Effects of CREB phosphosite mutations on CREB–CRTC2–DNA ternary complex formation. HEK 293T cells transfected with CREB^WT, CREB^S114A, CREB^S117A, CREB^S121A or CREB^3A were treated with 2 nM CLM for 1 h and cell lysates incubated with GST-CRTC2^CBD and either CRE-containing or scrambled oligonucleotides in the presence of GSH-agarose beads. Immobilized CREB–CRTC2–DNA ternary complexes were washed under indicated buffer conditions analyzed by western blotting with α-CREB and α-GST antibodies. (C) Densitometric analysis of CREB binding to GST–CRTC2^CBD–DNA complexes. *P < 0.05 (Student's t-test). (D) Intramolecular interaction between Q2-bZIP and amino-terminal regions of CREB. Recombinant 6×-His-CREB lacking the bZIP domain (CREB 1–197) was incubated with cell lysates from HEK 293T cells transfected with GFP or GFP fused to a minimal CREB bZIP domain (GFP-bZIP) or an extended bZIP domain containing a portion of the adjacent Q2 domain (GFP-Q2-bZIP). The 6×- His tag pull-downs were analyzed by immunoblotting with α-GFP antibody. (E) Interaction between Gal4-CREB and Q2-bZIP is unaffected by DNA damage. GFP, GFP-Q2-bZIP or GFP-bZIP were immunoprecipitated (IP) from transfected HEK 293T cells with α-GFP antibody. The IP samples were incubated with cell lysates from HEK 293T cells separately transfected with Gal4-CREB with or without treatment of 2 nM CLM. Co-immunoprecipitated proteins were analyzed by western blotting with α-GFP or α-CREB antibodies.

Figure 5.

CREB S111 phosphorylation contributes to the bidirectional regulation of gluconeogenic genes by cAMP and IR in vivo. (A) CREB^+/+ and CREB^S111A mice were injected with either PBS or 200 μg/kg glucagon and expression levels of G6P and PEPCK at 1 h analyzed by qPCR. Each bar represents averaged results for three biological replicates, assayed three times each. Error bars indicate SEM *P < 0.02 (Student's t-test). (B) CREB^+/+ and CREB^S111A primary hepatocytes were treated with glucagon for 60 min, irradiated with 20 Gy IR for 120 min (20 Gy) or treated with glucagon for 60 min after 20 Gy IR for 30 min (20 Gy-glucagon). *P < 0.05 (Student's t-test). (C) CREB^+/+ and CREB^S111A primary hepatocytes were treated with Fsk for 90 min, irradiated with 20 Gy IR for 120 min (20 Gy) or treated with Fsk for 90 min after 20 Gy IR for 30 min (20 Gy-Fsk). *P < 0.05 (Student's t-test). (D) CREB^S111A mice exhibit hyperglycemia. Fasting blood glucose levels were measured in 19-week-old male mice (n = 7 CREB^+/+ and 10 CREB^S111A, respectively) under fasted (16 h) conditions. (E and F) CREB^S111A mice maintained on a high fat diet exhibit increased body weight (E) and adiposity (F). (G) Working model for phosphoregulation of CREB DNA binding activity in response to cAMP and DNA damage.