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. 2016 Jul 18;44(20):9667–9680. doi: 10.1093/nar/gkw643

Figure 2.

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 CREBS111A 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) CREBS111A protein exhibited increased affinity for the CBP KIX domain. GST-KIX pull-downs were performed using spleen extracts from CREB+/+ or CREBS111A 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 CREBS111A MEFs. CREB+/+ and CREBS111A 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 CREBS111A MEFs determined by microarray analysis. (F) Defective cAMP-induced expression of CREM in CREBS111A MEFs. CREB+/+ and CREBS111A 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).