Journal: The Journal of Neuroscience
Article Title: p38 MAP Kinase Mediates Both Short-Term and Long-Term Synaptic Depression in Aplysia
Figure Lengend Snippet: Ap-p38 kinase activates CREB2 and induces histone deacetylation. A, Ap-p38 kinase phosphorylates CREB2. GST or the GST-CREB2 fusion protein was incubated with activated Ap-p38 and γ-labeled 32P-ATP at 30°C for 30 min. The products were analyzed by running a gel and then exposing to an x-ray film. The kinase phosphorylated the GST-CREB2 fusion protein but not GST. B, Ap-p38 kinase activity is critical for recruiting CREB2 and HDAC5 to the C/EBP promoter. With ChIP assays, a basal level of CREB2 and HDAC5 was detected at the promoter in unstimulated neurons. Treatment with FMRFa (10 μm) for 90 min induced more CREB2 and HDAC5 to bind. SB 203580 (1 μm) lowered the basal amount of both CREB2 and HDAC5, an effect similar to that of a 90 min treatment with 5-HT. The inhibitor also blocked the FMRFa-induced recruitment of CREB2 and HDAC5. As control, chromatin samples were also analyzed before immunoprecipitation (Input) to show that equal amounts of starting material were applied. C, Ap-p38 kinase activity is necessary for FMRFa to induce histone deacetylation. Basal histone H4 acetylation (Acetyl H4) was detected at the C/EBP promoter. Although treatment with 5-HT (S) increased H4 acetylation, FMRFa, either alone (F) or together with 5-HT (FS), decreased the acetylation. SB 203580 blocked the FMRFa-induced histone deacetylation at the promoter (Acetyl H4/SB 203580). In addition, 5-HT (S) induced the specific acetylation of lysine 8 of H4 [Acetyl H4 (K8)], and FMRFa cotreatment (FS) blocked this acetylation. SB 203580 blocked this inhibitory effect of FMRFa [Acetyl H4 (K8)/SB 203580]. As control, chromatin samples were also analyzed before immunoprecipitation (Input). All of the samples were also analyzed with the primers specific to the promoter of Aplysia histone H4, a gene with a strong basal expression but no response to either 5-HT or FMRF (Guan et al., 2002). No changes were observed after any of the treatments (data not shown).
Article Snippet: Human ATF2 (2 μg) (Cell Signaling Technology) was incubated with phosphorylated Ap-p38 kinase in kinase buffer (Cell Signaling Technology) supplemented with 0.2 m m ATP at 30°C for 30 min. Proteins from the reaction mixture were separated by SDS-PAGE, and the phosphorylated transcription factor was quantified by immunoblotting using anti-phospho-ATF2 (Cell Signaling Technology).
Techniques: Incubation, Labeling, Activity Assay, Immunoprecipitation, Expressing